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PCR METHODS IN FOODS

FOOD MICROBIOLOGY AND FOOD SAFETY SERIES

Food Microbiology and Food Safety publishes valuable, practical, and timely
resources for professionals and researchers working on microbiological topics
associated with foods, as well as food safety issues and problems.

Editor-in-Chief
Michael P. Doyle

Regents Professor and Director of the Center for Food Safety
University of Georgia
Griffin, Georgia

Editorial Board
Francis F. Busta

Director

National Center for Food Production and Defense

University of Minnesota

Minneapolis, MN

Bruce R. Cords

Vice President

Environment, Food Safety & Public Health

Ecolab Inc.

St. Paul, MN

Catherine W. Donnelly

Professor of Nutrition and Food science
University of Vermont
Burlington, VT

Paul A. Hall

Senior Director Microbiology & Food Safety
Kraft Foods North America
Glenview, IL

Ailsa D. Hocking

Chief Research Scientist
CSIRO

Food Science Australia
North Ryde, Australia

Thomas J. Montville

Professor of Food Microbiology
Rutgers University
New Brunswick, NJ

R. Bruce Tompkin

Formerly Vice President-Product Safety
ConAgra Refrigerated Prepared Foods
Downers Grove, IL

PCR METHODS IN FOODS

Edited by
John Maurer

The University of Georgia, Athens

Athens, GA, USA

Springer

Dr. John Maurer

252 Poultry Diagnostic and Research Center

College of Veterinary Medicine

The University of Georgia

Athens, GA 30602

USA

ISBN-10: 0-387-28264-5
ISBN- 13: 978-0387-28264-0

Printed on acid-free paper.

2006 Springer Science+ Business Media, Inc.
All rights reserved. This work may not be translated or copied in whole or in part without the written
permission of the publisher (Springer Science+Business Media, Inc., 233 Spring Street, New York, NY
10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection
with any form of information storage and retrieval, electronic adaptation, computer software, or by similar
or dissimilar methodology now know or hereafter developed is forbidden.

The use in this publication of trade names, trademarks, service marks and similar terms, even if they are
not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject
to proprietary rights.

Printed in the United States of America. (SPI/SBA)

987654321

springeronline. com

Preface

This book will introduce non-molecular biologists to diagnostic PCR-based tech-
nologies for the detection of pathogens in foods. By the conclusion of this book,
the reader should be able to: 1) understand the principles behind PCR including
real-time; 2) know the basics involved in the design, optimization, and imple-
mentation of PCR in food microbiology lab setting; 3) interpret results; 4) know
limitations and strengths of PCR; and 5) understand the basic principles behind
a new fledgling technology, microarrays and its potential applications in food
microbiology. This book will provide readers with the latest information on PCR
and microarray based tests and their application towards the detection of
bacterial, protozoal and viral pathogens in foods. Figures, charts, and tables will
be used, where appropriate, to help illustrate concepts or provide the reader with
useful information or resources as an important starting point in bringing
molecular diagnostics into the food microbiology lab. This book is not designed
to be a "cookbook" PCR manual with recipes and step-by-step instructions but
rather serve as a primer or resource book for students, faculty, and other
professionals interested in molecular biology and its integration into food safety.

Table of Contents

Preface

Chapter 1. PCR Basics

Amanda Fairchild, M.S., Margie D. Lee DVM, Ph.D.,

and John J. Maurer, Ph.D 1

Chapter 2. The Mythology of PCR: A Warning to the Wise

John J. Maurer, Ph.D

Chapter 3. Sample Preparation for PCR

Margie D. Lee, DVM, Ph.D. and Amanda Fairchild, M.S 41

Chapter 4. Making PCR a Normal Routine of the Food Microbiology Lab

Susan Sanchez, Ph.D 51

Chapter 5. Molecular Detection of Foodborne Bacterial Pathogens

Azlin Mustapha, Ph.D. and Yong Li, Ph.D 69

Chapter 6. Molecular Approaches for the Detection of Foodborne Viral

Pathogens

Doris H. D'Souza and Lee-Ann Jaykus 91

Chapter 7. Molecular Tools for the Identification of Foodborne Parasites

Ynes Ortega, Ph.D 119

Index 147

Vll

CHAPTER 1

PCR Basics

Amanda Fairchild 1 , M.S., Margie D. Lee 12 DVM, Ph.D.,

and John J. Maurer 1 ' 2 *, Ph.D.

Poultry Diagnostic & Research Center 1 , College of Veterinary Medicine,

The University of Georgia, Athens, GA 30602
Center for Food Safety 2 , College of Agriculture and Environmental Sciences,

The University of Georgia, Griffin, GA 30223

Introduction

Using Molecular Methods to Identify Microbial Pathogens
The Theory Behind PCR
Thermocycler Technology
Detection
Advanced PCR Technologies

Real-Time PCR

Multiplex PCR

Terminal Restriction Fragment Length Polymorphisms

Microarrays
Design and Optimization of Diagnostic PCR as Applicable to Food
Microbiology

Systematic Approach to Creating Your Own PCR
Access DNA Databases to Retrieve Sequences or Search for DNA Matches
References

INTRODUCTION

The safety of your food supply is an important goal of the U.S. government and
diagnostic food microbiologists across the country. Up to 5,000 deaths and 76
million illnesses in the U.S. each year are associated with the consumption
of foods laced with pathogenic bacteria (53), costing the U.S. an estimated
$6.5-$34.9 billion annually (8). Even though bacteria have been shown to be the
cause of the majority of food-related illnesses, the government does not have a
mechanism for detecting and accounting for the losses due to other common
foodborne pathogens, such as viruses and protozoa. Detection, identification,
and quantification of foodborne pathogens are often made difficult by the low
numbers of pathogenic organisms and interference from the food matrix that is
being sampled. Bacterial pathogens of particular importance include Listeria,
Campylobacter, Escherichia coli, and Salmonella (53), and the norovirus and

Corresponding author. Phone: (706) 542-5071; FAX: (706) 542-5630; e-mail: jmau-
rer@vet.uga.edu.

PCR Basics

hepatitis A virus are currently regarded as important foodborne viruses (44).
However, since the advent of the polymerase chain reaction, finding these few
pathogenic microorganisms in otherwise innocent looking provisions is
becoming easier, mainstreamed, and second nature to many diagnostic labora-
tories. The polymerase chain reaction (PCR) is a simple way to quickly amplify
specific sequences of target DNA from indicator organisms to an amount that
can be viewed by the human eye with a variety of detection devices. A goal of
the present-day food microbiology research laboratory is to use the growing
database of bacterial genomic information, made available by researchers
mapping unique identifier genes of foodborne pathogens, to design monitor-
ing systems capable of analyzing various incoming samples for hundreds of
different organisms accurately and efficiently.

Using Molecular Methods to Identify Microbial Pathogens. Prior to the 1980s and
the advent of PCR, identification of microbial pathogens relied on bacteriological
methods to enrich and isolate the organism from clinical or food sample, and sub-
sequent biochemical and/or immunological tests to confirm the microbe's identity.
During the past 30 years, we have gained tremendous insights into how microor-
ganisms spread and cause disease. In several instances, pathology associated with
many bacterial illnesses is attributed to a single gene (4, 9, 18, 75, 97). Other
pathogens like Salmonella are more complex, requiring coordinate regulation of
several virulence gene sets to cause disease (49). Therefore, the organism's genetics
or genotype dictates its ability to cause disease, or the severity of the illnesses asso-
ciated with it. Many of these virulence genes are unique to the pathogen and sub-
sequently make useful markers for identifying said pathogen (37, 41, 86, 95). With
several bacterial genomes completed, we now know the genetic basis for pheno-
types that have been useful markers for distinguishing pathogens from closely
related commensals that inhabit the same niche. By identifying gene(s) associated
with phenotype (e.g., 0157 serotype), we have identified a marker with greater
specificity than afforded by the actual antigen itself, especially when confronting
cross-reactivity and false positive associated with the immunological test (50).
Although quite specific, the early molecular-based method, DNA: DNA
hybridization had limited utility due to its limited sensitivity, time length, or safety
issues associated with the use of radioactive probes (77, 80). Even with the intro-
duction of nonradiometric methods for detecting hybridization of probe to its tar-
get gene, there was still the limitation of sensitivity, [i.e., the ability to detect the
fewest cells possible (80)]. How could one amplify the target gene enough to detect
its presence in the sample contaminated with organism X?

THE THEORY BEHIND PCR

The concept of the PCR was first described by Panet and Khorana in 1974(64)
and owes its name to Dr. Kary Mullis and colleagues, who developed the process
over the course of 4 months in 1983 at the Cetus Corporation. While driving
down Highway 128 in Mendocino County, California, Dr. Mullis let his mind

The Theory Behind PCR

slip back to the lab and a burning question that could not escape his mind. How
could someone go about reading the sequence known as DNA, the language of
our genes and blueprint for our existence? Like perfectly crafted ball and socket
joints, the oligonucleotide base pairs within the DNA molecule bond to one
another as the entire length of the ladder-shaped molecule twists into a
corkscrew shape. Dr. Mullis referred to DNA structure as something like a mass
of "unwound and tangled audio tape on the floor of the car in the dark" (58).
In 1953, Drs. Watson and Crick had mentioned the biological significance of
the DNA molecule with its complementary base pairing that suggested "a pos-
sible copying mechanism" for genetic material (91). One strand of DNA could
be a template for the formation of a new complementary chain, and in the end
you could have two DNA ladders, identical in every way. Dr. Mullis' develop-
ment of PCR has extrapolated the copy machine theory one step further. He
stated this simply as an analogy to a "'Find' sequence in a computer search"
(58). This technique would have equivalent power to the latest computer dis-
playing results of finding a document that consisted of just one word taking up
20 kilobytes of space on a hard drive the size of 150 Gigabytes littered with files
of different types and sizes; like finding the code for blue eyes — and that code
only — within the code that sums up every single trait for a person.

The second aspect Dr. Mullis had to account for in this process would be the
ability of the chemical program to display the located sequence in a large
enough fashion to be detected by the human eye. Dr. Mullis knew that if he
could produce a short piece of DNA to find a sequence flanking a gene of inter-
est and then start a process that could make the sequence reproduce itself over
and over (hence a chain reaction), the concept of PCR could be realized. After
all, it was already known that DNA innately makes a copy of itself when cells
divide, so that each daughter cell can have a copy. If he introduced his "find"
search strings, or primers, into a tube with DNA encouraged to uncoil from its
natural double helix by heating and a biological glue (polymerase) to attach
deoxynucleoside triphosphates (dNTPs) to the freshly uncoiled DNA at the
location, where the primers bonded, a new copy of the DNA would be pro-
duced as specified by the primers that included the gene(s) of interest as the
temperature decreased. The DNA polymerase that is used in some PCR reac-
tions is made from the bacteria Thermus aquaticus, which was found originally
in Yellowstone thermal water reservoirs. It is stable in temperatures above that
which denatures DNA, making it a perfect enzyme for the job of attaching free
dNTPs to make a new strand of DNA. There are other polymerases available
that can actually proofread the addition of dNTPs, so there will be no errors
made in the synthesis of longer PCR products. After each cycle in a PCR assay,
the amount of DNA present doubles, so repeat the cycle and there are 4 copies
of the gene, repeat again and there are 8 copies. With 30 cycles of this process,
there would theoretically be just over a billion copies of the sequence in ques-
tion (2 30 = 1.07 billion). Find a way to tag each copy to make it visible to the
human eye, with more copies making a stronger visible signal and you have
proof of the presence of the small sequence embedded within the large DNA
molecule you started with. A widely used method for viewing PCR products

PCR Basics

involves running them on an agarose gel, staining the gel with ethidium bro-
mide, and observing and photographing the gel on ultraviolet (UV) light source
(56). The process is relatively fast, dictated by the amount of time it takes to
heat the DNA strands until they will separate, the time to reduce the tempera-
ture so the primers bind to the single-stranded DNA, and the time allowed for
the polymerase to add individual deoxynucleoside triphosphates to extend the
forming DNA molecule. Dr. Mullis stated that scientists claimed that PCR
made DNA research boring (57). Even though PCR is often considered "cook-
book chemistry" because of its simplicity, his suggestion could not be further
from the truth. For example, PCR has been one of the most important genetic
tools available to those mapping the human genome and for those attempting
to detect pathogenic bacteria, viruses, and protozoa. The PCR has made its way
from the research lab to forensic and diagnostic laboratories worldwide. There
have been considerable efforts to validate and standardize this tool (17, 38); to
become a normal routine task/service performed by reference laboratories
(3, 48, 76), and clinical diagnostic and food microbiology laboratories (1, 33, 38,
45, 54, 79, 87).

THERMOCYCLER TECHNOLOGY

Since the technique of PCR was developed, there have been many evolutions of
the equipment that makes the process possible, based on the concept that
strands of DNA denature, or unwind, and anneal, or wind again back into the
helical corkscrew, in response to fluctuations in temperature. The first success-
ful PCR reaction took place using water baths at the appropriate temperatures
for each step in the procedure, with the technician moving vials by hand from
bath to bath at the appointed time, for 30 or more cycles to get adequate
amounts of DNA copies that could be detected. Nowadays, thanks to automa-
tion, PCR reactions can be set up in thermocyclers that over the course of min-
utes to a few hours reliably yield high numbers of a specific DNA sequence if
present in a sample.

The standard thermocycler uses a large heating block, into which microcen-
trifuge tubes are placed. This type of thermocycler through its computer con-
trols the heating and coolings of the blocks through the cycles of each reaction.
Sometimes an oil or wax overlay is put on the samples within the microcen-
trifuge tubes to keep the sample from escaping the bottom of the tube during
the heating for the denature step of the PCR reaction. This type of machine is
not as desirable because of the time it takes to heat and then cool the entire
block to the appropriate temperature within each cycle. The time required for
the heat block to uniformly reach each temperature coupled with the slow heat
transfer rates to the microcentrifuge tubes makes this type of thermocycler vir-
tually inadequate with today's demand for high-speed accurate amplification of
PCR products.

The RapidCycler, manufactured by Idaho Technologies, is an example of
equipment designed to provide the quick temperature cycling necessary for

Thermocycler Technology

PCR reactions. This type of thermocycler uses heat transfer through blasts
of high-velocity hot air to accomplish the temperature transactions from the
initial heating of the DNA sample through the annealing of the primers and the
extension of the new double strand of DNA by the polymerase. There is overall
temperature uniformity within the cavity of the reaction vessel and rapid heat
exchange within the sample because individual samples are loaded into micro-
capillary tubes or thin walled microcentrifuge tubes for the reactions to take
place. This also allows for a smaller overall volume in each reaction tube, thus
saving valuable amounts of reaction components such as polymerase and
primers. After the PCR cycles are complete, the samples are loaded on an
agarose gel that contains ethidium bromide, and viewed under a UV light source.

A gradient thermocycler allows the clinician to optimize each of the three
temperatures needed for the denaturing, annealing and extension of new DNA
products. Optimization might be required if an existing PCR cycle program
cannot be located for detection of a particular gene sequence. Optimizing the
PCR reactions is critical to the success of the production of amplicons and is
not always the easiest thing to do. The melting temperature can be calculated
for the primers when they are made, but the denaturing and annealing temper-
ature of the cycle might have to be determined by educated guess with some
trial and error, possibly rerunning the same reaction with many different tem-
peratures before the best fit temperature is found. Luckily, most machines have
the ability to reach the different temperatures at the same time (thus the gradi-
ent), so the reactions are run at the same time with results from each tempera-
ture trial collected at the same time. The block used to hold the samples in this
type of machine can be programmed to heat over a gradient of about 20° C
range with the annealing temperature for the PCR reactions increased by an
increment of 1 or 2°C.

Another thermocycler type offers PCR product detection at the same time
as each cycle of the PCR reaction progresses. It allows the technician to track

Figure 1.1. Example of real-time PCR output graph showing amount of DNA
sequences produced over 40 PCR cycles.

PCR Basics

the increase in products during a PCR reaction as displayed on a graph
(Fig. 1.1). Higuchi and colleagues introduced this feature, dubbed "Real-Time"
PCR, and described how the number of cycles necessary to produce a detectable
fluorescence was "directly related to the starting number of DNA copies" in a
sample (32). There are a number of companies that offer this technology, which
combines the rapid cycle polymerase chain reaction with fluorescent detection
of amplified PCR product in the same closed vessel as the reaction mix. The
primers are usually labeled with fluorogenic probes, or a DNA-binding dye is
included in the PCR reaction, which fluoresces under light emitted at a certain
wavelength.

DETECTION

Detection of the PCR product or "amplicon" can be accomplished several
ways. Following PCR, the sample is loaded into an agarose gel, and the DNA
fragment(s) or amplicon if present in the sample is separated by electrophoresis
based on size. Molecular weight, DNA standards are included to estimate size
of amplicon(s) present in positive samples and positive control. The agarose gel
and electrophoresis buffer contain a dye, ethidium bromide that binds double-
stranded DNA and fluoresces upon excitation with UV light. This dye is used
to visualize the DNA in an agarose gel. As the primers bind to fixed position
within the target sequence, the expected size for our PCR product/amplicon is
the distance between the forward and reverse primers. For example, if forward
and reverse primers bind target gene X at positions 850 and 1000, respectively,
then one expects to observe an amplicon of 150 bp for positive control or any
sample that bears organism that contains gene X. The size of the amplicon is
extrapolated from the DNA standards included in the gel. The sample MUST
produce an amplicon of expected size predicted for the primers used and cor-
robated by the positive control before it can be considered positive by PCR.
There is an inverse linear correlation between the log 10 size of the DNA frag-
ment (bp) and the distance migrated by the DNA fragment in the agaraose gel.
The smaller the DNA fragment the farther it migrates through the agarose gel
during electrophoresis. Therefore one can estimate the amplicon's size from
DNA standards included with the agarose gel. As most PCRs produce small
size amplicons (100-1,000 bp), one must use DNA standards that accommo-
date this size range and agaraose concentration (1.5%) that resolves small DNA
fragments sufficiently to accurately determine the size for DNA band X. The
PCR result is recorded photographically with a polaorid or digital camera with
the appropriate lens filters and exposures for capturing images illuminated by
the UV light.

Detection systems are slowly moving towards nongel methods for detecting
and recording PCR results. Enzyme-linked immunosorbent assay (ELISA) has
been developed for detecting amplicons (37). In the PCR reaction mix, the stan-
dard nucleotides have been substituted for chemically "tagged" nucleotides
(e.g., dioxygenin or DIG). During PCR, the "DIG'-labeled nucleotides are

Detection

dsDNA

94°C

Heat (Denature DNA)

regular nucleotide bases
Dig - labeled nucleotide

DNA polymerase
primer

ssDNA

C~ ft*) °C^\a P runer Annealing

65°C~ 72 °C / DNA polymerase(Extension)

LLi

A A

V V

A A

VV V V

Denature

94°C

Anneal

50 °C

►

A A A A

Enzymatic
detection

digoxigenin
: biotin
: strepavidin

IJ : p -nitrophenyl phosphate

anti-digoxigenin -alkaline phosphatase

Figure 1.2. PCR-ELISA. Digoxygenin (DIG) labeled, nucleotides are incorporated
into amplicon during PCR (A). An internal, 3' biotinylated oligoprobe anneals to
denatured, single-stranded amplicon following PCR. The strepavidin, coating the
wells, binds to the biotin moiety of the oligoprobe and thus captures the amplicon.
The amplicon is then detected using anti-DIG antibody enzyme conjugate (B). The
oligoprobe adds additional specificity to this PCR test.

incorporated into the amplicon as it is synthesized (Fig. 1.2A). Following the
last round of PCR, the sample is denatured and allowed to anneal with 5',
biotin-labeled, internal oligonucleotide. This oligoprobe binds to the comple-
mentary sequence present within the amplicon. This amplicon-oligoprobe
hybrid is captured in strepavidin-coated 96 well microplate through the interac-
tion of the biotin group with the strepavidin (Fig. 1.2B). The bound amplicon
is visualized colormeterically using anti-DIG antibody enzyme conjugate, usu-
ally either horseradish peroxidase or alkaline phosphatase. The advantage of

8 PCR Basics

this "PCR-ELISA" is that it easy to scale-up for high throughput of samples
and lends itself quite well to automation. Another nongel method for detecting
PCR amplicons involves detecting fluorescent dyes bound to or released from
the amplicon using a fluorometer. This detection method is the basis of real-
time PCR discussed below.

ADVANCED PCR TECHNOLOGIES AND MICROARRAYS

Real-Time PCR. Real-time PCR technology is based on the ability for the detec-
tion and quantification of PCR products, or amplicons, as the reaction cycles
progress. Higuchi and colleagues introduced this technology (32) and it is made
possible by the inclusion of a fluorescent dye that binds the amplicon as it is
made (Fig. 1.3 A). There are several ways to detect the PCR products under flu-
orescent detection. In TaqMan PCR, an intact, "internal" fluorogenic oligo-
probe binds target DNA sequence, internal to the PCR primer binding sites.
This oligprobe possesses a reporter dye that will fluoresce and a suppressor dye
known as a quencher that prevents fluorescent activity via fluorescence reso-
nance energy transfer (FRET). After each PCR cycle, when the double-
stranded DNA products are made, a measure of fluorescence is taken after the
fluorogenic probe is hydrolytically cleaved from the DNA structure by the
exonuclease activity of the Thermus aquaticus DNA polymerase (29, 36). Once
cleaved, the probe's fluorescent activity is no longer suppressed (Fig. 1.3B).
FAM (6-carboxyfluorescein) and TAMRA (6-carboxy-tetramethyl-rhodamin)
are most frequently used as reporter and as quencher, respectively. This PCR is
often refered to as 5'exonuclease-based, real-time PCR or TaqMan PCR (55).
When a DNA-binding dye is used, as more DNA copies are made with each
successive cycle of the PCR, they are all bound, or intercalated, with the dye,
and the fluorescence increases (Fig. 1.3A). SYBR Green I is the most frequently
used DNA-binding dye in real-time PCR.

Two additional advanced methods of amplicon detection are hybridization
probes and molecular beacons. The hybridization method uses fluorescence reso-
nance energy transfer from one probe to another after annealing of the primers
to the template strand of DNA. One probe has a donor dye at the 3' end of the
oligonucleotide and the other probe has an acceptor dye at the 5' end. When both
probes anneal to the target sequences, they are situated to have the dyes adjacent
to one another one base apart. While in that configuration, the energy emitted by
the donor dye excites the acceptor dye, which emits fluorescent light at a longer
wavelength. The ratio between the two fluorescent emissions increases as the PCR
progresses and is proportional to the amount of amplicons produced. Molecular
beacons are short segments of single-stranded DNA. They use a hairpin shape to
facilitate quenching of the fluorescent signal until the probe anneals to the com-
plementary target DNA sequences, produced from PCR.

Some advantages of real-time technology include high sensitivity with the
use of an appropriate probe or DNA-binding dye, ability for detection of rela-
tively small numbers of target DNA copies, and ease of quantification because

Advanced PCR Technologies and Microarrays

dsDNA

940C

Heat (Denature DNA)

— : regular nucleotide bases

DNA polymerase

primer

fluorescent dye

ssDNA

300 o 650 C

Primer Annealing

1*1

dsDNA

650C- 720 C

DNA polymerase (Extension)

^1 M

Nffi \y [o^

•■k k^l xK

-^s >is >-■-

XN M^ X>

frfr [S?| >p

94°C

Heat (Denature DNA)

— : regular nucleotide bases
+ : internal TAQMAN probe

: DNA polymerase

: primer

ssDNA

▲ : quencher

30°O 65° C

Primer Annealing

fluorescent dye

MT1

65°C~72°C

♦

DNA polymerase (Extension)

■/.

♦

Figure 1.3. "Real-time" PCR detection of amplicons. Using fluorogenic dyes, ampli-
cons can be detected by a fluorimeter as they are synthesized with each PCR cycle.
Intially, a fluoresent dye, SYBR Green (A), was used to detect the amplicons. In this
PCR, SYBR Green binds the double-stranded, DNA amplicon and fluorescences
upon illumination with ultraviolet light (UV). Subsequently, real-time PCR was devel
oped using an internal oligoprobe for detecting the amplicons. In TaqMan PCR (B),
the oligoprobe contains a fluorscent marker and a chemical group that quenches fluo
rescence of the oligoprobe until the dye is liberated by 3' exonuclease activity of the
Taq DNA polymerase. This can only occur if the oligo binds the complementary
sequences present in the target gene and amplicon.

PCR Basics

of the lack of post-PCR detection measures. Molecular beacons can even be
used to detect single nucleotide differences (27). Disadvantages of real-time
PCR technology lie with the detection of the amplicons. If the DNA-binding
dye is used, then any double-stranded product is labeled and fluoresces, includ-
ing primer-dimers, and nonspecific amplicons, whether they are close to the tar-
get DNA in sequence or have erroneous secondary structure. The effectiveness
of the fluorogenic probe is also influenced by the creation of primer-dimers,
and both methods of detection are susceptible to less than optimal design of
primers for use in the PCR and primer concentration in the master mix. To
compensate for the unspecific binding of the DNA dye, real-time PCR equip-
ment has the capability of running a melting curve after the PCR assay, which
increases the temperature of the vessels in tiny increments until the fluorescence
is lost due to the DNA denaturing. When the melting temperature of the target
DNA sequence is reached, a sharp loss of fluorescence will be recorded. If addi-
tional losses are recorded, there may be PCR contamination, or the parameters
of the PCR assay were not stringent enough, such as suboptimal primer design
or temperature choice for the program. This feature of the equipment makes
DNA-binding dyes a feasible and often cheaper alternative to the other meth-
ods available. The melting temperature of the amplicons should be known when
designing primers for the assay and are usually referenced when programming
the annealing step of the PCR reaction. Accurate primer design and optimiza-
tion of the PCR reaction conditions for the primers are required in any PCR
application, but especially with real-time technology.

Multiplex PCR. Multiplex PCR is a way to amplify two or more amplicons in a
single PCR reaction. For multiplex PCR, each primer set is designed to its tar-
get gene to amplify a PCR product of a size unique to the target gene. To per-
form a multiplex PCR, the concentrations of primers, Mg 2+ , free dNTPs and
polymerase are altered to allow for the synthesis of the genes of interest, while
the PCR reaction temperature parameters are optimized to the best average for
amplicon production for all primer sets. This technique saves time and labor
since more than one target DNA sequence is detected for each reaction, but
might not be optimal if the PCR products are close in size and detection
requires viewing an agarose gel stained with ethidium bromide. In a single PCR
reaction, one can determine the identity of the organism (28, 39) or genotype
(21), as the amplicon(s) size is unique to specific organism or gene. Therefore, it
is possible to detect multiple pathogens in a sample from a single PCR test (65).

Terminal Restriction Fragment Length Polymorphisms (TRFLP). We can use

PCR to characterize microbial communities and identify member species using
a single PCR primer set. This PCR targets the 16S rDNA, a gene that is uni-
versally conserved among all bacterial species and amplifies a single ~1,500 bp
amplicon. We can resolve diversity of 16S rDNA amplicons generated from
this PCR using restriction enzyme(s) that recognize restriction sites within
genus or species specific sites within this gene and produce DNA fragments,
whose size corresponds to a specific genus or species (46). This PCR involves

Advanced PCR Technologies and Microarrays

using universal 16s rDNA primers in which one of the forward or reverse
primers is fluorescently tagged (Fig. 1.4). Following PCR, the amplicons are
digested with a restriction enzyme and subsequently loaded onto capillary bed
of an automated DNA sequencer. This method has been refined and applied to
automated DNA sequencers to resolve minor (x bp) differences between DNA
fragments, monitoring and measuring fluorescence associated with the various
sized DNA fragments as they elute from the sequencers' capillary bed (Fig. 1.4).
Fluorescently labeled molecular weight standards are included to calibrate col-
umn in order to demarcate and identify the molecular weight for each DNA
fragment separated by on the sequencer's capillary column. Each peak corre-
sponds to specific genus/species present within the sample. The identity is deter-
mined from comparisons to an established database of restriction fragments
predicted from 16S rDNA sequences (47). This database can be generated in
house, from cloning and sequencing your 16S rDNA library or comparing it
against an ever-expanding Web-based 16S rDNA database (Michigan State
University Center for Microbial Ecology; http:ll35.8.164.52lhtmllTAP-
trflp.html\ 47). The latter has a tool for analyzing your TRFLP profile against
this database, for various restriction enzymes. Depending on the restriction
enzyme used, one may not be able to resolve various species or genera with a

PCR

(16 S rDNA)

^B Restriction ^B Separate DNA fragments
r enzyme digest r column chromatography

Peak's position: Genus or species

Fluorescence

( =

Elution Time
DNA fragment size)

Change in composition of the microbial community

Sample 1

Sample 2

Fluorescence

OU£l^

Elution Time

Figure 1.4. Characterizing microbial communities and identification of pathogens in
foods from terminal restriction fragment length polymorphisms (TRFLP) of total
microbial community 1 6S rDNA. (A) Concept behind TRFLP. (B) Interpretation of
TRFLP.

12 PCR Basics

single restriction enzyme. This is because they produce the same size DNA
fragment with restriction enzyme X. It may take a number of different TRFLP
profiles of the same community, generated with different restriction enzymes,
before genera and/or species differences can be resolved (47). This method is
currently used in assessing stability and structure of microbial consortiums,
and it has been recently applied to analyzing changes in the community struc-
ture of gastrointestinal microflora in response to diet or probiotics (34, 42).
TRFLP can also identify signature peaks for microbial pathogens (14, 60),
where differences in 16S rDNA can be discerned between them and closely
related commensal organisms, exceptions E. coli vs. Salmonella (61).
Theoretically, TFLP and other molecular ecology tools (e.g., DGGE) will
prove useful towards analyses of microbial communities present in foods, gas-
trointestinal tracts of food animals, probiotics and starter cultures and deter-
mine the impact certain food processes have on their composition, with regards
to the food's safety for consumers.

Microarrays. Macroarrays, microarrays, high-density oligonucleotide arrays, and
microelectronic arrays are all part of a new technology that allows one to screen
for gene(s), sequence(s) or specific mRNA among myriad of possible sequences
or genes in a single test (22). DNA hybridization arrays are based on specific posi-
tioning of a myriad of oligonucleotides or PCR amplicons, representative of a
complete bacterial genome, on nylon membrane (macroarray), glass slide
(microarray), or electronic microchip (microelectronic array). Each position on
this solid support contains an oligonucleotide or PCR product unique to a par-
ticular gene. Total mRNA or genomic DNA from an organism is fluorescently or
radioactively labeled and used in hybridization with solid support. The bound
oligonucleotides or amplicons on the solid support serve to capture labeled probe
in the RNA: DNA or DNA: DNA hybridization (Fig. 1.5). The labeled nucleic
acid hybridizes to the position or "spot" on the solid support that contains com-
plementary sequence for the labeled probe to bind. Identity of gene or sequence
relates back to the original positioning of the oligonucleotides or amplicons on
the solid support (Fig. 1.5). This technology has already been applied towards the
study of bacterial gene expression (30, 71), host-microbe interactions (15, 73, 84),
bacterial evolution and population genetics (6, 11, 23, 70, 85, 96). Currently,
microarrays have been applied towards PCR-based detection of pathogens in the
environment (2, 43, 65, 88). At present, this methodology is experimental, per-
formed primarily by research laboratories. However, advancement in technologies
and manufacturing will someday make microarrays affordable and practical for
use in diagnostic setting, as PCR has now become.

DESIGN AND OPTIMIZATION OF DIAGNOSTIC PCR
AS APPLICABLE TO FOOD MICROBIOLOGY

To perform PCR in any microbiological application, the DNA sequences of an
infectious agent must be known, and the target sequences must be unique to the
organism(s) to be detected. For example, if a food sample is suspected to be

Design and Optimization of Diagnostic PCR as Applicable

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defined region on glass side or nylon membrane (A, B). The positioning of this cap-
ture probe on this solid matrix defines gene or signature sequence for organism X. If
any of the genes present on slide or membrane are present, then it will be amplified
during PCR and labeled with fluorescent nucleotide (C) and subsequently bound to
the complementary sequence present on the solid support (D, E). Position of fluores-
cent signal (F) identifies gene or organism present in the sample.

contaminated with bacteria "X", such as E. coli 0157, then a PCR can be used
to determine the presence of the bacteria if there is a gene that only that bacte-
ria possesses, such as an identifier gene "XI", or in the case of E. coli, the 0157
antigen biosynthesis gene (50). If the gene was found in more than one bacteria
type, say gene "XY4," additional PCRs would have to be performed to separate
bacteria that harbor that gene (bacteria X and bacteria Y) by looking for a
unique identifier gene of the target bacteria X, but at additional work, cost and
time for the clinician. The case of identifying E. coli 0157:H7 might require a
multiplex PCR approach because of the closely similar genes of the different
antigen subunit serotypes (21, 26). There are many genes that are shared within
the same genus and species of bacteria, such as the genes shared among patho-
genic E. coli strains. Instead of differentiating between bacteria X and Y, the
researcher is met with finding a uniqueness of bacteria XI versus bacteria X2.

14 PCR Basics

Systematic Approach to Creating Your Own PCR. The development and valida-
tion of PCR is a long and arduous journey from concept to application. It
involves identification of a candidate marker or allele for pathogen X, whose
distribution among microbes is strongly associated with the pathogen in ques-
tion, and the cloning and sequencing of the cognizant gene(s) associated with
the marker or allele (50). For antigenic variable, surface proteins like flagellin,
PCR, using primers that recognize conserved sequences flanking sequence vari-
able regions (19, 81, 83), and subsequent sequencing of the PCR amplicon has
identified sequences unique to serovar (26, 31) or pathogen (63), which subse-
quently led to development of serovar or pathogen-specific PCR (26, 31, 63).
Design and development of PCR is the pursuit of researchers and if a PCR is
available, commercially or otherwise, it is best to adapt this PCR to your lab
than having to start from "scratch". Therefore, for most our readers, the inter-
net, www.ncbi.nlm.nih.gov and the PUBMED search is the best place to look for
PCRs and protocols for screening foods.

In the past, PCR design was based on gene(s) or DNA sequences obtained
from screening plasmid clone (50) or transposon libraries (5, 16) for relevant
marker, subcloning and sequencing DNA inserts. This approach took consid-
erable time and resources. Now, in less time, we can sequence the entire genome
of a single bacterial species, and spend the remainder of our time at the com-
puter annotating its sequence, searching for signature sequences unique to
pathogen X. In 1995, the first organism was completely sequenced (20). Since
that time, 91 bacterial genomes of several, important human pathogens have
been sequenced, annotated, and published (www.tigr.org; accessed 2/16/05),
including several foodborne pathogens (10, 12, 35, 52, 59, 66, 69, 74, 82, 92).
From comparisons of these bacterial genomes, especially between closely
related commensals and pathogens, several regions within the chromosome
have been identified that appear to be unique to organism X that is tied to its
virulence (7, 62, 93), or metabolism (69). With the growing number of bacter-
ial genomes present in public accessible DNA databases, identification and
design of PCR for organism can be done in silico, on your desktop computer.
A priori, of course is that organism X's genome has been sequenced and
accessible to the user. With advances in PCR and in silico analyses of bacter-
ial genomes, we can amplify, clone and sequence large regions of the bacterial
chromosome to quickly identify target DNA sequences for PCR primer design
(89, 90)

Access DNA Databases to Retrieve Sequences or Search for DNA Matches.

For the researcher, the most important resource, second only to the library
and PUBMED, is the DNA database, GenBank at the National Center
for Biotechnology Information, National Institutes of Health, Bethesda,
Maryland. This database can be accessed via the internet at the following
Website: www.ncbi.nlm.nih.gov, go to the ENTREZ selection at the top of the
page, and then go to GenBank on the next Web page. One can then search the
database of sequences by typing in keywords or combination of words for a
specific organism, serovar, or gene(s). Prior to this search, it is important to do

Design and Optimization of Diagnostic PCR as Applicable 15

your initial research in the library, so that your GenBank search is refined and
specific to pull out select sequences from the millions, probably billions, of data
base entries present at this Website. The next step is to access a specific
GenBank accession, for this exercise we will examine the Salmonella enterica
Typhimurium LT2 genome at NCBI, GenBank Accession # NC 003917
(Fig. 1.6A, B) and search the annotated genome for the invasion gene invA (24,
25) by using the search function in Netscape Navigator for the word "invA" to
find the beginning and end of each gene's open reading frame (ORF)
(Fig. 1.6C, D). We write down this information and scroll down to the complete
sequence to find and copy these sequences (Fig 1.7 A). We can paste this
sequence for the time being into MSWORD, MS WORDPERFECT, or WORD
Notepad and save this file, giving it the organism/gene name. The first three
nucleotides should start with ATG, the start codon or rare start codon GTG,
and end with TAA, TAG, or TGA, the stop codons. It should be noted, espe-
cially with genome sequences, the gene may be in the opposite orientation on
the chromosome, requiring inversion of DNA sequence and transcribing the
opposite DNA strand to identify start and stop of our ORF Many DNA soft-
ware analysis programs can do this for us. We chose the ORF rather than
flanking or intergenic regions, because we expect greater selection pressure
and less chance for sequence divergence among strains of organism X than
these intergenic regions. This is especially important if we are to identify all
members of organism X. Now that we have these sequences, we need to deter-
mine, in silico, whether these sequences are unique to genus Salmonella and
specifically, the serovar Typhimurium. This can be determined going to
BLAST on www.ncbi.nlm.nih.gov Website. Click on BLAST and under
Nucleotide, click on "nucleotide-nucleotide BLAST (blastn) (Fig. 1.7B)." This
will take you to a new site within NCBI that has a box beside "Search". Paste
your sequence into this box, and click on the BLAST button (Fig. 1.7B). On the
next page, select under the "Format" section, the box titled "or select from" and
chose "Bacteria [ORGN]" and click on the FORMAT button (Fig. 1.7C).
Allow the BLAST search time to search the database. The time it takes for the
search is dependent on gene and the amount of "traffic" at this Website; it is a
very popular site with researchers. The results are returned, outlining how many
matches there are to your gene sequence. As of 8 February 2005, there were
222 matches with the closest matches, (>90%) to S. enterica invA representing var-
ious serovars. Other matches are identified, most notably in homologues, genes
with similar function, present in Escherichia coli. This is expected, as invA is part
of the type III secretion system present in many human and plant pathogens (40).
More importantly, the BLAST results identify for us region of the invA sequence
to focus on in our primer design. This database search using BLAST is the same
approach one would use in analyzing DNA sequence generated from the sequenc-
ing of plasmid clones or PCR amplicons. There are however, no guarantees
that your gene or sequence will prove useful as a diagnostic marker for organism
X, based solely on this database search. You find only what is available on the
database, at the time of your search. It is therefore important experi-
mentally to determine the distribution of your candidate gene or allele among

PCR Basics

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18 PCR Basics

a sampling of strains, serovars, and closely and distantly related microbes.
Despite the presence of invA homolog in other genera and species, sequences
are divergent enough for this to be a useful genetic marker for detecting
Salmonella (68, 72).

Now that you have determined in silico your candidate genetic marker,
you can proceed to analyze your sequence(s) for the best PCR primer pairs.
There are several commercially available, as well as Web-based (13;
http://dbb.nhri.org.tw/primer/) DNA analysis software packages for designing
PCR primers that vary in price, utility, ease, options, or familiarity to the
authors. Therefore, we will only provide the reader with general design consider-
ations. First, let us consider in our design the size we want for our amplicon. This
consideration is especially important in the development of multiplex PCR
where the size of the amplicon identifies the gene or organism present in our
sample. Also PCRs sensitivity is influenced by the size of the amplicon. For sen-
sitive, real-time PCR, small amplicons, 75-200 bp are preferred. Next thing to
consider is where to concentrate our search for specific PCR primers. From our
BLAST search, it appears that the 1st 750 bp of Salmonella invA is ideal for our
analysis. Also to improve the specificity of our PCR, we need to consider the
length of each primer (94). Generally, the minimum default value for many of
the PCR primer design algorithms is 18 bp. This value is generated from the
probability of finding this exact sequence within the bacterial genome, where for
this example; we are dealing with an organism with 50% GC content and
4,000,000 bp genome. The probability of a specific 18 bp sequence is present is
(1/4) 18 x 4,000,000 = 6 x 10~ 5 . The smaller the sequence, the greater the likelihood
of finding sequence not just once but multiple times within the genome. That is
why short 10-mer oligonucleotides have become useful tools for typing bacteria
by random amplified polymorphic DNA (RAPD) PCR (51), because based on
size and using our calculations we expect to find these sequences at least 4 times
within the bacterial genome. Now, having run our analysis, we are presented with
all possible primer pairs. Our next step is to select primers for the amplicon size
that we want and screen these primer sets further, to identify those that do not
form "hairpins" or primer-dimers. We especially want to avoid primers that form
hairpins at the 3' end as this will interfere with the primers annealing precisely to
its target sequence and participation of the primer in the DNA extension step in
PCR. Primer-dimers and hairpins can affect the specificity and sensitivity of
PCR and should be avoided if possible (78). Once the appropriate primer set(s)
has been identified, search the GenBank DNA database for match with our
primers. With the BLAST search, it is recommended with searches of short
sequences to select Bacteria under "or select from" option. This is to limit con-
fusion with random and insignificant matches with the larger animal and plant
genomes (10 9 bp) that sometimes occur. Beyond this point, we generally empiri-
cally optimize our PCR, using appropriate positive and negative controls, and
identifying the magnesium concentration and PCR annealing temperature with
the sensitivity and specificity that is best for detecting organism X. We then ver-
ify the specificity of our PCR by comparing same strains, serovars, or species
against different strains, serovars, and closely and distantly related microorgan-

References 19

isms to see if same size PCR amplicon is produced only for those groups of bac-
teria to which the PCR was intended to identify. Ideally, once our PCR has been
optimized, PCR amplicon, of the expected size is only observed among select
bacteria that possess the target gene and nothing for all other microorganisms
that do not possess this gene. It is at this point too that we verify that our ampli-
con, with size expected based on the primers designed, is the target gene to which
our primers were intended to amplify. We accomplish this by sequencing the
PCR amplicon and match resulting sequence against GenBank DNA database
using the BLAST algorithm. Our amplicon's sequence should match the invA
sequences present on the DNA database.

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22 PCR Basics

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CHAPTER 2

The Mythology of PCR:
A Warning to the Wise

John J. Maurer 1 ' 2 *, Ph.D.

Poultry Diagnostic & Research Center 1 , College of Veterinary Medicine,

The University of Georgia, Athens, GA 30602
Center for Food Safety 2 , College of Agriculture and Environmental Sciences,

The University of Georgia, Griffin, GA 30223

Introduction
Interpretation

Conventional PCR

Real Time PCR
Validation
Problems and Their Solutions

False-Positives and Dead vs. Live Bacterial Cell Debate

PCR Inhibitors, Limits of Detection and False-Negatives
Conclusions
References

INTRODUCTION

Most diagnostic PCR tests are a qualitative yes or no, presence or absence of
pathogen X. We know what it means if our sample is positive by PCR, report-
ing back presumptive positive for organism X and a negative PCR result was
the end-point for that sample. Were these assumptions correct? The decisions
we make based on these PCR results require that we know how to interpret
these results and like any other diagnostic test, know its limitations with regards
to sensitivity and specificity. Even if your laboratory is only interested in adapt-
ing existing PCR methods for identification of pathogens in foods, it is impor-
tant that you know what the results mean, and know how to recognize and
troubleshoot problems as they occur. You can safe guard or at least be prepared
to recognize these problems, as they appear, by implementing standard operat-
ing procedures and including controls recommended by authors in the chapters
discussed in this book. In this section, I will specifically delve into interpreta-
tion and understanding of PCR results as well as discuss the limitations, prob-
lems, and erroneous assumptions associated with PCR and other PCR based
technologies (e.g., real-time PCR).

Corresponding author. Phone: (706) 542-5071; FAX: (706) 542-5630; e-mail: jmau-
rer@vet.uga.edu.

28 The Mythology of PCR: A Warning to the Wise

INTERPRETATION

Conventional PCR. A sample is positive, by PCR, if an amplicon is produced
with the size expected for the primers used. What if the sample yields an ampli-
con larger or smaller than the size expected for our PCR primers? Is this sam-
ple considered positive by PCR? NO!!! This result is referred to in PCR parlance
as a nonspecific amplicon, it is ignored, AND if we do not observe an amplicon
with a size expected for primers used, the sample is considered PCR negative. It is
therefore a requirement to always include DNA molecular weight standards, in
the appropriate size range for accurately assessing the amplicon's size, and the
percentage of agaraose and electrophoresis time needed to adequately separate
the molecular weight standards. One needs to also consider other parameters
(electrophoresis buffer, buffer strength, voltage, etc.) that affect uniformity of
DNA separation across the entire width and length of the agarose gel. For wide
gels with many wells or lanes (>10), one may consider placement of the DNA
standards in the middle and the outermost wells. With appropriate gel docu-
mentation software, the user can, using these well placed molecular weight
standards, correct for electrophoresis migration anomalies that produce
"smiles" at high voltages. Avoiding electrophoresis at high voltages or circu-
late/cool the buffer during electrophoresis can prevent this electrophoretic
anomaly. With every PCR run, ALWAYS include a positive control so that you
can match your sample with the control, and allow adequate separation of your
DNA standards, samples, and control so that you do not erroneously report a
sample with a nonspecific amplicon as positive. If molecular biology is new to
your laboratory, it is advisable to purchase a general molecular biology manual,
that details the specifics of gel electrophoresis, includes theory and helps trou-
ble shoot problems commonly associated with the molecular technique (1, 62).
For the experienced molecular biologist, this is rather obvious, but for oth-
ers, especially the novice, it is easy to be lulled into believing the presence of any
PCR product, regardless of size, on the gel means the sample must be positive
for organism X. Most genes targeted by PCR have been selected based on their
conservation and uniformity within a species, subspecies, serovar or pathotype.
These genes are uniform in size. There are, however, exceptions, genes or DNA
segments containing repetitive elements or extragenic sequences, the number,
size, or presence of which varies within the bacterial population (10, 16, 22, 38,
57, 70). PCRs have been developed to exploit these genetically variable regions
for the purpose of genus/species identification (10, 16, 24, 35, 57) and strain typ-
ing (25, 56, 57). Here the different size amplicon identifies the genus or species
and/or distinguishes strain types. However, a requirement for using any of these
PCRs is first the isolation of the organism. For PCR screens of foods, it is
advisable to avoid those PCRs that produce, as designed, these variable size
amplicons. Unless, an internal probe is included in the PCR screens, for speci-
ficity, the technician may confuse a true, nonspecific amplicon in a sample as a
positive and erroneously report the sample as such.

Real-Time PCR. Results generated by real-time PCR are generally more
straightforward to interpret for a simple question like: is the organism present

Interpretation

in our sample? Rather than visualize the amplicon following PCR, we monitor
the increase in fluorescence over time as newly synthesized, amplicon binds to
SYBR Green® or the chemically quenched, fluorescent dye is liberated as the
amplicon displaces an internally bound, dye-labeled probe. Fig. 2.1 illustrates
kinetics of real-time PCR. Note the points on the x-axis, "threshold cycle" (C T )
where the log-linear phase of fluorescence begins for the different target DNA
concentrations (43). There is a linear correlation between C T and DNA con-
centration, making the PCR quantitative. A sample is considered positive pro-
vided it falls within the range of C T values, demarcated by the PCR's limit of
detection, and the background fluorescence associated with the negative or no
DNA controls. While real-time PCR surpasses conventional PCR in speed and
sensitivity, nonspecific amplicons can result in our erroneously reporting a pos-
itive result. SYBR Green® binds to double stranded DNA, regardless of
whether it is the expected amplicon, nonspecific amplicon, or primer-dimers.
Gradient thermocyclers have become a useful tool in rapidly identifying anneal-
ing temperature best for PCR amplification of the target gene while avoiding
primer-dimers. We can distinguish nonspecific amplicon(s) from a true positive
based on their distinctive DNA melting curves (Fig. 2.2) (59).

35000

S-H

30000

25000

20000

15000

10000

5000

7 10 13 16 19 22 25 28 31 34 37 40

Cycle #

Figure 2.1. Detection of foodborne pathogen X in foods by real time PCR. As ampli-
con is synthesized, the thermocycler continuously measures fluorescences with each
cycle. The PCR product fluoresces due to binding of fluorescent dye, SYBER Green
to the double stranded DNA, amplicon as it is formed. When the PCR amplicons are
first detected during real time, is a function of the target DNA concentration: ■ (100
pg), ▲ (10 pg), • (1 pg), (0.1 pg), O (0.01 pg), and + NO DNA control. Arrows iden-
tify the "threshold cycle," C T on the x-axis, # PCR cycles where the log-linear phase of
fluorescence begins. The cycle numbers the target DNA concentration was plotted rel-
ative to C T and as shown in the inset, there is a negative, linear correlation between
DNA concentration and C

The Mythology of PCR: A Warning to the Wise

400000

Tm 54°C

350000

300000

250000

200000

150000

100000

50000

oj> o$> t>> & ^ ^ S % <^ b b ^ <\* ^ <*> % b

Tm (°C)

Figure 2.2. Identifying nonspecific PCR amplicons in real time PCR. We can distin-
guish nonspecific from specific amplicons by measuring the melting temperature (Tm)
for each amplicon following real-time PCR. The melting temperature is a reflection of
the amplicons's nucleotide sequence, therefore one looks to see if the DNA melting
curve for the putative, PCR-positive sample (□) overlaps with that of the positive con-
trol (♦) or produces a different melting curve (A), that is indicative of a nonspecific
amplicon.

When we do not observe, directly or indirectly, any PCR product or ampli-
con of the expected size, the finding is reported as negative. What does a nega-
tive result mean? For a pure culture, it means our isolate does not possess the
gene or gene allele to which our PCR was designed to detect. PCR has become
an important diagnostic tool not only in identifying medically important gen-
era (40, 58), but it has been used to identify an organism to species (9, 19, 23,
40), or serotype level (6, 21, 26, 42, 50, 66) as well as determine its antibiotic
resistance (20, 27) or virulence potential (2, 55). Depending on the organism
and gene(s) or gene alleles associated with resistance to drug X, PCR negative
result may indicate: (1) the organism is susceptible to the antibiotic in question
(e.g., mycobacterium and isonazid resistance; 27); or (2) PCR negative only
means the organism does not possess this gene (e.g.,enterococci and strep-
togramin resistance; 69) and susceptibility cannot be inferred. Gene screens to
assess, genotypically, drug resistance is challenging due to multiple genes and
gene alleles associated with resistance to certain antibiotics (8, 15, 64). With
regards to detection of multi-drug resistant (MDR) pathogens, while it is

Validation 31

tempting to select antibiotic resistance genes associated with the MDR as
probes in PCR screens (31), mobile genetic elements have disseminated these
drug resistance genes to innocuous commensals also contaminating foods
(32, 63, 74), providing potential for false positives. Gene screens for these MDR
loci should therefore be limited to the cultured pathogen. For detection of
pathogens in foods, it is imperative that we select a target gene or sequence that
is unique to pathogen X, uniform in its distribution within this bacterial popu-
lation and genetically stable.

If this target gene is strongly associated with genus, species or serotype, a
negative PCR translates to this isolate is NOT the species, strain, or serotype
identified by this PCR. However, if we apply this very same PCR to screen for
the presence of the organism that the PCR was designed to identify, does a neg-
ative result mean it is NOT present? We now are confronted with several ques-
tions relating to our PCR test's sensitivity and specificity (28, 39), important in
assessing, validating and finally standardizing our PCR for screening pathogens
in foods (30).

VALIDATION

In optimizing any PCR, we strive to design, identify and develop the primer
set(s) for discerning the one genus, species, or strain from multitude of micro-
bial species while being able to detect the fewest cells possible. This is the molec-
ular biology definition of specificity and sensitivity, respectively. To determine
specificity, we test our PCR against, many different bacterial strains, closely or
distantly related species and/or genera. A PCR specific for Salmonella, for
example, will produce positive results, amplicon of the expected size, for ALL
Salmonella species, strains, and serovars but will prove negative for all other
bacterial species, especially closely related species (28, 58). If we continue using
Salmonella as our example, sensitivity is measured by lowest Salmonella cell
density detectable by our PCR (28, 39). In its infancy, PCR's specificity and sen-
sitivity were determined using pure cultures and at best a food product was
spiked with the offending organism and PCR was performed to detect the
organism in the processed sample. Only recently have investigators vigorously
put PCR through its paces in the real world to validate its utility for rapid detec-
tion of pathogens in foods (30).

Validation of any diagnostic PCR involves comparison against another
test, considered the "gold standard" for detection. For food microbiologists,
the "gold standard" is the bacteriological approach of culture, isolation, and
the biochemical or serological confirmatory tests. From this comparison, we
determine statistical specificity (false-/?ositives) and sensitivity (false-
negatives) of our PCR test (28, 39). A false-positive is when the sample is
PCR positive but culture negative, while a false-negative is vice-versa: PCR
negative, culture positive. What is responsible for reporting false-positives
and false-negatives and what can we do to minimize this in our food microbiol-
ogy lab?

32 The Mythology of PCR: A Warning to the Wise

PROBLEMS AND THEIR SOLUTIONS

False positives can be attributed to several things, most you cannot control, but
at least one you can: PCR, template, or sample contamination. As discussed in
Chapter 4, "Making PCR a Normal Routine of the Food Microbiology Lab,"
preventative measures and standard operating procedures are essential to avoid
these contamination issues. These measures include physically separating DNA
and PCR preparation areas from each other as well as from the area where gel-
electrophoresis is performed; use of barrier tips, disposable gloves; and cleaning
the PCR preparation area with bleach and/or overnight, ultraviolet illumina-
tion. As mentioned earlier, PCR amplifies target gene 10 9 -fold, producing more
than enough molecules per pg-fg of template to serve as template in the next
PCR reaction. Following a PCR run and upon opening the tube, we create an
aerosol of amplicons that can quickly contaminate our hands, pipettes, and the
immediate bench area. Something as simple as disposing of our gloves follow-
ing the loading of our PCR sample in the agarose gel and before we set up our
next PCR reaction, can avoid future PCR "carry-over" contamination. PCR
contamination results in considerable down time for the diagnostic laboratory
due to the time it takes to identify the source of contamination, and subsequent
decontamination of the affected area or disposal of the contaminated
reagent(s). Alternatively, some labs substitute thymidine with uracil in the PCR
reaction mix and subsequent pretreatment of all PCR reaction mixes with
uracil DNA glycosylase prior to running these reactions in the thermocycler
(41). The principle behind this method is that during PCR, amplicons incorpo-
rate uracil; the amplicon is now susceptible to hydrolysis by uracil DNA glyco-
sylase, and eliminated prior to each subsequent PCR run. Therefore, erroneous
reporting of false-positives due to PCR contamination is eliminated.

As synthesis of the amplicons is identified in "real-time" with newer, PCR flu-
orescence-based detection technologies, tubes never have to be opened following
the initial PCR reaction set-up. With conventional PCR, we can identify PCR con-
tamination when negative or NO DNA controls turn positive. For an experienced
lab, something is amiss, when the number of PCR positives greatly exceeds fre-
quency the lab normally encounters for this PCR test or incidence reported in the
literature AND subsequent culture results do not correlate with the PCR (i.e.,
increase in false positives). This can be observed with real-time PCR as lower C T
than encountered for past PCR-positive samples, indicative of high-cell density or
template/target concentration, and fails to yield the organism upon culture. As
PCR can be extremely sensitive, great care must be taken in sample preparation to
avoid cross-contamination. Inclusion of a negative control, sample prep with every
PCR run will be useful in identifying cross-contamination, as a positive PCR for
this negative control would definitely be indicative of template/sample contamina-
tion. Anytime when its evident there is PCR contamination, discard the results for
that PCR run, discontinue any future PCRs, and identify and correct the problem
immediately before rerunning PCR on any past or future samples.

Nonspecific PCR amplicons can also result in erroneously reporting a sam-
ple positive for pathogen X. This can be especially problematic for real-time

Problems and Their Solutions 33

PCR using SYBR Green® to detect PCR products and multiplex PCR: the mul-
tiple gene screens, single PCR test where the size of the amplicon identifies:
genus, species, serotype, or strain. For real-time PCR, we can identify this prob-
lem by measuring the amplicon's Tm from the DNA melting peak as stated ear-
lier or run sample PCR on gel side-by-side with positive control to see any
differences between the two in their size. Tweaking the PCR conditions to
improve its stringency can sometimes eliminate these nonspecific amplicons.
This can be done by empirically identify annealing temperature or MgCl 2 con-
centration, that eliminates signal for our "false-positive" sample while not
affecting the positive control. To increase the stringency of the PCR, one
increases the annealing temperatures and/or lower the MgCl 2 concentrations in
the reaction mix.

Another way we can improve the specificity of our PCR and reporting false
positives, is to use PCR that incorporates internal: nested PCR primers (39);
DNA: DNA hybridization or capture probes (28, 45); molecular beacon (37); or
TaqMan probe (29). These PCRs have improved specificity because the internal
capture or detection probes can distinguish between the real amplicon and non-
specific amplicons, by binding to the complementary sequence within the target
amplicon during PCR or at DNA: DNA hybridization step. These internal probes
also heighten the sensitivity of the PCR at least 100-fold (28, 45).

False-Positives and Dead vs. Live Bacterial Cell Debate. Even when PCR is run-
ning optimally, there may not always be 100% agreement between PCR and cul-
ture. The reasons for false-positives are not completely understood. Several
explanations have been offered and include: (1) the bacteria are in a viable, non-
culturable state (61); (2) injury of the bacterial cells during food processing (52);
or (3) the bacteria are dead (48). One can obtain variable culture results alone
depending on: (1) whether to include a step(s) that allows for the recovery of
injured cells (13, 33); (2) the type of enrichment broth (18) and culture conditions
(12) used, or (3) the use of a delayed, secondary enrichment (72, 73) and may
explain the disconnect sometimes observed between PCR and culture results.

Depending on where samples are taken within the continuum of food pro-
cessing steps, especially at Critical Control Point (CCP) designed to reduce or
eliminate the pathogen, (e.g., heating), PCR may not be able to distinguish live,
dead or damaged cells. In fact, we routinely boil bacteria or wash cells in ethanol
to prepare template for PCR, conditions that readily and rapidly kill bacteria.
Therefore, one may consider where and when to use PCR in assessing product
contamination with pathogen X. For a process that readily ruptures or dissolves
the bacterial cell, pre-DNAse treatment step can remove residual DNA carried
over from dead, lysed cells (51). However, a significant proportion of heat-
treated cells remain intact, dead, and suitable as template for PCR (51). We still
need to know whether CCP was effective at eliminating the pathogen or reduc-
ing it to an acceptably safe level. PCR still affords us the opportunity to identify
the few cells still viable following CCP step, (e.g., pasteurization), by using RNA
as the template. Unlike DNA, RNA has short-half life in the bacteria cell (34),
as genes are turned on and off as the cell grows and responds to its environment.

34 The Mythology of PCR: A Warning to the Wise

Upon cell death, these mRNA transcripts are quickly degraded. There has been
considerable interest in using RNA as the template for diagnostic PCR to detect
the few viable cells remaining in the sample (17, 46, 52, 65, 75). This can be
accomplished by converting RNA to its complementary (c) DNA copy with the
retroviral reverse transcriptase, at which point the cDNA can serve as template
in standard PCR. This procedure is referred to as reverse-transcriptase PCR.
The challenge currently is identifying a constitutively, expressed gene that has
sequence unique to the organism and has a short, mRNA half-life, especially
upon death of the bacterial cell (75). RNA turnover in the bacterial cell is
dependent on its intracellular ribonucleases, and like any enzyme once denatured
it becomes inactive and the RNA therefore persists, which may explain the long
half-life of RNAs following thermal inactivation of the bacterial cell (47).
Therefore, there are times when culture continues to be necessary in assessing
microbial risk following food processing step at CCP and other instances where
PCR trumps culture in the detection of foodborne pathogens (see below).

Finally, we are left to consider viable but ^onculturable (VBNC) bacteria and
PCR. We know bacteria can enter a physiological state where, with the micro-
scope, we know they are present and viable, as determined using viability stains,
but we are unable to plate them from sample X. This VBNC state may result
from cellular injury (14), adaptation to a harsh, oxygen-poor or nutrient deplete
environment (5, 7, 71) or subsequent transformation from planktonic to sessile
state in biofilms (11). In foods, the VBNC state may be the consequence of cel-
lular injury/damage and may require a recovery period, in a preenrichment
broth, before the cells can be cultivated. Organisms like Vibrio and
Campylobacter can readily enter VBNC state, especially in aquatic environments
(54, 60). Although regarded as a foodborne pathogen, Campylobacter is also
recognized as the cause of several waterborne outbreaks in the United States (4,
36). With Campylobacter, the VBNC state may be due to physical or chemical
agent that damages the cell, or nutrient depletion or limitation triggers a physi-
ological change to a survival state. When Campylobacter enters the VBNC state,
its cell morphology changes from helical to coccoidal. Pathogens can revert back
from this nongrowing, VBNC state into actively growing; cultivatable state,
under the right conditions in vitro (7, 71) and cause disease in its animal host
(53). It may be that we are unable to detect it in this state using our current selec-
tive, enrichment media because of the antibiotics in the media that interfere with
cellular repair and changes to the cell wall necessary to resume growth (67, 68).
Where our culture-based approaches currently have failed, PCR offers the
opportunity for the pathogen's detection, especially in its VBNC state (3, 49, 52).

PCR Inhibitors, Limits of Detection, and False-Negatives. False-negatives, PCR-
negative, culture positive samples are attributed to two major factors: PCR
inhibitors or the PCR's limit of detection. PCR inhibitors may be attributed to
the food sample itself or the enrichment used to amplify the target organism.
We can often remove these inhibitors by using simple DNA affinity, spin
columns to produce clean DNA template for PCR, making samples generally
recalcitrant to PCR (e.g., soil) pliable for PCR-based screens and analyses.

Conclusions 35

Chapter 4, "Sample Preparation for PCR" will go into more detail concerning
sample preparation and preparation of template that is free of PCR inhibitors.
More recently, diagnostic PCRs for screening foods have been adapted to include
an "internal control" in the sample screened in order to eliminate possibility of
extraneous factors (e.g., PCR inhibitors) from factoring into interpretation of
PCR negative results. "Internal amplification control" is the cloned, positive-
control amplicon where an internal region has been removed (44). As template
in PCR, "internal amplification control" produces a smaller sized-amplicon. The
plasmid DNA bearing our "internal amplification control" is included with sam-
ple template in PCR. If the sample is negative for organism X, a single amplicon,
corresponding in size to that expected for the "internal amplification control."
However, for a positive sample, two amplicons are produced; one that corre-
sponds in size to that expected from amplification of the organism X's targeted
gene and the other corresponds in size to that expected for the internal control.
For most PCR beginners, false-negatives due to PCR's insensitivity to detect
a single-cell per sample appear to be a paradoxical, if not a heretical statement.
You have probably read many research papers and believe their claim that their
PCR can detect a single cell/ml of a sample. Is this really possible? With PCR,
we are generally dealing with reaction mix volumes that range between 1 and
100 |il to which we may add 1 or 10 |il of the sample, once its been processed
for PCR. What is the probability that you detect lcell/ml by PCR, if you were
to take 0.001 ml or 1 |il, once from that sample? Knowing Poisson distribution,
we know that odds are very small that we can detect it. However, if we took
multiple aliquots from this same sample, a most-probable number approach, we
would improve our chances of detecting this organism by PCR. The reality is
that for most PCRs the limit of detection is 1-1000 cells per 1 |il sample tem-
plate run, which translates to 1,000-1 x 10 6 cells/ml. Therefore, if we relied on
PCR alone, and discounting PCR inhibitors, does a PCR negative sample mean
the organism is NOT present? Ideally, one wants to use the PCR that is the most
sensitive for identifying pathogen X in our food product. How might we
improve our chances of detecting our pathogen knowing these limitations and
assuming the organism might be present in our specimens at levels <1000
cells/ml? One approach is to concentrate cells into a smaller volume, or include
an enrichment step that amplifies what few cells are present to levels above the
PCR's threshold for detection (Chapter 3). For the latter, short enrichment
period may be sufficient to bring cell density of the pathogen above the detec-
tion limits of the PCR. Enrichments have been especially adapted to PCR pro-
tocols for foods due to the necessity of processing the large sample volumes
associated with screening foods from pathogens.

CONCLUSIONS

One must be aware of the limitation of any diagnostic test, and PCR is no
exception. Will PCR soon be the substitute for current culture or immunologi-
cal tests for foodborne pathogens? Probably not for all pathogens, but it will

36 The Mythology of PCR: A Warning to the Wise

become standard tool for detecting foodborne protozoans and viruses,
pathogens that are currently recalcitrant to culture-based methods of detection.
PCR will become an important tool in identification of serotypes and patho-
types. It can be a useful part of any detection scheme, helping with decisions as
to which samples and enrichments to focus our efforts towards (39). Of course,
acceptance and implementation of PCR in the diagnostic laboratory requires
an understanding of its mechanics, meaning of results, the test's limitations, and
being able to recognize problems and trouble-shoot them as they arise.

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Curtiss III, and C.L. Gyles. 1992. Amplification of invA gene sequence of
Salmonella typhimurium by polymerase chain reaction as a specific method of detec-
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59. Ririe, K.M., R.P. Rasmussen, and C.T. Wittwer. 1997. Product differentiation by
analysis of DNA melting curves during the polymerase chain reaction. Anal.
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60. Rollins, D.M. and R.R. Colwell. 1986. Viable but nonculturable stage of
Campylobacter jejuni and its role in survival in the natural aquatic environment.
Appl. Environ. Microbiol. 52:531-538.

61. Sails, A.D., F.J. Bolton, A.J. Fox, D.R. Wareing, and D.L. Greenway 2002. Detection
of Campylobacter jejuni and Campylobacter coli in environmental waters by PCR
enzyme-linked immunosorbent assay. Appl. Environ. Microbiol. 68:1319-1324.

62. Sambrook, J, E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory
Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

63. Sanchez, S., M.A. McCrackin Stevenson, C.R. Hudson, M. Maier, T. Buffington, Q.
Dam, and J.J. Maurer. 2002. Characterization of multi-drug resistant Escherichia
coli associated with nosocomial infections in dogs. J. Clin. Microbiol. 40:3586-3595.

40 The Mythology of PCR: A Warning to the Wise

64. Shaw, K.J., P.N. Rather, R.S. Hare, and G.H. Miller. 1993. Molecular genetics of
aminoglycoside resistance genes and familial relationships of the aminoglycoside-
modifying enzymes. Microbiol. Rev. 57:138-163.

65. Sheridan, G.E.C., E.A. Szabo, and B.M. Mackey 1999. Effect of post-treatment
holding conditions on detection of tufA mRNA in ethanol-treated Escherichia colt
Implications for RT-PCR-based indirect viability tests. Lett. Appl. Microbiol
29:375-379.

66. Shi, F., Y.Y. Chen, T.M. Wassenaar, W.H. Woods, P.J. Coloe, and B.N. Frye. 2002.
Development and application of a new scheme for typing Campylobacter jejuni and
Campylobacter coli by PCR-based restriction fragment length polymorphism analy-
sis. J. Clin. Microbiol. 40:1791-1797.

67. Signoretto, C, M.M. Lleo, and P. Canepari. 2002. Modification of the peptidogly-
can of Escherichia coli in the viable but nonculturable state. Curr. Microbiol.
44:125-131.

68. Signoretto, C, M.M. Lleo, M.C. Tafi, and P. Canepari. 2000. Cell wall chemical
composition of Enterococcus faecalis in the viable but nonculturable state. Appl.
Environ. Microbiol. 66:1953-1959.

69. Simjee, S., D.G. White, J. Meng, D.D. Wagner, S. Qaiyumi, S. Zhao, J.R. Hayes, and
PR McDermott. 2002. Prevalence of streptogramin resistance genes among
Enterococcus isolates recovered from retail meats in the Greater Washington DC
area. J. Antimicrob. Chemother. 50:877-882.

70. Spinaci, C, G. Magi, C. Zampaloni, L.A. Vitali, C. Paoletti, M.R. Catania, M.
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by analysis of RD2 region of the prtFl gene. J. Clin. Microbiol. 42:639-644.

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liquid media for resuscitation of starvation- and low-temperature-induced noncul-
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72. Waltman, WD., A. Home, C. Pirkle, and T. Dickson. 1991. Use of delayed second-
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73. Waltman, W.D., A.M. Home, C. Pirkle. 1993. Influence of enrichment incubation
time on the isolation of Salmonella. Avian Dis. 37:884-887.

74. White, D.G, C.R. Hudson, 11 Maurer, S. Ayers, S. Zhao, M.D. Lee, L.F. Bolton, T.
Foley, and J. Sherwood. 2000. Characterization of chloramphenicol and florfenicol
resistance in bovine pathogenic Escherichia coli. J. Clin. Microbiol. 38:4593^4598.

75. Yaron, S. and K.R. Matthews. 2002. A reverse transcriptase-polymerase chain reac-
tion assay for detection of viable Escherichia coli 0157:H7: Investigation of specific
target genes. J. Appl. Microbiol. 92:633-640.

CHAPTER 3

Sample Preparation for PCR

Margie D. Lee 12 *, DVM, Ph.D. and Amanda Fairchild 1 , M.S.

Poultry Diagnostic & Research Center 1 , College of Veterinary Medicine,

The University of Georgia, Athens, GA 30602

and
Center for Food Safety 2 , College of Agriculture and Environmental Sciences,

The University of Georgia, Griffin, GA 30223

Introduction

How Do You Get Started?

What Conditions Affect the Success of the PCR?

What Are PCR Inhibitors?

Potential Solutions to the Challenges of Using PCR to Detect Pathogens in

Foods

References

INTRODUCTION

Adding PCR-detection to a laboratory's repertoire of tools can improve sample
turn-around time and accuracy. Yet, PCR is not a universal solution for pathogen
detection problems. For pathogens that are rapidly growing and contaminate
foods in high numbers, culture onto selective and differential media may actually
be more rapid and cost-effective than PCR. However, PCR can greatly improve
turn-around time in instances of slow-growing pathogens and can improve detec-
tion of pathogens present at low concentrations. Nevertheless, like any other pro-
tocol, correct preparation of the samples is key to PCR's success. Figure 3.1
shows a sample processing strategy for PCR. Specific steps in the processing strat-
egy will vary depending on pathogen and foodstuff. Prior research may have
shown whether pathogen amplification steps, such as enrichment culture, are
needed prior to PCR, so check the published literature for relevant protocols.

HOW DO YOU GET STARTED?

Sample preparation serves several functions for PCR detection (14, 19). It ini-
tially decreases sample volume and concentrates the PCR template into a work-
able volume. The first challenge in choosing a good sample preparation protocol
is to know whether the pathogen contaminates food at high levels or whether it

Corresponding author. Phone: (706) 583-0797; FAX: (706) 542-5630; e-mail:
leem@vet.uga.edu.

Sample Preparation for PCR

Collection of
food sample

Process food
sample

Homogenization
Washing

Concentrate Pathogen

Enrichment

Immunocapture

Buoyant density centrifugation

Template Extraction

Heat

Detergent

Chemical

Solvent

will be necessary to amplify the bacteria
with an enrichment culture. For exam-
ple, very few Listeria cells may be pres-
ent on a slice of deli meat but these few
bacteria may be enough to cause serious
illness in a pregnant woman. It may be
impossible to directly collect a few bac-
terial cells and detect them using PCR.
An enrichment culture can amplify the
bacterial cells and the PCR can detect
the bacteria in the enrichment broth
more rapidly than they can be identified
using standard bacteriological methods.
In this instance PCR can aid in the rapid
detection of Listeria and the sample
preparation protocol will include per-
forming the enrichment culture, collec-
tion of bacteria from the enrichment
broth, extraction of DNA from the bac-
terial cells and then performing the PCR
test.

Once the pathogen is collected,
PCR template must be prepared from
its DNA (or RNA). The first step in
preparing template from a pathogen
requires lysis (rupture) of the cells (or
viruses) to release the nucleic acids
(DNA and RNA). Specific organisms
may require specific protocols for effi-
cient template extraction. For example,
there are a few basic approaches to
extracting nucleic acids from bacteria
but their effectiveness depends on sev-
eral features of the bacterial cell wall. Gram-negative bacteria lack a thick cell
wall, thus heat or detergent can lyse the cells. Many of the published protocols for
E. coli, Salmonella, and Campylobacter use this approach for lysis of the cells (see
Table 3.1 for applications). Gram-positive bacteria have a thick cell wall that must
be removed or disrupted in order to lyse the cells. Lysozyme (plus lysostaphin for
Staphylococcus) digestion is commonly used, prior to detergent treatment, for
nucleic acid extraction from gram-positive and gram-negative bacteria. A third
method of bacterial cell lysis involves a high salt/chemical lysis with guanidium
salts. This method is most commonly used for gram-positive bacteria but will
work for gram-negative cells as well. Solvent extraction of nucleic acids, with
organic solvents such as ether, can be used for bacteria, viruses, and protozoa.
Commercial PCR detection kits will incorporate one or some derivation of these
methods, but the methods have to be optimized for the specific organism.

Concentrate Template

Alcohol precipitation
Binding matrices

PCR

Figure 3.1. Sample preparation for PCR
detection of foodborne pathogens.

HOW DO YOU GET STARTED ?

Table 3.1. Sample preparation of foods for PCR

Method to

DNA (RNA)

Food Category

Concentrate

Extraction

Pathogens

( Challenges)

Sample

Pathogen

method

(Reference)

Dairy

PCR inhibitors

Skim milk,

Differential

Solvent-based

E. coli 0157

(fat, protein,

pasteurized

centrifugation

nucleic acid

(16)

calcium, chelators),

milk, dry

or none

extraction or

Listeria (12)

dead cells, low

milk, hard and

guanidinium

Staphylococcus,

numbers of

soft cheese,

isothiocyanate

Yersinia (20)

pathogen cells,

reconstituted

extraction

Campylobacter

other bacteria

whey powder

(24)

Raw milk

Centrifugation

Boiled cells
with Chelex-
100 removal
of inhibitors;
Tth polyme-
rase improved
sensitivity

Staphylococcus
(10)

Raw milk

Enrichment

and

centrifugation

Commercial
kits

Salmonella (5)

Soft cheese

None

Detergent lysis
with Nal
extraction
of DNA

Listeria (15)

Meat and poultry

rinses

PCR inhibitors (fat,

Chicken

Enrichment

Commercial

Listeria

protein, collagen,

carcass rinses,

and

kits

Salmonella

blood), small num-

red meat

centrifugation

E. coli (4, 5)

bers of bacteria,

Homogenates

Buoyant

Guanidinium

Campylobacter

of chicken skin,

density

isothiocyanate

(24)

whole chicken

centrifugation

and detergent

leg, chicken

extraction

sausages, turkey

leg meat,

ground beef,

mince meat,

beef, pork

Raw whole

Buoyant

Boiled cells

Campylobacter

chicken rinses

density centri-
fugation and
enrichment
culture

(27)

Chicken and

Enrichment

Multiple meth-

Salmonella (6)

turkey muscle,

ods firmed

skin, internal

including boiled

organs; raw

cells, alkaline

carcasses

lysis, and
commercial kits

Continued

Sample Preparation for PCR

Table 3.1. Sample preparation of foods for PCR — cont'd

Food Category
( Challenges)

Sample

Method to

Concentrate

Pathogen

DNA (RNA)

Extraction

method

Pathogens
(Reference

Meat and poultry

rinses

Ground beef

Buoyant

density

centrifugation,

immunoma-

gnetic

separation,

enrichment

Boiled cells
or Chelex-
extraction

E. coli (25)

Ham

Immuno-
magnetic
separation

Lysozyme
and detergent
extraction

Listeria (8)

Minced Pork

Enrichment

Commercial

Yersinia (13)

meat, raw

and buoyant

extraction

whole pork leg

density
centrifugation,

buffer and
heat

Ground pork

Enrichment

Chelex resin-
based

commercial
kit

Yersinia (26)

Sausage and

Homogeni-

Commercially

Clostridium (11)

meat rolls

zation of

available kits

(Korean ethnic

food then

but increased

foods)

filtration and
centrifugation

Mg++ levels in
samples

Deli meats:

None

Commercial

Norwalk-like

ham, turkey,

extraction

virus

roast beef

solution

Hepatitis A
virus (22)

Seafood

PCR inhibitors

Smoked

Homogeni-

Detergent

Listeria (23)

(phenolics,

salmon

zation of

extraction

cresols, aldehydes,

food

and Tween

proteins, fats),

20 facilitator;

low numbers of

PCR

bacteria

inhibitors
removed by
solvent
extraction
or column
purification

Fish cakes,

Enrichment

Detergent and

Listeria (1)

fish pudding,

boiling for

peeled frozen

extraction

shrimp, salted

herring,

marinated and

sliced coalfish

in oil

What Conditions Affect the Success of the PCR

Produce
PCR inhibitors
(chelators), few
bacteria

Shellfish:

Homogeni-

Guanidinium

Norwalk-like

muscles and

zation of

thiocyanate

virus,

oysters

food then

and silica

Adenovirus

high-speed

purification

Enterovirus,

centrifugation

Hepatitis
A virus (7)

Raw Oysters

Homogeni-

Commercial

Norovirus (17)

zation then

kit

buoyant

density

centrifugation

Whole

Column

Commercial

Protozoa (18)

raspberries

filtration and

kit (FTA

centrifugation

filter)

Lettuce

Homogeni-

Commercial

Hepatitis A

zation, centri-

kits

virus

fugation, and

Norwalk virus

precipitation

(21)

with

polyethylene

glycol

The next step in sample processing is to concentrate the template and reduce
the concentration of PCR inhibitors. The specific approach will vary depending
on whether DNA or RNA is desired as template and the chemical composition
of the PCR inhibitors present in the sample. Phenol/chloroform extraction
steps can reduce protein and lipid inhibitors. Other chemical inhibitors can be
diluted by washing bound (silica beads or column matrices) or precipitated
(ethanol or propanol) nucleic acids. An effective protocol for removing
inhibitors must be developed for each specific food. Then the specific PCR can
be performed for the pathogens of interest.

WHAT CONDITIONS AFFECT THE SUCCESS OF THE PCR?

The purpose of PCR is to detect the organism's specific nucleic acids in the sam-
ple so that time-consuming biochemical and immunological assays are not
needed. PCR causes the synthesis of DNA using an enzymatic reaction that
cycles over and over due to the temperature cycling of the thermal cycler.
Enzymes, including the polymerases that are used in PCR, must have specific
chemical conditions in order to do work effectively. One of the major concepts
of PCR is that the polymerase can exponentially increase the amount of DNA
in the sample because of the temperature cycling (9). However, if the conditions
are not optimal, the polymerase may not be able to synthesize enough DNA for
the reaction to be detected as positive; these are called "false-negative" reac-
tions. There are many situations where the PCR reaction can be suboptimal and

46 Sample Preparation for PCR

produce false negative results. Incorrect primers, buffer composition, cation
(Mg++) concentration, nucleotide concentration (dNTPs), the wrong annealing
temperature, extension cycles that are too brief, and incorrect template can
cause the reaction to be falsely positive or negative. Always include two nega-
tive controls: a different organism's DNA and a control with no DNA template.
These will help you determine the specificity of your PCR and whether you
have sample contamination. In addition, always include a positive control with
DNA template that you know will amplify in the PCR. These controls can help
identify the problem when the PCR is inhibited. For example, different poly-
merases need different cations in the buffer in order to synthesize DNA. Taq
polymerase uses magnesium (Mg++) therefore too little Mg++ in the master mix
will result in a negative PCR reaction. The nucleotide concentration is impor-
tant as well; they will chelate the Mg++ if you use too much of the dNTP mix.
However, too much Mg++ will also result in a false negative reaction because
there is a narrow window of effectiveness for the PCR to work. Every PCR
reaction must be tested to determine the optimal concentration of Mg++ for the
specific primers, buffer, and cycling temperatures. A similar situation also exists
for the template concentration, too much or too little will result in a false neg-
ative reaction. You should optimize the PCR, by running different concentra-
tions of cation and template to find the concentrations that will produce the
amplicon of the correct size (or melting temperature if you are using real-time
PCR). If you are setting up a new PCR that you found in published literature,
do not just assume that the published conditions will work for you. If you get
into the habit of optimizing your PCR reaction conditions, you will seldom
have a problem with your routine PCRs that you cannot quickly solve.

WHAT ARE PCR INHIBITORS?

The PCR reaction can be inhibited when substances bind (chelate) or degrade
a component in the reaction and prevent it from participating in the synthesis
of DNA (9, 28). These substances are called "PCR inhibitors" and include
chelators of cations and substances that bind or degrade the polymerase or the
DNA template. When pathogens are grown to high levels in culture, PCR tem-
plate can often be made directly by chemical or enzymatic lysis of the organ-
ism's cells. For example, DNA can easily be extracted from gram-negative
bacteria by boiling the cells in water and using the boiled lysate as template. One
important caveat, most enrichment broths and selective agars contain sub-
stances that inhibit PCR so it will be important to wash the cells collected from
an enrichment or agar plate. You can do this with bacteria by pelleting the cells
using centrifugation, removing the liquid and resuspending the cells in saline or
water for the DNA extraction.

The real challenge is to isolate the pathogen and/or its DNA directly from a
food matrix. The great variety of foodstuffs complicates any quest to produce a
single sample preparation protocol that will work for every application. Unique
PCR inhibitors are found in just about any food type including meat, milk,

Potential Solutions to the Challenges of Using PCR to Detect Pathogens 47

cheese, produce, and spices (28). Many of these have not been identified but
some are known substances. For example, milk contains high levels of cations
(Ca++), proteases, nucleases, fatty acids, and DNA. In addition, heme, bile salts,
fatty acids, antibody, and collagen are PCR inhibitors that may be present in
meat or liver. The inhibitors have variable effects on the PCR reaction but in
general, they will make it more difficult to detect low numbers of bacterial cells
or viruses. A good sample preparation protocol will focus on collecting the
pathogen, removing the inhibitors present in the foodstuff (or culture medium)
and concentrating the template for PCR. In addition, use of a polymerase that
is less susceptible to the effects of inhibitory substances is a possible solution to
some PCR problems. For example a number of the newer polymerases, such as
Tfl and rTth, are more reliable than Taq polymerase when using PCR template
made from meat or cheeses (2). Moreover, the activity of the polymerases, in the
presence of inhibitors, can be improved with the use of some facilitators such
as bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), Tween 20 and
betaine (2, 3, 19, 28). If you are trying to adapt a published PCR to a different
food type, you may have to consider adding a facilitator or using a different
polymerase to enhance sensitivity of the reaction.

POTENTIAL SOLUTIONS TO THE CHALLENGES
OF USING PCR TO DETECT PATHOGENS IN FOODS

Foods differ greatly in their composition. The presence of fats, proteins,
enzymes, chemical additives, fiber, and bacteria as well as ranges of pH influ-
ence your ability to isolate the organism, its nucleic acids, and amplify its nucleic
acids using PCR. In addition, nonpathogenic organisms present in fermented
foods, the contaminating soil and manure organisms present on produce, and
fecal contamination of meat will produce competing DNA that may reduce the
sensitivity of the PCR reaction. Unless you are experienced in developing PCR
reactions, you may not want to solve all of these problems yourself. Use proto-
cols developed and validated by reputable labs. Note specific steps in the proto-
col. Are enrichment steps such as immunomagnetic capture or enrichment
culture needed to collect the organism from the sample? How are the organisms
collected from the sample or the enrichment? How is the DNA extracted from
the organism? What are the specific conditions of the PCR reaction? How will
you detect the PCR amplicon? Do you have a positive control organism (or tem-
plate) for the reaction? Once you have dissected out these important compo-
nents from the publication or protocol, you can determine, which components
can be modified for your specific needs.

Table 3.1 illustrates some of the challenges and solutions for PCR detection
of pathogens that contaminate foods. If the challenges are acknowledged, then
possible solutions become feasible. If the pathogen contaminates the food in low
numbers, then the pathogen must be amplified in some way. The important thing
to know is whether the PCR can detect very low numbers of bacteria.
Theoretically PCR can detect 1 pathogen in the reaction. Yet realistically,

Sample Preparation for PCR

because of PCR inhibitors and other factors, you will usually need a substantial
amount of template, from a few hundred to thousands of pathogen cells or
viruses, in the PCR reaction in order to reliably detect the presence of the
pathogen. In addition, if you consider that some food samples may only contain
a few hundred pathogen cells per gram of food, the need for an enrichment step
becomes apparent. Figure 3.2 shows how common pathogen amplification
methods work to concentrate the pathogen in a volume that can be used for the
next steps in the sample preparation . Enrichment culture is commonly used to
amplify the bacterial numbers although immunological capture can theoretically
be used for bacteria, viruses, and protozoa. Nevertheless, immunocapture
requires the availability of an antibody that is specific for the pathogen. An
immunocapture system works by binding one end of the antibody to a handling
apparatus (such as magnetic beads in immunomagnetic separation) then expos-
ing the other end of the antibody to the contaminated food. If the pathogen is
present in the food, many of the cells or virus particles will be bound to the anti-
body. This means that the immunocapture system can concentrate the pathogen
onto the magnetic beads, which can then be used for enrichment or directly
processed in a DNA extraction for PCR. Similarly, pathogens can be isolated
from liquid samples by using centrifugation protocols that either float the
bacteria or virus particles out of the sample (buoyant density centrifugation) or
pellet the cells in the tube. Filtration of liquid samples may be effective for

Centrifugation

8000x£

►

Suspension of Pellet of
pathogen cells pathogen cells

Enrichment culture

Food sample
(at least lgr)

Incubate

►

Enrichment
broth

Suspension of
pathogen cells

Buoyant density centrifugation

Immunocapture

Homogenized
food sample

■* f- r
; = = =

1-5000xp

►

Layer of

pathogen cells

and food sample

-i - r

<— Layer of
pathogen cells

Homogenized
food sample

Add antibody-y
coated beads^P^

►

Suspension of

food sample and

pathogen cells

Pellet of
pathogen cells

Figure 3.2. Approaches to concentrate or amplify pathogens.

References

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Campylobacter and virus particles). For some pathogens where the infectious
dose is very low, more than one concentration step may be needed in order to
amplify the pathogen to a detectable level. Many studies have addressed these
detection issues in developing PCRs for specific applications (Table 3.1).

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50 Sample Preparation for PCR

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19. Radstrom, P., R. Knutsson, P. Wolffs, M. Lovenklev, and C. Lofstrom. 2004. Pre-
PCR processing strategies to generate PCR-compatible samples. Mol. Biotechnol.
26:133-146.

20. Ramesh, A., B.P. Padmapriya, A. Chandrashekar, and M.C. Varadaraj. 2002.
Application of a convenient DNA extraction method and multiplex PCR for the
direct detection of Staphylococcus aureus and Yersinia enter ocolitica in milk samples
Mol. Cell. Probes 16:307-314.

21. Sair, A.I., D.H. D'Souza, C.L. Moe, and L.A. Jaykus. 2002. Improved detection of
human enteric viruses in foods by RT-PCR. J. Vir. Meth. 100:57-69.

22. Schwab, K.J., F.H. Neill, R.L. Fankhauser, N.A. Daniels, S.S. Monroe, D.A.
Bergmire-Sweat, M.K. Estes, and R.L. Atmari. 2000. Development of methods to
detect "Norwalk-Like Viruses" (NLVs) and Hepatitis A Virus in delicatessen foods:
Application to a foodborne NLV outbreak. Appl. Environ. Microbiol. 66:213-218.

23. Simon, K.C., D.I. Gray, and N. Coo. 1996. DNA extraction and PCR Methods for
the detection of Listeria monocytogenes in cold-smoked salmon. Appl. Environ.
Microbiol. 62:822-824.

24. Uyttendaele, M., J. Debevere, and R. Lindqvist. 1999. Evaluation of buoyant den-
sity centrifugation as a sample preparation method for NASBA-ELGA detection of
Campylobacter jejuni in foods. Food Microbiol. 16:575-582.

25. Uyttendaele, M., S. van Boxstael, and J. Debevere. 1999. PCR assay for detection of the
E. coli 0157:H7 eae-gene and effect of the sample preparation method on PCR detec-
tion of heat-killed E. coli 157:117 in ground beef. Int. J. Food Microbiol. 52:85-95.

26. Vishnubhatla, A., D.Y.C. Fung, R.D. Oberst, M.P. Hays, T.G. Nagaraja, and S.J.A.
Flood. 2000. Rapid 5' nuclease (TaqMan) assay for detection of virulent strains of
Yersinia enter ocolitica. Appl. Environ. Microbiol. 66:4131-4135.

27. Wang, FL, J.M. Farber, N. Malik, and G. Sanders. 1999. Improved PCR detection of
Campylobacter jejuni from chicken rinses by a simple sample preparation procedure
Int. J. Food Microbiol. 52:39-45.

28. Wilson, I.G 1997. Minireview: Inhibition and facilitation of nucleic acid amplifica-
tion. Appl. Environ. Microbiol. 63:3741-3751.

CHAPTER 4

Making PCR a Normal Routine
of the Food Microbiology Lab

Susan Sanchez, Ph.D.

Athens Diagnostic Laboratory, and the Department of Infectious Diseases,
College of Veterinary Medicine, The University of Georgia, Athens, GA 30602

Introduction

Setting up Your Laboratory for PCR
Physical Set-up of PCR
Personnel

Real-Time vs. Standard Format PCR
Equipment

Reagents and Disposables
Quality Control and Quality Assurance
Where to Locate Vendors
Noncommercial Tests for Foodborne Pathogens
Validation
Standardization
Available Commercial PCR Tests for Foodborne Pathogens
Real Time PCR

BAX Dupont-Qualicon
Food-proof Roche
IQ-Check BioRad
PCR-ELISA
Probelia BioRad

AnDiaTec Salmonella sp. PCR-ELISA
References

INTRODUCTION

In food microbiology, polymerase chain reaction (PCR) should not be consid-
ered a substitute for conventional microbiology techniques. The rationale of
employing PCR technology, as well as any other molecular diagnostic tech-
nique, should be founded on the following key consideration: (1) simplicity, (2)
throughput, (3) cost, (4) speed, and (5) appropriateness (37). Conventional
bench microbiology is often considered to be less technically demanding than
polymerase chain reaction, however, in reality, the techniques of PCR are eas-
ier to master and usually requires less time to achieve competence than conven-
tional microbiology. Experience in our laboratory shows that personnel of
varied technical and educational backgrounds, and absolutely no training in
microbiology, can master polymerase chain reaction in no more than a week. By

52 Making PCR a Normal Routine of the Food Microbiology Lab

contrast, competence in conventional microbiology techniques and their inter-
pretation requires significant training and experience. Polymerase chain reac-
tion can be mastered in less than a week with very little or no previous training
in microbiology, whereas conventional microbiological techniques will require
several weeks of training and a background in microbiology. A technique, as
simple as PCR, can be applied in more laboratories and it is more amenable to
field applications, which could allow for data collection right at the food-pro-
cessing plant. The food microbiologists' constant aim is to make our food sup-
ply safer; this is only achieved with large-scale screening of foodstuffs.
Polymerase chain reaction is a test process that allows for high throughput and
is amenable to automation. Over the past few years, molecular reagents have
become more affordable, and easier to obtain, with longer shelve life (37).
Naturally, the cost and accessibility varies greatly with location. Except for
primers and probes, most PCR reagents can be used and shared among differ-
ent PCR tests, as well as for other molecular-based techniques used in food
microbiology (e.g., strain typing) (37). The decision to use PCR needs to be
made by the laboratory based on clients' requirements for quick turnaround,
reliability, and confidence in the laboratory for correctly reporting results. Real-
time PCR allows not only the detection of suspect pathogen in food but its
enumeration as well (36, 37, 38). These molecular tools are essential to a con-
temporary food laboratory. Although the cost to equip a laboratory is high,
PCR complements and enhances the traditional microbiological methods; by
increasing speed, sensitivity, and, specificity for detecting pathogens in foods.
Polymerase chain reaction can be performed rapidly in the field, and limits the
number of cultures and isolations to the few samples identified as positive by
PCR (28). This reduces labor, conserves resources, and holds down costs.

SETTING UP YOUR LABORATORY FOR PCR

The PCR is a very sensitive, exceptionally powerful, and relatively simple
method that can unlock the door to many a genetic mystery after a few rounds
of cycling temperatures. However, PCR can return false positives or negatives if
care is not taken in the proper setup, standardization, and implementation of
quality controls. Careful planning needs to be given to standardizing a routine
protocol(s) for processing and testing of samples by PCR. This includes: the
physical setup of the PCR laboratory; quality control; storage and selection of
reagents; deciding which PCR detection platform to use; and finally, a critical
component — well-trained personnel. With good laboratory practices, the diag-
nostic laboratory avoids the possible pitfalls with PCR that lead to erroneous
reporting of laboratory results.

Physical Setup of PCR. Avoiding sources of PCR contamination is paramount
when dedicating laboratory space to PCR. Ideally there should be 4 to 5 distinctly
separate rooms or areas in the laboratory for: (1) sample preparation, which
includes sample enrichment, clean reagent preparation, and DNA extraction; (2)
PCR setup; (3) thermocycler; and (4) detection of PCR products or amplicons by

Setting Up Your Laboratory for PCR

agarose gel electrophoresis, or enzyme-linked immunosorbent assay (ELISA).
Realistically, having this much room may not be possible in existing food micro-
biology laboratory, although it should be kept in mind if future renovations are
planned, or a new building is being designed. In most instances, this physical sep-
aration can be accomplished by the development of one-way traffic flow (Fig. 1)
within a laboratory, assigning particular areas to each step in the process. This will
hopefully prevent cross-contamination from sample to sample and PCR to PCR.
The assignment of areas and/or rooms needs to be intuitive and easy to follow so
that it does not influence the speed and normal work flow of the laboratory.
Ideally, personnel will start the day in the clean area and move to those areas
where there is higher risk of aerosols that could be transported to clean areas and
become sources of PCR contamination. In those laboratories that process a very
large number of samples, dedicated personnel to each area is a good idea with
rotation of these people to a different area every week to avoid boredom and
maintain full competence in all aspects of the PCR protocol. Laboratory coats or
scrubs should be worn always, but these garments need to remain in each area
except when cleaned. DNA present on a bench top can easily contaminate a
sleeve. If a technologist later uses the same laboratory coat, then the jacket could
serve as the source of PCR carryover contamination when the individual sets up
the next PCR reaction. Gloves should be used at all times and should never be
worn in the different PCR areas. The sample preparation area is one of the most
important areas in the laboratory and it should be divided in three individual areas
for: ( 1 ) food sample preparation, (2) clean reagent preparation for DNA extraction,
where no sample or template should ever be present; and (3) processing and extrac-
tion of DNA from foods or enrichments. This final step should be conducted in a
biological safety cabinet to avoid sample cross-contamination. The technician can
inadvertently contaminate sample(s), from which PCR template is prepared by

Sample
handling and
enrichment
steps

Clean reagent for PCR
preparation area

Clean reagent
for DNA
extraction
preparation
area

PCR product
detection

Figure 4.1. Chart delineating the workflow in the PCR laboratory. Some of the PCR
work areas need to be physically separated in order to minimize the potential for PCR
carryover contamination, or sample cross-contamination. DNA template or PCR
product should never enter the clean reagent preparation area.

54 Making PCR a Normal Routine of the Food Microbiology Lab

touching a small droplet from an enrichment broth, positive for the microbe in
question, and subsequently can transfer this to another tube containing a differ-
ent sample, by touching the second sample with contaminated gloved hand. This
cross-contamination is insidious as it will not be detected by our PCR controls
and, even if contamination is suspected, retesting of the contaminated sample
template will only yield the same spurious false-positive results. The sample will
have to be reextracted, adding time and cost to the process. The PCR can be
exquisitively sensitive, even detecting a few cells that may cross-contaminate a
"negative" sample. To avoid this scenario, care should be taken in cleaning the
outside of the sample tubes with a disinfectant to kill bacteria and DNAse, or
10% bleach solution to destroy any contaminating DNA. It is also recommended
that the cabinet and the pipettes are wiped with bleach solution to eliminate sam-
ple cross-contamination, and subsequently wiped clean with ethanol or water to
avoid corrosion by the bleach, before the next DNA extractions (13).
Contamination by aerosols can also be minimized if supernatants are aspirated
and not decanted. When large volumes of sample need to be handled, disposable
individually wrapped sterile pipettes should be used. DNA extraction from
already enriched samples is one of the PCR steps that is amiable to automation,
which reduces the risk for intersample cross-contamination and sample exchange
(30, 49). The PCR setup and thermoycler area can be physically separated but they
can also be placed together as long as there is a biosafety cabinet in this area, or
a PCR box for the PCR setup. "PCR clean hoods" are relatively inexpensive, sit
on a table or bench and they are easy to clean. Their relatively small size allows
for their placement in most laboratories. PCR clean hoods also have germicidal
ultraviolet (UV) lights, which aid in maintaining sample purity by denaturing
DNA contaminants (13). The use of UV light to decontaminate the PCR setup
area, where reagents are openly handled can minimize the risk of carryover con-
tamination (40).

The detection area itself, where tubes or capillaries are opened and loaded
into agaraose gels or ELISA microplates, can serve as a source of PCR con-
tamination. PCR can amplify DNA from very small amounts of template to
large quantities of amplicon, which once aerosolized, aspirated, or spilled, can
easily contaminate surfaces. The glass capillaries used with the hot-air thermo-
cyclers can break within their carrier during transport to and from thermocy-
cler and detection area, or required glasscutter to free it from its block.
Breakage can result in contamination of this block with amplicon. Therefore,
pipettes as well as racks and carriers, used to transport samples from PCR setup
to thermocycler to detection area and back, can provide an opportunity for
contamination of the next scheduled PCR run. This issue is largely avoided with
thin-walled microcentrifuge tubes and other specialized microtubes for real-
time PCR. These tubes are not likely to break and contaminate the holders, the
only piece of equipment that will go back in the PCR setup area for future use.
Barrier tips can prevent inadvertent suction of fluids into the barrel of pippet-
tors. Also a separate set of pipettors also eliminates pipettors as vehicles for
introducing PCR carryover contamination during setup. Real-time PCR avoids
carryover contamination due to the "real-time" detection of the amplicons as

Real-Time vs. Standard Format PCR 55

they are produced, and thus eliminates the necessity from ever having to open
the tube once the PCR reaction has been set up to run.

With each additional PCR run, the risk of false-positives increases for that
test. Therefore, minimizing the risk using the best work flow and work practices
possible is paramount. The size and the number of working areas that can be
used simultaneously at one given time will have to be anticipated based on the
number of samples that the laboratory plans to process daily, and how many
personnel will be working in each area at the same time.

Personnel. The PCR is simpler than traditional microbiology, not only regarding
the procedure itself, but also because mastering this new technology takes less
time than required to learn conventional microbiology. It can be mastered in less
than a week with very little or no previous training in microbiology, whereas con-
ventional techniques will require several weeks of training and a background in
microbiology. Competent conventional microbiologists can be easily trained to
perform PCR and will quickly discover the advantages of this methodology. It
can provide rapid same or next day results and, as an initial screen, it can stream-
line culture and isolations to the few presumptively positive samples (Fig. 2). If
we only check PCR-positive samples, we decrease our workload. For the labora-
tory, this reduces labor, resources, and finally cost. Labor costs rise exponentially
as the number of microorganisms to be ruled out increases when comparing
PCR to bacterial culture methods (47). Multiplex PCR and microarrays dis-
cussed in Chapter 1, reduces these pathogen screens to a single test (12, 26, 41,
46). Unfortunately, several of the commercial tests discussed later in this chapter
are detection tests for single foodborne pathogen, and single multipathogen-
detection systems are in their infancy. While detection and final confirmation of
foodborne pathogen may take up to 6 days with standard microbiology proto-
cols, PCR can provide clientele with preliminary results while the microbiology
lab continues to work up the submission. Laboratory supervisory personnel need
to be knowledgeable in interpreting PCR results, recognizing and correcting
problems as they develop, and in explaining the significance of PCR results to
the clientele. Chapter 2 discusses interpretation of PCR results. Personnel with
enough molecular training needs monitor result output and keep track of the
number of positive samples, to request the retesting of positive samples sus-
pected of being cross-contaminated. The personnel should also keep abreast of
the literature regarding PCR to discover improvements and problems with cur-
rent tests and identify new primers, PCR assays, or methodology available for
foodborne pathogens confronting the food industry.

REAL-TIME VS. STANDARD FORMAT PCR

The PCR amplicons are detected either in "real time" as they are synthesized,
or following PCR run on an agarose gel, or in an ELISA microplate. The suit-
ability of either real-time or standard format PCR will differ according to the
laboratory's needs, resources, and expertise.

Making PCR a Normal Routine of the Food Microbiology Lab

Tetrathionate broth
41 C; 24 h

* *...

►

24 h

BGN

XLT4 & BGN

37 C; 24 h

Salmonella Poly-O
Antisera

Culture-positive"

AND

48 h

longer

Biochemical Verification
Serotyping

Salmonella
PCR

+ --- + - - - +

^ 2h

24 h

48 h

Salmonella Poly-O
Antisera

Figure 4.2. PCR as tool to identify samples or enrichments, which need to be
processed further. (A) Steps and time required to identify sample(s) contaminated with
PCR. (B) Incorporation of PCR into screen of samples, or culture enrichments to
identify samples requiring additional culture/platings to identify the foodborne
pathogen, in this example Salmonella enter ica.

Amplicon detection can be done without agarose gels. In PCR-ELISA, the
amplicon hybridizes with a specific, internal oligo probe tagged with biotin,
which is subsequently bound and captured by streptavidin-coated ELISA
microplate. As the amplicon was labeled with digoxygenin-tagged nucleotides
during PCR, the bound amplicon-oligoprobe complex is detected coloromet-
ricly with antidigoxygenin antibody conjugated either to the enzymes alkaline
phosphatase or horseradish peroxidases. Color change is subsequently recorded
using an ELISA plate reader. With real-time PCR, a fluorimeter is built into the
thermocycler to monitor fluorescence as the machine cycles through its dena-
turing, annealing, and extension step. As the double stranded amplicon is

Real-Time vs. Standard Format PCR 57

produced, a fluorescent dye, called SYBR Green, or specific probes labeled with
a fluorescent dye bind to the PCR product and the detector measures the result-
ing fluorescence. The advantageous of either PCR-ELISA or real-time PCR
over agarose gels in the detection of amplicons is their amendability to 96-well-
microplate formats and automation (31, 43, 46). However, the need for agarose
gels for detecting amplicons is not eliminated with either PCR detection format,
as it still has a use in validation; the occasional trouble shooting, as problems
arise (see Chapter 2); and implementation of real-time PCR or PCR-ELISA
into the laboratory's routine.

Equipment. When talking about the laboratory adopting PCR, the first and in
most cases the only piece of equipment that comes to mind to most readers is
the thermocycler. Although, by far it is the most expensive individual piece of
equipment, we should not lose sight of other expenses accrued in setting up the
laboratory to perform PCR. Several sets of pipettes are needed for DNA extrac-
tion and PCR setup, a minimum of four sets. If the laboratory does not own
enough biosafety cabinets, PCR clean hoods can be used. Electrophoresis
equipment is required for amplicon detection in gels, the microwave oven
needed for melting agarose, and enough -20°C freezer space to keep all the nec-
essary PCR reagents separated from the sample template to be tested. For PCR-
ELISA, it will be necessary to also possess an ELISA reader, although this is an
essential component already in place for any laboratory that does serology (46).
Finally, the laboratory will need to record PCR results. For standard PCR for-
mat, where agarose gels are used to detect amplicons, the laboratory will need
UV transilluminator and a camera to capture the gel image.

There are a number of factors that will influence which type of thermo-
cycler is purchased. The overall cost of the machine itself might be a major
deciding factor in a laboratory's ability to take advantage of PCR technology at
all. A simple, hot-air, or conventional heating block thermocycler cost a few
thousand dollars. However, the price can vary depending on user's needs and
requirements regarding sample throughput (48 vs. 96), sample format (tube vs.
96-well microplate), and PCR reaction volume. The laboratory also needs to
consider the cost of the PCR assay with regards to the individual reagents, dis-
posables and ancillary components (e.g., wax beads or mineral oil) in selecting
a thermocycler. For example, thermocyclers with heated lids allow for smaller
PCR reaction volumes and eliminates the need for mineral oil in the PCR reac-
tion. The speed of a 30-cycler program, 10 vs. 90 min, may be another factor in
the purchase of a thermocycler. The laboratory may want to consider the ben-
efits of a gradient thermocycler, which allows users to perform, simultaneously,
several PCR tests that require different PCR cycle parameters. These gradient
thermocylers also have the advantage of identifying optimal-annealing temper-
ature for a single or multiple PCR primer sets in a single run, due to the
machine's ability to assign separate programs to each well. The price for real-
time PCR technology jumps to between $30,000 and $140,000, depending on
the components of the unit such as 96-well format and automation module. In
selecting a thermocycler, the laboratory also needs to determine if the PCR will

58 Making PCR a Normal Routine of the Food Microbiology Lab

be an in-house validated assay or a commercial assay, as the latter will require
specific PCR equipment. The following questions need to be addressed before
purchasing a PCR thermocycler: Will there be many samples submitted on a
regular basis or only periodically that require this type of technology? Will the
samples only be a part of the usual submitted workload? Will the samples
require detection of only one gene of interest, or are there various genes to iden-
tify, which would require different programs for the thermocycler? The PCR
format chosen by the laboratory is a big deciding factor to determine the needs
for equipment as we have seen before, because initial cost of equipment and
applications of the equipment vary.

All instruments, from pipettes to freezers, must be calibrated and certified
according to the respective institutional standard operating procedures, which
in most cases are dictated by the institutions accreditation agency. For most
institutions it is at least once a year. Most large equipment manufacturers offer
yearly contracts for calibration of their equipment.

Reagents and Disposables. Reagents for the PCR consist of those used for the
reaction that takes place in the thermocycler, and those used for the detection
of amplicons. Purchasing of reagents should be done from a reputable company
and molecular grade should be requested for any reagent that is going to be
used for PCR. Primers and probes as well as Taq DNA polymerase can be con-
taminated with extraneous DNA. Top quality components should only be used
and when handling these reagents, gloves should be worn as to prevent the
introduction of metal ions, nucleases, or other contaminants to the PCR reac-
tion (48). Water is an important component. Always use it as aliquoted, single
use volumes that have been filtered (0.22 |im), and autoclaved. If money is not
a problem, then ideally DNA-free and DNAse-free water should be purchased
for PCR. We must not forget that the PCR reaction requires a DNA template,
therefore; the required reagents for extraction or cell lysis need to be taken into
account. Consumption and cost of reagents is related to the machine and
the DNA extraction method that is used and the number of samples to be ana-
lyzed. Depending on the machine, volumes for each reaction can vary from 10
to 100 |il. The average volume used in PCR reactions is 25-50 |il. The smaller
the PCR reaction volume, the cheaper the cost per PCR test is. Costs for Taq
DNA polymerase, dNTPs, and other components of the PCR can turn PCR
into an expensive venture for a laboratory very quickly. Lastly, one must con-
sider the cost of labor for PCR setup as well as for sample preparation. It has
been reported that the overall costs of the reagents and materials involved in
identifying specific bacterial agents by PCR were 2 to 5 times higher than the
costs involved with bacterial culture identification (17). As molecular reagents
are continuously getting better in quality and longer in shelf life making them
overall less expensive, this may not hold true. A good example of this is the
reduction in cost of oligonucleotide synthesis.

In cost analysis, one must consider whether certain alternative traditional
microbiological methods are feasible or practical with the laboratory's current
resources and manpower. For example, Salmonella serotyping requires the lab-

Real-Time vs. Standard Format PCR 59

oratory to keep an extensive battery of antisera to identify the thousands of
different Salmonella serovars. Considering the limited shelf-life of the neces-
sary serotyping reagents, the laboratory's sample volume, and time it takes to
identify the Salmonella serovar, a PCR-based approach might be a more time-
and cost-effective approach (19, 21). Likewise, several of foodborne pathogens
described in Chapters 6 and 7 are either recalcitrant to current culture, or iso-
lation methods and PCR is more cost effective compared to the alternative iso-
lation procedures. Where PCR trumps culture-based detection methods is that
it can provide rapid preliminary results for making important decisions (15, 39,
41). For the laboratory, the decision as to which samples to work up further, and
for the clientele, which lots to hold and which ones to ship.

The thermocycler and its throughput capacity will dictate the type of con-
tainer used to hold PCR reaction: glass capillary tubes vs. thin-walled plastic;
PCR microfuge tubes vs. 96-well microplate. Pipette tips must have filter barri-
ers to avoid contamination of the inside of the pipette.

All newly prepared and purchased PCR reagents require quality control
before use. This is necessary for any food microbiology laboratory to become
successfully proficient at PCR.

Quality Control and Quality Assurance. Food microbiology laboratories are
used to working under standard operating procedures (SOPs), as their labora-
tories are accredited to perform food microbiology. Each SOP not only contains
the information on precisely how to conduct the procedure, but will also have
information regarding when and how much quality control (QC) needs to be
performed. A QC program usually requires all products and reagents (from
DNA extraction to PCR) of each lot received to be tested to make sure they
meet the same standard as previous lots so they can be utilized with confidence
in the procedures. A good practice is to aliquot all reagents in single-use format
after QC testing. Sometimes the testing of several aliquots is a good idea, espe-
cially if the laboratory does large volume of PCR routinely. Preparation of an
aliquoted ready-to-use PCR master mix, that has been QC tested, is a good
practice because this minimizes technician error resulting from miscalculation,
or from forgetting to add a key component in the master mix. Commercially
available kits should come with all reagents previously tested and this informa-
tion if not in the packet insert should be available upon request. SOPs must
include information regarding the required positive and negative amplification
controls, as these will determine the stringency and accuracy of our PCR tests.
The basic PCR controls are: DNA extraction controls, sample purposely spiked
with the organism of interest, and another spiked with a different, unrelated
organism; and PCR controls, pure, known amount of DNA from the organism
of interest, and negative, no DNA, control. A QC for PCR can be made even
more robust if an internal amplification control is included to check for the
presence of PCR inhibitors in our samples (22, 23, 34). For more information
about PCR inhibitors (see Chapters 3 and 4). Good SOPs and a good QC pro-
gram will help minimize mistakes due to bad reagents and human error. Every
laboratory should run a quality control program that is applicable and relevant

60 Making PCR a Normal Routine of the Food Microbiology Lab

for this methodology, and which is capable of detecting deficiencies at any level.
This process can be made simpler most times by consulting with other labs per-
forming similar PCR assays and purchasing reagents from suggested reputable
vendors, although this will not eliminate the need for QC testing. SOPs are good
training guides for new staff. Good record keeping is essential to any laboratory,
where a bound laboratory notebook is kept with detailed and dated descriptions
of protocols, reagents (including lot numbers, purchase, and expiration dates),
controls, and results (32). Furthermore, a good quality assurance (QA) program
at the institution ensures that there is compliance with SOPs for the PCR assays
and consistency is achieved. Additional measures to consider are addressed in
Chapter 2, and for a more thorough review of PCR laboratory setup, see
Methods in Molecular Medicine, Vol. 16: Clinical Applications of PCR (29).

Where to Locate Vendors. For setting up your laboratory to do diagnostic PCR,
you will need equipment and reagents to routinely perform PCR. Reagent-wise, you
will need the enzyme, buffers, nucleotides, and barrier tips for dispensing
reagents into thin- walled PCR tubes that contain the PCR reaction. In addition
to this, you will need a source for custom synthesis of your oligonucleotides and
probes needed for PCR. You will need to purchase agaraose gel electrophoresis
and photo documentation equipment (film or digital-based), for analysis and
documentation of conventional PCR results, as well as source for agarose, elec-
trophoresis buffers, loading dye, and molecular weight (MW) standards. We
have listed in Table 1, several sources for reagents and equipment listed above.
This list of companies is not an endorsement of the companies or their products;
rather, the table provides the reader an idea of what will be needed to implement
PCR into food microbiology laboratory

NONCOMMERCIAL TESTS FOR FOODBORNE PATHOGENS

Your laboratory after much consultation has decided that you need PCR as an
additional method to evaluate your food samples. Now you need to make a
decision which PCR format you want, provided you have a good physical infra-
structure that will allow you to have the required physically separated areas in
your laboratory, and you also have capable and trained personnel and a good
QC program. Your laboratory has also decided that the use of a commercial test
is not applicable, but instead the laboratory is going to use currently published
primers, or even design for a much better PCR primer set. Published data by
one laboratory can sometimes be difficult to reproduce due to the nature of the
reagents, the variation in equipment, and the personnel training. Validation
based on consensus criteria, detection limit, diagnostic accuracy (the degree
of correspondence between the response obtained by the PCR method and
the response obtained by the reference method on identical culture samples
[AC = (PA + NA)/total number of samples; where PA = positive agreement;
NA = negative agreement], diagnostic sensitivity, diagnostic specificity, and
robusteness, is a must for a successful microbiology laboratory (24, 25, 33). Your

Noncommercial Tests for Foodborne Pathogens

Table 4.1. Molecular biology vendors of PCR

Vendor

Web Address

Pro duct (s

BIO-RAD

www.bio-rad.com

Dupont Qualicon www.qualicon.com

EPICENTRE

Fisher Scientific
Co.

Idaho Technology
Inc.

Invitrogen

MO BIO

Laboratories Inc.

Molecular Probes,
Inc.

Promega

Roche Applied
Science

SeqWright

Sigma-Genosys

Thermocyclers, Electrophoresis
apparatus, buffers, and MW standards,
Photo documentation system

PCR diagnostic tests (e.g., Bax
Salmonella)

www.epicentre.com PCR reagents, DNA cloning reagents

www. fisher sci . com

www.idahotech.com

PCR tubes, barrier tips, etc.
Electrophoresis apparatus, Photo
documentation, Agarose, Electro-phoresis
buffer, Micropipettors,
-20 °C Freezer

Thermocyclers, design and synthesis
of primers and probes

www.invitrogen.com PCR reagents, PCR cloning vectors

www.mobio.com

Nucleic acid extraction kits

www.probes.com

Fluorescent dyes

www.promega.com

www.roche-applied-
science.com

PCR reagents, PCR cloning vectors,
MW standards, gel loading dye

PCR reagents, Thermocycler

www.seqwright.com DNA sequencing

www.sigma-
genosys.com

Custom oligonucleotide synthesis

USA/Scientific Inc. www.usascientific

com

PCR clean hood

laboratory will need to implement and then validate the PCR tests, bench-
marking performance by demonstrating that the new method can generate
results that are equal, or better, than those obtained by the current gold stan-
dard for detection. For a commercial PCR test that has already been validated,
implementation is the only step required by your laboratory (34). One last
point, when deciding which PCR format your laboratory is going to select,
keep in mind the compatibility of the PCR tests chosen with the laboratory's
current instrumentation and training. Molecular diagnostic tests that require
new equipment, more laboratory space, and more training may not be the best
choice. Therefore, a concerted effort at the initial planning stages should be
made to foresee future demands.

Validation. There is no perfect PCR test and interlaboratory variation in per-
formance of a PCR does occur. However, before diagnostic labs accept a PCR,

62 Making PCR a Normal Routine of the Food Microbiology Lab

there is a requirement for multilaboratory confirmation of the tests, specificity,
sensitivity, and reproducibility. (See Chapter 2, discussion of validation.) Spiked
samples as well regular samples should be used in the validation process as to
mimic as much as possible everyday samples and situations. The extent your lab
plays in the validation process of a PCR will be directly related to the target
organism, food matrix, and previous work describing validation of PCR in
peer-reviewed publications.

Currently, there is not a single harmonized validation protocol available. In
1999, the European Union (EU), through an initiative titled "the FOOD-PCR
Project" (http://www.PCR.dk), set out to validate and standardize PCR for the
detection of pathogenic bacteria in food using nonproprietary primers. The
intended outcomes of this project were production of guidelines and kits for
proficiency testing of different brands and types of thermocyclers, method for
DNA extraction and purification, production of reference DNA material, and
an online database containing validated PCR protocols. These protocols and
results are available from their website. A further attempt has been made by the
EU through the ISO/TC34 committee in collaboration with CEN/TC275,
through the proposal EN ISO/FIDS 16140 "Microbiology of food and animal
feeding stuffs — Part 42: Protocol for the validation of alternative methods" (1).
Validation through this method seems more suited for commercially developed
tests as the process is costly for nonproprietary, "home brew" PCR (34). Several
commercial diagnostic tests have been validated in a very extensive manner
and have been accredited by organizations on standards both at the interna-
tional and national level (14, 20, 44). Current commercially available tests for
the detection of foodborne pathogens will be reviewed later in this chapter.

Standardization. Standardization of PCR tests and extraction protocols at the
national and international level will allow for accurate interlaboratory com-
parison. This can be achieved either with commercial tests, or with validated
published primers. Standardization allows for fast implementation of tests, war-
ranted accuracy, and detection limits, as well as known strength to allow for
some variation in the tests procedure without giving misleading results.
Standardization will also guarantee continued research and improvement of the
PCR assay and protocols. Thus, the PCR test will fulfill its promise of being
simple, high-yield, fast, appropriate, and even cheaper than the traditional
culture (24, 34).

AVAILABLE COMMERCIAL PCR TESTS FOR FOODBORNE
PATHOGENS

There is a wealth of ready-to-use PCR-based tests for the most common food-
borne pathogens: Salmonella spp., Listeria sp. and E. coli 0157:H7. The avail-
ability of commercial PCR tests for other foodborne pathogens is sparse and
laboratory "home-brewed" PCR tests may be a better, if not the only, option.
Chapters 5-7 describe several published PCR tests for bacterial, viral, and

Available Commercial PCR Tests for Foodborne Pathogens

protozoal-foodborne pathogens. Several real-time and standard format PCR
are commercially available. These two formats need to be studied carefully by
each individual laboratory introducing PCR into their routine, chiefly to deter-
mine whether the equipment cost in comparison with potentially improved
diagnostic ability is a worthwhile endeavor to pursue. One or more national and
international agencies have validated commercial PCR tests described in this
section (14, 20, 44). Their sensitivities and specificities are well known (14, 18,
20, 35, 41, 44, 45), and information is readily available form the products
websites (www.andiatec.com; www.bio-rad.com; www.qualicon.com/bax.html;
and www.roche-applied-science.com). Once a decision has been made on a PCR-
detection format, then the choice of brands should be determined by the individ-
ual laboratories based on following consideration: (1) simplicity, (2) throughput,
(3) cost, (4) speed, and (5) appropriateness (37). Table 2 lists the currently avail-
able PCR-based diagnostic tests and identifies which type of format they are
based. The commercial PCR tests described in the next section is not an endorse-
ment of any one product, but rather presents the reader with the commercial kits
available for PCR detection of pathogens in foods.

Real-Time PCR. BAX Dupont-Qualicon This PCR test is based on the use of
pathogen-specific primers combined with a dye that allows for detection
of amplicon formation during each cycle. A selective enrichment step appropri-
ate for each food is required before the DNA extraction step. Testing is carried

Table 4.2. Commercial validated and approved test for the detection of bacterial
pathogens in food

Test Format

Brand Name

Certification Agency

Organisms Detected

Real-Time

BAX Dupont-

AOAC-RI

Salmonella, E. coli

PCR

Qualicon

0157:H7,

USDA-FSIS

Listeria monocytogenes

AFNOR

NorVal

Food-proof

AOAC-RI

Salmonella, E. coli

Roche

0157:H7, Listeria

IQ-Check BioRad

AFNOR

Salmonella

PCR-ELISA

Probelia BioRad

AFNOR

Salmonella, E. coli
0157:H7, Listeria monocy-
togenes, Campylobacter
jejuni and Campylobacter
coli.

AnDiaTec

Salmonella

Listeria monocytogenes

Notes: AOAC-RI: Association of Official Analytical Chemists-Registration International; AFNOR:
Association Francaise de Noramlisation; NorVal: Nordic Validation Organ; USDA-FSIS: United
States Department of Agriculture- Animal and Food Safety and Inspection Service.

64 Making PCR a Normal Routine of the Food Microbiology Lab

out in a 96-well type matrix and up to 94 samples can be tested at once with one
positive and one negative control. The product amplification is detected real
time by including the fluorescent SYBR Green I. This fluorogenic reporter dye
is not specific for the desired target molecule, therefore, post-PCR melting curve
analysis is required in the protocol, and spurious, nonspecific amplicons are
easy to identify. The BAX system has been developed for the detection of
Salmonella spp., Listeria monocytogenes, E. coli 0157:H7, and Campylobacter
spp(12, 16, 20) (Table 2).

The BAX Salmonella spp has been accepted as an official method by several
accreditation bodies; the Association of Official Analytical Chemists (AOAC)
has accepted it as an official method (#2003.09) for use in raw beef, raw chicken,
raw frozen fish, cheese, frankfurters, and orange juice (5). The AOAC-RI
Performance Tested Method license #100201 applies to food: tested on milk,
black pepper, chilled ready meal, chipped ham, chocolate, cooked chicken,
cooked fish, custard, dry pet food, elbow macaroni, frozen peas, hot dogs, non-
fat dry milk, orange juice, peanut butter, pizza dough, seafood-prawns, alfalfa
sprouts, ground beef, and liquid egg (3). The USDA-FSIS (MLG 4C.00) has
adopted the BAX Salmonella spp. for use in ready-to-eat meat, poultry, and
pasteurized eggs (6). The AFNOR (certificate QUA- 18/3- 11/02) applies to all
human and animal food (2). The NordVal (certificate 2003-2-5408-00023)
applies to all foods and animal feed (7).

The BAX Listeria monocytogenes has been approved by the AOAC. It has
been accepted as an official method (#2003.12 AOAC-RI Performance Tested
Method license #070202) for use in a wide variety of foods including raw meats,
fresh produce/vegetables, processed meats, seafood, dairy cultured/noncultured,
egg and egg products, and fruit juices (4). The USDA-FSIS (MLG 8A.00) has
adopted the BAX Listeria monocytogenes for use in red meat, poultry, egg, and
environmental samples (11). The BAX E. coli 0157:H7 has been approved by
the AOAC, and has accepted as an official method (#2004.8 AOAC-RI
Performance Tested Method license #010402) for use in apple cider, orange
juice, and ground beef (9). The USDA-FSIS is currently in the process of vali-
dating this technology.

Food-proof Roche This PCR test is based on real-time detection of either
Salmonella spp. or Listeria monocytogenes DNA in raw materials and food
samples through the use of a combination of primers and sequence-specific taq-
man probes with hot start methodology. An internal control is added to each
sample prior to extraction, in order to assess the presence of PCR inhibitors.
Additionally, this commercial test contains uracil-DNA glycosidase to avoid
PCR carryover contamination. The Salmonella spp. test method is certified by
the AOAC-RI with license #12030 (8), as a performance-tested method for
detecting Salmonella in food products. Some raw materials are highly inhibitory
for the PCR reaction and the use of a proprietary sample preparation kit (High
Pure Food-proof kit; Roche; Indianapolis, IN) seems to ensure DNA of high
quality for PCR. The Listeria monocytogenes test method has also been certi-
fied by the AOAC-RI with license #12030 (10) as a performance-tested
method for the detection Listeria monocytogenes in food products when used in

References 65

combination with ShortPrep foodproof II Kit. These foodstuffs include peanut
butter, dried whole eggs, dry whole milk, dry pet food, milk chocolate, melon
cubes, white cabbage, pizza, vanilla ice cream, paprika emulsion dye, spaghetti,
sausage, gravlax, "harzer" cheese, raw ground chicken, raw ground pork, bean
sprouts, parsley flakes, ham, and Pollack fillet.

IQ- Check BioRad This commercial test (BioRad; Hercules, CA) uses
primers and a molecular beacon probe tagged with a fluorescent label specific
for the target organism. Amplified products are detected real time by detection
of the fluorescence. This system also contains an internal control, present in the
amplification mix that assesses the presence of PCR inhibitors. The internal
control is detected real time using another florescent beacon labeled with a dif-
ferent fluoroprobe (27, 45). The BioRad's IQ-Check Salmonella detection kit
has been approved by AFNOR as a valid method for the detection of
Salmonella in all human and animal food products, and environmental samples.

PCR-ELISA. Probelia BioRad This PCR test is based on the enzymatic detec-
tion of a PCR product that combines DNA: DNA hybridization with its cap-
ture and in a microtitier plate with an internal oligoprobe. For the detection of
Salmonella, this PCR-ELISA can detect 3 CFU/25g sample with 99.6% speci-
ficity, following an 18 h of preenrichment step (14, 18). It includes an internal
control to evaluate PCR inhibitors in samples, which are monitored in a paral-
lel well. Results depend on the optical density obtained on the detection
microplate relative to the internal control well. Salmonella and Listeria applica-
tions have been approved AFNOR for all foodstuffs.

AnDiaTec Salmonella sp. PCR-ELISA This commercial kit (AnDiaTec
GmbH & Co.; Kornwestheim, Germany) comes in two modules. Module one
includes all reagents needed for DNA extraction, amplification mixture in a
ready-to-use format, and negative and positive controls. The second module con-
sists of a microtiter plate, probes, the peroxidase conjugate, and all the buffers
required for DNA: DNA hybridization and enzymatic detection of the amplified
PCR products. For Salmonella spp. detection, this test has a demonstrated 98%
agreement with bacterial culture when it is conducted according to the ISO 6579
standards. Only samples that had high levels of inhibitors, such as bitter choco-
late and herbs required a different extraction method than the one included in
the tests kit (35). There is also a kit for the detection of Listeria monocytogenes.

REFERENCES

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66 Making PCR a Normal Routine of the Food Microbiology Lab

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25. Josefsen, M.H., S.T. Lambertz, S. Jensen, and J. Hoorfar. 2003. Food-PCR.
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CHAPTER 5

Molecular Detection of Foodborne

Bacterial Pathogens

Azlin Mustapha*, Ph.D. and Yong Li, Ph.D.

Food Science Program, University of Missouri-Columbia, 1 Columbia, MO 65211
l Food Science and Human Nutrition Program, University of Manoa, Honolulu,

HI 96822

Introduction

Conventional PCR Detection of Foodborne Pathogens

Multiplex PCR Detection of Foodborne Pathogens

Reverse Transcriptase PCR Detection of Foodborne Pathogens

Real-Time PCR Detection of Foodborne Pathogens

Conclusions

References

INTRODUCTION

Since its discovery in the mid 1980s, the Nobel prize-winning polymerase chain
reaction (PCR) has gained acceptance as a powerful microbial detection tool.
The application of automated PCR technology in the medical and pharmaceu-
tical industries has a longer history than its use in the food industry. In recent
years, however, PCR technology has become more recognized for its potential at
becoming a powerful alternative to cultural methods of pathogen detection in
foods. The BAX PCR system manufactured by Dupont Qualicon, Inc. and the
TaqMan Pathogen Detection kits by Applied Biosystems Co. are two examples
of automated PCR systems that have found application in the food industry. The
BAX PCR is currently an AOAC-approved PCR-based method that can be used
to detect Listeria monocytogenes, Listeria species, Salmonella and Escherichia
coli 0157:H7 in food products, and is used by the USDA Food Safety and
Inspection Service (FSIS) for detecting L. monocytogenes and Salmonella in
meat, poultry, egg, and ready-to-eat meat products.

The advantage of using PCR techniques for food products is the specificity
and rapidity of the tests as compared to traditional cultural techniques.
However, the sensitivity of a PCR-based test for detection of pathogens in foods
will depend on the type of food matrix involved. Because of the complexity of
food matrices, many compounds in foods can prove to be inhibitory to PCR
reactions (74). Thus, in most PCR assays, including the automated BAX and

Corresponding author. Food Science Program, 256 William Stringer Wing, University of
Missouri-Columbia, Columbia, MO 65211, Phone: (573) 882-2649; FAX: (573) 882-0596;
e-mail: MustaphaA@missouri.edu.

70 Molecular Detection of Foodborne Bacterial Pathogens

TaqMan systems, food samples are enriched prior to the amplification steps in
order to overcome this hurdle. In addition to increasing the number of target
microorganisms, and thus the detection sensitivity, enrichment is also helpful
at reducing the risk of amplifying nucleic acids from dead or nonculturable
cells (11). Following PCR, target species can be detected by agarose gel elec-
trophoresis or hybridizations with labeled DNA probes (60).

Despite its limited use in the food industry, numerous studies have been
reported in the literature on the development of PCR-based detection methods
for foodborne pathogens. The following text will discuss the various PCR detec-
tion methods that have been successful at detecting various pathogens in different
types of foods. Table 1 illustrates the target genes, primer sequences, tested foods,
detection limits, specificity, and references of certain published PCR protocols.

CONVENTIONAL PCR DETECTION OF FOODBORNE
PATHOGENS

Conventional PCR relies on the amplification of nucleic acids via a single pair
of primers to detect one pathogen at a time, and the PCR reaction is optimized
for the specific food product tested. With enrichment of tested samples, con-
ventional PCR assays can detect Clostridium perfringens at 10 cfu/g in meat,
milk, and salad (17), enterohemorrhagic E. coli 0157:H7 at ca 10" 1 cfu/g in beef
(55), enterotoxigenic E. coli at 1 cfu/ml in milk (70), L. monocytogenes at ca 10" 2
to 10° cfu/g in various foods (19, 30, 48, 63), Salmonella at ca 10 _1 to 10 1 cfu/g
in milk and meat products (11, 22, 43, 44), Shigella at 2 cfu/g in mayonnaise
(71), Staphylococcus aureus at 5-15 cfu/g in skim milk and cream (65), and
10 cfu/ml in raw milk and curd (16), and Vibrio parahaemolyticus at 10 cfu in
fish (31). However, other studies have found much higher detection limits of
PCR assays for foods, without enrichment, including 4 x 10 2 to 4 x 10 3 cfu/g for
C. perfringens in Korean ethnic foods (32), 10 cfu/ml for L. monocytogenes in
milk (3), 5 x 10 1 to 5 x 10 2 cfu/ml for Shigella in various produce washes (36),
10 2 cfu/g for S. aureus in skim milk and cheddar cheese (67), 3 x 10 2 cfu/g for
V. parahaemolyticus in shellfish (74), and 4 x 10 4 cfu/g for Yersinia enterocolitica
in pork (37). A PCR based on degenerate primers targeting known nonribo-
somal peptide synthetases (NRPS) has also been successfully developed for
detecting emetic strains of Bacillus cereus (15).

MULTIPLEX PCR DETECTION OF FOODBORNE
PATHOGENS

In multiplex PCR, two or more gene loci are simultaneously amplified in one
reaction. This technique has been used widely to characterize pathogenic bac-
teria on the basis of their virulence factors and antigenic traits. Fratamico
et al. (21) employed primers for a plasmid-encoded hemolysin gene (hlyA 933 ),

Multiplex PCR Detection of Foodborne Pathogens

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84 Molecular Detection of Foodborne Bacterial Pathogens

chromosomal flagella (fliC H1 ; flagellar structural gene of H7 serotype), Shiga
toxins (stx v stx 2 ), and attaching and effacing (eaeA) genes for specific identifi-
cation of E. coli 0157:H7. Similar protocols were reported for concurrent
determination of multiple toxin genes in C. perfringens (70) and S. aureus
(4, 10). Further, species or serotype differentiation can also be achieved via mul-
tiplex PCR. Denis et al. (14) selected 16S rRNA, map A, and ceuE as the target
genes for simultaneous detection of Campylobacter jejuni and Campylobacter
coli. A similar assay was established for simultaneous identification of
Salmonella sp., S. Enteritidis, and S. Typhimurium in one reaction (64). In multi-
plex PCR, bacterial pathogens belonging to different genera can also be screened
in the same amplification system. Li and Mustapha (41) and Li et al. (42)
established a multiplex PCR for simultaneous detection of E. coli 0157:H7,
Salmonella, and Shigella in apple cider, produce and raw and ready-to-eat meat
products. In most situations of multiplex PCR, the optimal conditions for dif-
ferent primer sets may be unique and interference among different primer pairs
may occur, resulting in uneven amplification of different target sequences and
limited sensitivity (68). Thus, adjusting the concentration of Taq DNA poly-
merase, MgCl 2 , or dNTPs, as well as the concentration ratio among the differ-
ent primer pairs is needed in the optimization of the amplification system.
Although to design a robust multiplex PCR assay for foods can be challenging,
once optimized for the specific pathogens and food products, this method has
the advantage of being cost effective and highly efficient. Because of its selec-
tivity, sensitivity, and efficiency, a multiplex PCR protocol is very applicable and
suitable for comprehensive testings of specific foods.

REVERSE TRANSCRIPTION-PCR DETECTION
OF FOODBORNE PATHOGENS

The use of reverse transcription PCR (RT-PCR) in foods is limited due to
the difficulty of extracting undegraded mRNA from pathogens in complex
food matrices. By amplifying the iap mRNA, a RT-PCR was successfully
developed for detecting viable L. monocytogenes cells in cooked ground beef,
artificially contaminated with ca. 3 cfu/g, following a 2-h enrichment step (34).
Mclngvale et al. (52) established a similar protocol for Shiga-toxin-producing
E. coli with optimal growth medium, incubation temperature, and aeration.
The assay was validated in artificially contaminated ground beef. Viable E. coli
0157:H7 at an initial inoculum of 1 cfu/g was detectable in the meat after a
12-h enrichment. In addition, a RT-PCR was developed for detecting mRNA
from the sefA gene of S. Enteritidis (66). The sensitivity of the assay depended
on the physiological state of the cells under different temperatures and pH.
With the RT-PCR, it was possible to detect 10 cells of S. Enteritidis PT4 in
contaminated minced beef and whole egg samples following a 16-h enrich-
ment step. Although not cost-effective for routine testings of pathogens in

Conclusions 85

foods, these reports highlight the potential of RT-PCR for the detection of
viable bacterial pathogens in foods.

REAL-TIME PCR DETECTION OF FOODBORNE
PATHOGENS

Recent advances in fluorescent chemistries and detection instruments allow fur-
ther development of PCR technology as a more efficient and sensitive tool for
"real-time" microbiological analysis of foods. The use of nonspecific fluorescent
double-stranded DNA-binding dyes (such as SYBRGreen or SYBRGold), or
specific fluorescence resonance energy transfer technology (such as 5'-nuclease
assay [TaqMan], or molecular beacon) has resulted in PCR assays with quanti-
tative capability in a real-time manner (53). A number of real-time PCR assays
have been described for the detection and quantification of C. jejuni (59),
enterohemorrhagic E. coli serotypes 0157, Olll, and 026 in ground beef (62),
L. monocytogenes in cabbage (25), Salmonella serotype D in egg (61), pathogenic
Vibrio species in oyster (7, 50, 57), and Y. enterocolitica in ground pork (29, 72).
Further, a sensitive multiplex real-time PCR has been developed for the simulta-
neous detection of E. coli 0157:H7, Salmonella, and Shigella in pure culture and
in ground beef (A. Mustapha, unpublished data). In addition to maintaining all
the advantages of conventional PCR, real-time PCR has added speed and sensi-
tivity. This technique can quantify a target DNA with greater reproducibility,
which is very valuable in the quantitative assessment of microbial risks and the
execution of HACCP programs in the food industry. The current drawback for
using real-time PCR for routine food testing is the cost involved, not only in the
equipment but the reagents.

CONCLUSIONS

The PCR has come a long way since its discovery, evolving from a tool used
mainly in forensic, medical, pharmaceutical, and plant sciences to food sci-
ence and the food industry. It may be one of the most remarkable discoveries
of the 20th century and has opened new doors in a wide array of fields that
would never have been possible prior to its utilization. Although research
have shown that PCR can be a powerful method for detection of foodborne
pathogens in pure culture as well as in certain foods, much more work needs
to be done to truly make it the best alternative detection technique to con-
ventional cultural methods. Until the enrichment steps can be eliminated, the
rapidity of PCR assays can still be argued. Foods also are so different in their
composition, resulting in a multitude of compounds that may be inhibitory to
the detection of some pathogens while not affecting others, thus making it
more challenging to design a one-size-fits-all PCR assay for foods.

86 Molecular Detection of Foodborne Bacterial Pathogens

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J. Food Sci. 68:1459-1466.

CHAPTER 6

Molecular Approaches for the Detection

of Foodborne Viral Pathogens

Doris H. D'Souza* and Lee-Ann Jaykus

Department of Food Science, College of Agriculture and Life Sciences,
North Carolina State University, Raleigh, NC 27695-7624

Introduction

General Detection Considerations and the Challenges

Virus Concentration

Virus Concentration Methods for Shellfish

Virus Concentration Methods for Other Than Shellfish

Nucleic Acid Extraction

Detection

RT-PCR Detection of Viruses in Foods
Alternative Nucleic Acid Amplification Methods

Confirmation

Real-Time Detection

Conclusions

Acknowledgments

References

INTRODUCTION

The human enteric viruses are now recognized as major causes of acute non-
bacterial gastroenteritis throughout the world. Those of primary epidemiolog-
ical significance include hepatitis A virus (HAV) and the noroviruses, formerly
known as the Norwalk-like viruses (NLVs), or the small round structured
viruses (SRSVs) (reviewed in 88). Currently, the noroviruses consist of five
genogroups: GI (prototype Norwalk virus); Gil (prototype Snow Mountain
Agent); GUI (prototype bovine enteric calicivirus); GIV (prototype Alphatron
and Fort Lauderdale virus); and GV (prototype Murine norovirus) (Vinje, per-
sonal communication). The sapoviruses (previously called Sapporo-like viruses)
are genetically related to the noroviruses and have occasionally caused viral gas-
troenteritis in humans. Both the noroviruses and the sapoviruses are members
of the Calicivirideae family, an antigenically and genetically diverse group of
gastrointestinal viruses (9, 39, 40). Other viruses that can cause food and water-
borne disease include the adenoviruses, astroviruses, the human enteroviruses
(polioviruses, echoviruses, groups A and B coxsackieviruses), hepatitis E virus,

Corresponding author: Phone: (919) 513-2076, FAX: (919) 513-0014 e-mail:
ddsouza@unity.ncsu.edu

92 Molecular Approaches for the Detection of Foodborne Viral Pathogens

parvoviruses, and other relatively uncharacterized small round viruses. The
rotaviruses, which are the leading cause of infantile diarrhea worldwide, are
transmitted primarily by contaminated water but can on occasion be foodborne.

The human enteric viruses replicate in the intestines of infected human hosts
and are excreted in the feces. Their primary mode of transmission is the
fecal-oral route through contact with human fecal matter, although they may
also be shed in vomitus. These viruses are readily spread by person-to-person
contact, which is frequently responsible for the propagation of primary food-
borne outbreaks. Contamination of foods may occur directly, through poor
personal hygiene practices of infected food handlers, or indirectly via contact
with fecally contaminated waters or soils (49, 50). Since viruses must survive the
pH extremes and enzymes present in the human gastrointestinal tract, they are
regarded as highly environmentally stable, allowing virtually any food to serve
as a vehicle for their transmission (50). Although enteric viruses are unable to
replicate in contaminated foods, they are able to withstand a wide variety of
food processing and storage conditions. When present in contaminated food,
their numbers are usually quite low, but since their infectious doses are also low,
any level of contamination may pose a public health threat.

In spite of their initial recognition decades ago, the human enteric viruses
can be considered "emerging" agents of foodborne disease, mainly because only
recently have scientists been able to reliably detect these pathogens. In fact, prior
to the advent of molecular biological techniques, epidemiological criteria were
the best means, by which cases of enteric viral illness were recognized. Over the
last 10 years, significant advances in nucleic acid amplification methods have
made detection of enteric viruses in human clinical samples, specifically feces,
all but routine.

The opposite is the case for the detection of viruses in foods. Historically,
this has been done by infectivity assay using susceptible, live laboratory hosts.
Host systems employed were mainly mammalian cell cultures of primate origin.
Unfortunately, the epidemiologically important human enteric viruses, includ-
ing the noroviruses and wild-type hepatitis A virus, cannot be propagated in
mammalian cell culture systems, and so these are not viable detection options
(49, 50). In the absence of in vitro virus propagation methods, nucleic acid
amplification has been a promising alternative. In this chapter, we will discuss
existing technologies that can be applied to the detection of viruses in foods,
and we will address new developments and research needs for the application of
these methods on a routine basis.

GENERAL DETECTION CONSIDERATIONS
AND THE CHALLENGES

The development of effective virus detection methods from food commodities
poses several challenges. Like many bacterial pathogens, these agents are typi-
cally present at low levels in contaminated foods. However, unlike bacterial
pathogens, viruses cannot replicate in foods, making the use of traditional

Virus Concentration 93

food microbiological techniques of cultural enrichment and selective plating
inapplicable per se. Therefore, the first goal in developing virus detection meth-
ods for foods is to separate and concentrate the agents from the food matrix. It
is also necessary to sample relatively large volumes to assure adequate sample
representation, thereby optimizing detection assay sensitivity.

VIRUS CONCENTRATION

Sample preparation prior to detection is of key importance when applying
molecular methods to detect viral contamination in foods. In this regard, spe-
cific challenges include high sample volumes in relation to small amplification
volumes, low levels of contamination, and the presence of residual food com-
ponents that can later compromise detection (31, 37, 86, 97).

The main goals of virus concentration methods are to decrease sample vol-
ume and eliminate matrix-associated interfering substances, while simultane-
ously recovering most of the viruses present in the food sample. In order to
achieve these goals, sample manipulations are undertaken that capitalize on the
behavior of viruses to act as proteins in solutions, and to remain infectious at
extremes of pH or in the presence of organic solvents. Because of their frequent
association with viral foodborne disease outbreaks, early work on virus concen-
tration and purification from foods focused mainly on bivalve molluscan shell-
fish. Recent research endeavors have included a broader range of at-risk foods.

Two major approaches to virus concentration, particularly as applied to shell-
fish commodities, are termed extraction-concentration and adsorption-
elution-concentration (49). Both methods utilize conditions that promote the
separation of viruses from shellfish tissues, through the use of filtration, centrifu-
gation, adsorption, elution, solvent extraction, precipitation, and/or organic floc-
culation. The procedure generally begins with sample blending in a buffer, usually
containing amino acids and an elevated pH. A common example of elution buffer
is 0.1 M glycine-0.14 N saline, pH 9.0. A crude filtration step through a mesh
material such as cheesecloth may be done to remove large sample particulates.
Viruses do not sediment unaided, even at centrifugation speeds approaching
10,000 x g. Therefore, centrifugation can be used to sediment large food parti-
cles, with the recovery of the virus-containing supernatant. The next step usu-
ally involves pH manipulation or the addition of precipitation agents, creating
conditions such that viruses adsorb to the remaining shellfish tissues. Upon
subsequent centrifugation, the adsorbed viruses sediment with the tissues and
the supernatant is discarded. Elution, whereby the viruses are desorbed from
the tissues by further pH and/or ionic manipulations is then carried out. On
subsequent centrifugation, the precipitated tissue is discarded, again retaining
the supernatant. Using sequential steps of adsorption, elution, filtration, pre-
cipitation, and centrifugation, viruses are concentrated to small sample volumes
and simultaneously purified, with the removal of large proportions of the food
matrix and matrix-associated organic materials that may later compromise
detection of the viruses.

94 Molecular Approaches for the Detection of Foodborne Viral Pathogens

Precipitation of viruses can be achieved by lowering pH, so called acid precip-
itation, or by the addition of polyethylene glycol (PEG). Both methods capitalize
on the property that viruses behave as proteins in solution. The viruses, along with
some of the matrix-associated proteins, will precipitate when the pH is lowered to
that approximating the virus isoelectric point. Polyethylene glycol causes removal
of water, allowing proteins to fall out of solution. Another method similar to
precipitation is organic flocculation. Flocculating agents interact with organic
material in the matrix, causing the formation of a gelatinous "floe," to which
the viruses absorb (49, 50). In the case of acid and PEG precipitation, and in
organic flocculation, the virus-containing solid materials can be readily harvested
by centrifugation, usually done at fairly low speeds (e.g., <5000 x g).

Further removal of matrix-associated organic materials can be done using a
variety of agents. Since viruses remain infectious even after exposure these
organic solvents such as chloroform, trichloro trifluoroethane (Freon) and
more environmentally friendly solvents such as Vertrel (Dupont), these chemi-
cals can be used to remove polar food components such as lipids. Alternative
commercial virus purification agents such as ProCipitate and Viraffinity
(LigoChem, Inc., Fairfield, NJ 07004) (31, 51, 66) are known to eliminate poly-
saccharides, an important matrix-associated inhibitor in shellfish and produce.
The cationic detergent cetyltrimethylammonium bromide (CTAB) (6, 7, 52)
also aids in the removal of polysaccharides. The use of Sephadex (23), cellulose
(110), or Chelex (98) is helpful in the elimination of salts and small proteins.
Ultrafiltration, a method that is frequently applied at the latter stages of a
virus concentration scheme, provides reduction in volume while simultaneously
purifying the sample.

VIRUS CONCENTRATION METHODS FOR SHELLFISH

The early molecular work aimed at detecting viruses in the food matrix focused
almost exclusively on shellfish. The most common method utilizes some sort of
virion concentration step prior to release or extraction of nucleic acid, followed
by amplification. A second, less commonly used method resorts to direct
nucleic acid extraction of a previously untreated food matrix, which circum-
vents the need for virus concentration.

The objective of the virion concentration approach is to concentrate viruses
and remove inhibitors prior to nucleic acid amplification, with or without prior
nucleic acid extraction. In early work by Atmar et al. (6, 7), investigators
processed artificially contaminated shellfish samples using an initial concentra-
tion and utilized a purification scheme that consisted of solvent extraction
and PEG precipitation steps. This was followed by nucleic acid extraction and
subsequent amplification. Cetyltrimethylammonium bromide (CTAB) was
added to remove residual inhibitors from crude nucleic acid extracts, and the
resulting eluant was amplified using reverse transcriptase-polymerase chain
reaction (RT-PCR) (6, 7). Dissecting the oysters, discarding the muscle tissue
and processing only the digestive diverticula improved the PCR's detection

Virus Concentration Methods for Shellfish 95

limits. This sampling approach is now the method of choice (11, 65, 67, 69, 90, 92).
Figure 6.1 illustrates a representative sample-preparation protocol for shellfish.
Note that second generation protocols frequently employ sequential PEG
precipitation in addition to adsorption, elution, and solvent extraction steps
(8, 16, 19, 20, 25, 26, 30, 59, 60, 63, 64).

Some investigators apply an antibody capture step to further concentrate
and purify viruses from shellfish extracts prior to detection using RT-PCR.

25 g OYSTER MEAT

HOMOGENIZATION

Add 175 ml of sterile cold deionized water (ratio of meat: water is 1:7)

t
VIRUS ADSORPTION

Adjust pH to 4.8 and with conductivity of < 2000 jiS using water

CENTRIFUGATION

Collect pellets at 2,000 x g for 20 min

t
VIRUS ELUTION

Add 0.75 M glycine-0.15 M NaCl, pH 7.6 (1:7 ratio of meat: eluant buffer)

pH adjustment to 7.5 to 7.6, Vortex at room temp for 15 mins

Centrifuge at 5,000 x g for 20 min at 4 C

Collect supernatant (A)

t
RE-ELUTE VIRUS FROM PELLET

Add 0.5 M threonine-0.15 M NaCl, pH 7.6 (1:7 ratio)

Collect supernatant (B)

Combine/Pool supernatant A and B

t
PEG PRECIPITATION

Add 8% PEG 8000-0.3 M NaCl

Incubate at 4 C for 4 h or overnight

Centrifuge 6,700 x g for 30 min

Collect pellets and resuspend in 10 ml of Phosphate-buffered saline

SOLVENT EXTRACTION OF VIRUS

Add 10 ml of chloroform (ratio of eluant: chloroform is 1:1)

Centrifuge at 1, 700 x g for 30 min

Collect supernatant 1

t
Reextract from bottom layer with half volume of 0.5 M threonine

Collect supernatant 2

Combine/pool supernatant 1 and 2

t
PEG PRECIPITATION

Add 8% PEG-0.3 M NaCl at 4 C for 2-4 h
Centrifuge at 14,000 x g for 15 min, Collect pellets

t
RNA EXTRACTION

Figure 6.1. Concentration of viruses from oysters using virus adsorption, glycine-
saline buffer and threonine-saline extraction, PEG precipitation, chloroform extrac
tion, and PEG concentration as modified by Shieh et al. (95).

96 Molecular Approaches for the Detection of Foodborne Viral Pathogens

Desenclos et al. (29) were the first to implicate hepatitis A virus in oyster out-
break specimens by immunocapture of the virus, heat release of viral nucleic
acids, and subsequent RT-PCR detection. Capitalizing on the work of Jansen
et al. (48), other investigators have coated paramagnetic beads with anti-HAV
IgG and used these to capture HAV from oyster extracts initially processed for
virus concentration using a combination of elution, poly electrolyte floccula-
tion, filtration and/or ultrafiltration (28, 71). Sunen et al. (100) and Schwab
et al. (94) also used antibody capture as a final virus concentration step. More
recently, Kobayashi et al. (61) used magnetic beads coated with the antibody to
the baculovirus-expressed recombinant capsid proteins of the Chiba virus
(rCV) to capture noroviruses from food items implicated in an outbreak of
acute gastroenteritis in Aichi Prefecture, Japan, detecting the virus in these
foods by RT-PCR. Abd El Galil et al. (1) have used immunomagnetic separa-
tion for the detection of hepatitis A virus from environmental samples using
real-time nucleic acid amplification methods.

The alternative approach of direct nucleic acid extraction and RT-PCR
applied to unprocessed food sample, involves extraction of total RNA from the
sample without any prior sample manipulations. This method is best suited for
simple sample matrices such as the surfaces of fresh fruits and vegetables.
However, Legeay et al. (65) recently reported a method that involved enzymatic
liquefaction of shellfish digestive tissues, followed by clarification using
dichloromethane extraction. In this case, the investigator reported that the sam-
ple could be directly processed for nucleic acid isolation and subsequent virus
detection by RT-PCR.

VIRUS CONCENTRATION METHODS FOR FOODS
OTHER THAN SHELLFISH

Gouvea et al. (36) first reported a systematic method for the detection of
norovirus and rotavirus from representative food commodities other than shell-
fish, including orange juice, milk, lettuce, and melon. The method involved
blending or washing, clarification by centrifugation, and removal of inhibitors
by Freon extraction followed by RNA extraction. Leggitt and Jaykus (66) devel-
oped a prototype method for the concentration of poliovirus, hepatitis A virus,
and Norwalk virus from 50 g samples of artificially contaminated hamburger
and lettuce. The steps used included homogenization, filtration through cheese-
cloth, Freon extraction (hamburger only) and two sequential PEG precipita-
tions. The sequential precipitations, which used increasing PEG concentrations,
resulted in a 10- to 20-fold sample volume reduction from 50 g to approximately
2.5 ml (66). The resuspended PEG precipitate could be assayed for virus recov-
ery by mammalian cell culture infectivity assay, when applicable, which allows
for direct comparison between virus infectivity and molecular detection (104).
Subsequent nucleic acid extraction resulted in an additional 100-fold sample
volume reduction with detection at initial inoculum levels of >10 2 infectious
units per 50-g food sample (66). A schematic overview of this procedure is

Virus Concentration Methods for Foods Other than Shellfish 97

provided in Figure 6.2. Schwab et al. (91) used TRIzol, a proprietary RNA
extraction method, as a surface wash for deli meats, including samples artifi-
cially contaminated with norovirus and ones implicated in an outbreak of
norovirus gastroenteritis. Although simple, the main drawback of the TRIzol
surface wash method was that nucleic acid amplification inhibition persisted
unless sample concentrates were diluted 10- to 100-fold. A flow diagram of this
protocol is depicted in Figure 6.3.

Bidawid et al. (12) reported an immunocapture method for the concentration
of hepatitis A virus from lettuce and strawberries. After surface washing to elute
the viruses, the wash solution was passed through a positively charged filter,
eluted, and concentrated by immunocapture. As few as 10 PFU of cell culture-
adapted hepatitis A virus per piece of lettuce or strawberry could be detected by
RT-PCR using this sample preparation method. However, this method too had
problems with residual matrix-associated amplification inhibition.

50 g FOOD SAMPLE

(Complex food such as hamburger)
Add 350 ml of 0.05 M Glycine/0.14 N Saline, pH 9.0

HOMOGENIZE (350-400 ml)

FILTER

Cheesecloth

SOLVENT EXTRACT

(Vortex gently with 60% Freon or Chloroform: Isobutanol (1:1) as needed, ~70 ml)

Centrifuge at 2,500 x g for 10 min.

1° PEG PRECIPITATION and ELUTION

To eluant add 6% PEG, pH to 7.2, incubate 4°C, 2 H.

Centrifuge 5000 xg for 15 min.
Resuspend pellet in 25 ml of 50mM Tris, 0.2% Tween 20, pH 9.0

Elute at room temperature for 1 H.
Centrifuge at 3500 x g for 15 min.

(30-35 ml)

2°PEG PRECIPITATION and RESUSPENSION

To eluant add 12% PEG; incubate 4°C, 2 H.

Centrifuge at 5000 x g for 15 min.

Resuspend pellet in 50mM Tris, 0.2% Tween 20, pH 8.0

(3-5 ml)

I I

RNA EXTRACTION and RT-PCR OR CELL-CULTURE ASSAY

(25-40 ul)

Figure 6.2. Virus concentration from hamburger/complex foods using glycine-saline
buffer, chloroform extraction and two PEG extractions as followed by Leggitt and
Jaykus (66)

98 Molecular Approaches for the Detection of Foodborne Viral Pathogens

25-50 g PRODUCE SAMPLE

TRIZOL® SURFACE WASH OR BUFFERED SURFACE WASH

(4 mis of TRIzol® Reagent, twice) 0.05 M Glycine-0.14M Saline buffer, pH 9.0

(4 ml of buffer, twice)

ELUTE AND CLARIFY SUPERNATANT

Centrifuge 8ml at 5000 rpm for 15 mins at 4°C

EXTRACT RNA

Add 1.6 ml of Chloroform to 8 ml of TRIzol/Glycine extract
Shake and incubate at room temperature for 2-3 min.

t
Centrifuge 9000 rpm for 15 min. at 4°C

Collect aqueous phase (~ 4.8 ml)

t
Add 4 ml of isopropanol and incubate at room temperature for 10 min.

t
Centrifuge at 9000 rpm for 10 min. at 4°C

Wash pellet with 8ml of 75% ethanol

Centrifuge 7000 rpm for 5 min. at 4°C

Air-dry for 5 min.
Dissolve pellet in 100 \il of sterile water and store at -70°C to -80°C

Figure 6.3. Virus concentration and extraction from produce using TRIzol® or
glycine-saline buffer (modifications of protocol by Schwab et al. (91)).

As a general rule, virus concentration methods result in sample volume
reductions ranging from 10- to 1000-fold. This means that a 25-g sample theo-
retically can be reduced to 25 |il-2.5 |Lil volumes with recovery of infectious
virus (50). The yields after virus concentration in a food matrix have ranged
from as low as 1-2% to as high as 90%. Recovery efficiency is almost always
virus-specific, usually hepatitis A virus recovery is quite low when compared to
recovery of other viruses such as poliovirus (reviewed in 50, 88).

Although virus concentration and sample purification steps prior to nucleic
acid amplification can achieve significant sample volume reduction with rela-
tively efficient virus recovery, there are some additional considerations when
selecting a virus concentration approach. The antibody capture methods are
often simpler than others as they may require fewer sample manipulations.
There is also speculation that antigen-associated viral nucleic acid is more
highly associated with infectious virus. However, these methods may be limited
by reagent availability and high specificity, which means that only a single virus
type is detected in a single assay. Other concentration and purification methods
that rely on steps such as PEG precipitation and solvent extraction usually
require manipulations, which may result in substantial virus loss during extrac-
tion. The direct nucleic acid extraction methods almost always result in residual
RT-PCR inhibitors and often times do not provide adequate sample volume
reduction.

Nucleic Acid Extraction 99

In short, there are many virus extraction and concentration approaches that
have been applied in a variety of instances. All of these methods have limita-
tions that ultimately hinder the ability to detect the relatively low levels of virus
that might be anticipated in naturally contaminated foods.

NUCLEIC ACID EXTRACTION

The earliest and simplest protocol for amplifying nucleic acids from shellfish
concentrates was direct heat release followed by RT-PCR, in which case, an
RNA extraction step was not applied (31, 51, 52). It soon became apparent,
however, that in many instances the additional volume reductions and sample
clean up provided by RNA extraction was critical to improving detection lim-
its and circumventing the effects of residual matrix-associated amplification
inhibition.

Before applying nucleic acid amplification, an efficient nucleic acid extraction
step is critical in most instances. This is important because the amplification effi-
ciency is dependent on both the purity of the target template and the quantity
of target molecules obtained from the sample. Accordingly, the main goals of
nucleic acid extraction are: (1) to extract and purify the nucleic acids, (2) to
provide additional sample concentration, and (3) to remove residual matrix-
associated inhibitory substances that could remain after the initial concen-
tration steps are completed. The components that can hinder molecular
amplification are diverse and include compounds such as divalent cations,
matrix-associated components such as proteoglycans (42), polysaccharides, (6, 7,
27), glycogen, (6, 7, 51, 52), and lipids (84, 87, 91), among others. In many cases,
inhibitory compounds have been largely uncharacterized. Matrix-associated
inhibitors usually act by degrading the target and/or primer nucleic acids, and/or
inactivating or inhibiting enzymes (86, 109, 111). Unfortunately, these inhibitory
substances are frequently coextracted during virus concentration protocols,
adding to the challenge of identifying reliable nucleic acid extraction processes.

Early studies used SDS-proteinase K digestion to release nucleic acids from
shellfish concentrates, followed by phenol chloroform extraction with, or with-
out the addition of cetyltrimethylammonium bromide (CTAB) to remove resi-
dual inhibitors (6, 7, 16, 90). In the last decade or so, guanidinium thiocyanate
(GuSCN)-based methods became the RNA extraction method of choice largely
because they are effective at deproteinization of nucleic acids while providing
ample protection of RNA against native RNases. Many commercial guani-
dinium based kits have been used in more recent studies (4, 12, 22, 24, 25, 32,
59, 60, 75, 87, 90, 91).

Combinations of multiple extraction methods can also be used to purify
nucleic acids. A simple and rapid protocol for the purification of nucleic acid
that utilizes a combination of the chaotropic agent GuSCN and silica particles
was first described by Boom et al. (13), and later used by others (41, 53). Other
methods include a GuSCN method followed by RNA binding to glass powder,
instead of silica, to provide further nucleic acid purification (63, 64).

100 Molecular Approaches for the Detection of Foodborne Viral Pathogens

Several investigators have compared various RNA extraction approaches
specifically aimed at preparing samples for the detection of human enteric
viruses. Hale et al. (44) compared four different RNA extraction methods for
RT-PCR detection of noroviruses in fecal specimens, finding that the
GuSCN/silica (13) method was the best at removal of inhibitors. Likewise,
another study compared seven RNA extraction methods to purify hepatitis
A virus RNA from stool and shellfish concentrates for RT-PCR detection (4);
again, the GuSCN-silica methods were found to be the most suitable from the
standpoint of speed, ease, and cost. Gouvea et al. (36) used deproteinization
with GuSCN followed by adsorption of RNA to hydroxyapatite and sequential
precipitation with CTAB and ethanol to purify RNA from shellfish and other
selected foods. Sair et al. (87) compared multiple RNA extraction methods after
concentration of noroviruses from model food commodities (hamburger sand-
wiches and lettuce). These included GuSCN, commercial microspin columns,
the QIAshredder Homogenizer and TRIzol alone and in their various combi-
nations. These investigators found that the use of TRIzol followed by further
sample preparation using the QIAshredder Homogenizer yielded the best detec-
tion limits (< 1 RT-PCR amplifiable units/reaction) for Norwalk virus precon-
centrated from food samples. Similar studies by Svensson (101) demonstrated
that the use of the metal chelating agent Chelex-100, or alternatively, Sephadex
G200 column chromatography, during RNA extraction, provided the best RT-
PCR detection limits. Others have had success using phenol-chloroform-based
methods followed by further selection for viral RNA using magnetic poly (dT)
beads (30, 35, 59, 60).

Despite all the efforts in identification of efficacious RNA extraction proto-
cols, food-related amplification inhibitors frequently remain. Multiple sample
manipulation steps can result in incomplete recovery and/or degradation of
RNA during the extraction procedure, the consequence of which is less than
optimal RNA yields (49, 50). A major problem with RNA extraction is the
necessity to destroy virion integrity, thereby losing the ability to directly corre-
late infectivity to RT-PCR detection limits, at least when effective cell culture-
based methods are available. The two main areas of active research in RNA
purification are increasing yield and improving the purity of the resultant prod-
uct for detection by PCR.

DETECTION

The progress in clinical detection of pathogens has always been ahead of detec-
tion in foods and many of our food methods rely on protocols initially devel-
oped for clinical samples. However, whether clinical, food, or environmental
sample, the sensitivity and the specificity of molecular amplification methods is
largely dependent on the choice of primers.

The genetic diversity in the Calicivirideae family makes primer design for the
detection of the noroviruses quite challenging. Initial studies used extremely spe-
cific primers such as NV 573' and NV 36/35, which were based on sequences in

RT-PCR Detection of Viruses in Foods 101

the prototype Norwalk virus genome (23, 74). With the availability of sequence
information from related viruses, more broadly reactive primers have been
designed. (2, 38, 57, 108). Most of the primer sequences reported are based on
the highly conserved RNA-dependent RNA polymerase region of the
noroviruses; occasionally the capsid region has been targeted (39, 40, 62, 81, 105).

Initially, the primer sets developed by Ando et al. (2) for the genogroup I
(GI) and genogroup II (Gil) Noroviruses were used as the "gold-standard" for
the detection of noroviruses in clinical (fecal) and food samples (47, 80, 112).
Later on, degenerate primers, or a mixture of oligonucleotide primers that vary
in nucleotide sequence but have the same number of nucleotides, were devel-
oped and used for the detection of the noroviruses (38, 68, 69). The use of
degenerate primers is advantageous in that all combinations of nucleic acid
sequence that code for the amino acid are used in the PCR amplification.
Combinations of the Ando et al. (2) GI and Gil primers and various degener-
ate sets are routinely used in norovirus epidemiological investigations as applied
to the detection of virus in fecal samples.

As with RNA extraction methods, investigators have compared the per-
formance of various primers for the detection of a broad range of noroviruses,
largely in fecal specimens. For instance, the NV110/NV36 primer set was the
found to be the most efficient of the nine primer sets tested in a comprehensive
study, even though it could not detect 100% of the norovirus strains tested (46).
When five laboratories in five countries evaluated different RT-PCR methods
on a panel of 91 fecal samples (106), no single assay was superior based on the
criteria of sensitivity, detection limit, assay format, and successful implementa-
tion. However, the Boom extraction method and the use of the JV12/JV13
primer set were recommended for norovirus diagnostics.

Nonetheless, there is some lack of consensus regarding the optimal primer
pair(s) to detect the noroviruses and different laboratories tend to use various
methods that were developed, or are optimally suited for their purposes (112).
Primers used for the detection of hepatitis A virus usually target the VP1/2A
junction sequence. In this case, the issue of diversity is not relevant as it is for
the noroviruses (48).

RT-PCR DETECTION OF VIRUSES IN FOODS

The choice of primers is even more critical when attempting to detect viral con-
tamination in foods. This is because the levels of contamination are typically
much lower in foods when compared to clinical samples, and even with optimal
concentration and nucleic acid extraction methods, residual inhibitors often
persist. Furthermore, the matrix can be responsible for nonspecific amplifica-
tion and false positive results. The primers selected for the detection of viral
nucleic acids derived from the food matrix should therefore have the following
criteria: (1) a reasonably high annealing temperature, (2) relative nondege-
neracy, and (3) broad reactivity. High stringency and primer specificity (hence
the relative absence of degeneracy) are necessary to prevent nonspecific

102 Molecular Approaches for the Detection of Foodborne Viral Pathogens

amplification. For the genetically diverse norovirus group, the use of primers
that are broadly reactive and can detect as many genetically distinct strains as
possible in a single assay is essential.

The various primers used in the RT-PCR detection of noroviruses from dif-
ferent food matrices are summarized in Table 6.1. As with primers used in the
clinical realm, these sequences correspond to mainly the viral RNA dependent
RNA polymerase or the capsid protein genome regions. Primer sets targeting
both the RNA polymerase and capsid genes, Mon38 1/383 and SR33/46, respec-
tively, were used by Shieh et al. (96, 97) to identify a Gil norovirus in oyster
samples implicated in a California outbreak. For the detection of the Gil
noroviruses in shellfish, the NI/E3 primer set has also been used (33, 37, 63).
Dubois et al. (32) used a newer primer pair to detect both noroviruses and
sapoviruses in artificially contaminated produce. In a systematic comparison of
four primer pairs, as applied to the detection of noroviruses in hamburger sand-
wiches and lettuce, Sair et al. (87) found the best detection limits using the
NVpl 10/NVp36 primer combination. Honma et al. (46) reported that this same
primer pair was broadly reactive for a range of noroviruses, eliminating the
need for separate amplifications for the two norovirus genogroups (GI and
Gil). These primers have also been used together or in combination with NI or
NVp69 for the detection of norovirus contamination in shellfish (59, 67, 69).

"Nested" RT-PCR or double amplification has been used for the detection
of noroviruses (36, 63, 91, 99) and hepatitis A virus (18, 19, 20, 43, 59) in foods.
This approach can improve assay sensitivity and also provide another method
for the confirmation of amplified product. However, a major disadvantage is
that these nested reactions are prone to carryover contamination. Novel single-
tube nested RT-PCR methods may help circumvent these issues. Ratcliff et al.
(81) pooled the reagents required for the nested amplification in a "hanging
drop" that could be introduced by centrifugation after the first RT-PCR ampli-
fication while Burkhardt et al. (14) compartmentalized the nested RT-PCR
cocktail in a "tube-within-a-tube" device by using inexpensive materials such as
a pipette tip and a microcentrifuge tube. Primers for the detection of hepatitis
A virus by RT-PCR are summarized in Table 6.2.

ALTERNATIVE NUCLEIC ACID AMPLIFICATION
METHODS

Nucleic acid sequence-based amplification (NASBA) is an amplification method
that specifically detects RNA to the exclusion of DNA. The transcription-driven
NASBA reaction is carried out at a single temperature (41 °C) and theoretically
amplifies the RNA target more than 10 12 -fold within 90 min (34, 53). The
final product of the amplification is single-stranded RNA, which can be readily
detected by hybridization. The system utilizes three enzymes, (1) a reverse
transcriptase (AMV-RT), (2) an RNase H, and (3) a T7 RNA polymerase, all of
which act in a stepwise (sequential) manner with two oligonucleotide primers
specific to the target (34, 53). One of the primers (PI) contains the T7 RNA

Alternative Nucleic Acid Amplification Methods

103

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Molecular Approaches for the Detection of Foodborne Viral Pathogens

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Confirmation 107

polymerase promoter sequence at 5' terminal and the other primer (P2) can be
designed with a generic sequence that facilitates probe capture for amplicon
detection by liquid hybridization (described in the Confirmation section
below).

NASBA assays have been developed for the detection of foodborne enteric
viruses such as hepatitis A virus, rotavirus and noroviruses. For instance,
Greene et al. (41) applied the NucliSens Basic Kit NASBA protocol for the
detection of norovirus RNA in stool using primers targeting the RNA poly-
merase region of the viral genome. Jean et al. (54, 55, 56) developed a NASBA-
based method to detect hepatitis A virus on artificially contaminated lettuce
and blueberry samples and also for the detection of human rotavirus. More
recently, Jean et al. (53) developed a multiplex NASBA method for the simulta-
neous detection of hepatitis A and noroviruses (GI and Gil) from lettuce and
sliced turkey (deli meats).

The NASBA method is isothermal, as rapid (if not faster than) as RT-PCR,
and demonstrates detection limits equal to if not better than RT-PCR (41, 53, 56).
However, NASBA technology has many of the same limitations as RT-PCR (e.g.,
contamination control, sample volume considerations, matrix-associated reaction
inhibitors). Nonetheless, it remains an important alternative method for the
detection of foodborne viruses.

CONFIRMATION

Since nonspecific products of amplification are a major issue when food and
environmental samples are tested, it is critical to confirm that the nucleic acid
amplification products obtained are specific to the target. Most often, the con-
firmation step also improves the sensitivity of the assay. The most common
confirmatory tool is Southern hybridization using specific oligoprobes internal
to the amplicon. These probes are usually enzyme labeled for colorimetric,
luminescent or fluorescent endpoints. When RNA products (for NASBA) are
tested, Northern hybridization with labeled internal oligonucleotide probes may
be used (45, 46). An oligonucleotide array dot-blot format for the simultaneous
confirmation of norovirus amplicons and strain genotyping has recently been
reported, offering the promise of providing both detection and strain typing in
a single test (107).

DNA enzyme immunoassay (DEIA) methods provide an alternative to
Southern hybridization. In these assays, a capture probe is immobilized to a
microtiter plate well and a labeled amplicon can then be detected directly, or
alternatively, an unlabeled amplicon can be hybridized to a second labeled detec-
tor probe followed by detection after the addition of an enzyme-conjugate and
appropriate substrate (sandwich assay). For colorimeteric, luminescent, or fluo-
rescent endpoints, absorbance is read using a conventional microtiter plate spec-
trophotometer or fluorescent plate reader. The intensity of the signal obtained
may be approximately proportional to the concentration of amplicon. The sensi-
tivity of the microtiter plate assay is generally equal to or better than Southern

108 Molecular Approaches for the Detection of Foodborne Viral Pathogens

hybridization and this approach has advantages including ease of interpretation,
rapid (4 h) amplicon confirmation, and the potential for automation (54, 55, 90).

A liquid electrochemiluminescence (ECL) hybridization technology has
been used commercially for the detection of NASBA amplicons. This technol-
ogy utilizes two specific oligoprobes; a capture probe complementary to the
sequence on primer P2, immobilized to streptavidin-labeled magnetic beads and
a detector probe complexed to a ruthenium chelate. The hybridized magnetic
particles are trapped on an electrode, and application of a voltage trigger to the
electrode induces the ECL reaction such that the amount of emitted light is
directly proportional to the amount of the amplicon. The signals are reported
as ECL units by the NucliSens Reader and associated software. The NASBA-
ECL system typically generates confirmed detection results in a day and has
been used by Fox et al. (34) for the detection and confirmation of enterovirus
from clinical samples and by Greene et al. (41) and Jean et al. (53) for the detec-
tion Norwalk virus in stool and food samples.

Other confirmation methods include specific "nested" PCR reactions (36,
43, 63, 99), which use a second pair of primers internal to the first amplicon
sequence; and restriction endonuclease digestion of RT-PCR products (36, 43).
Direct sequencing of the amplicon for the confirmation of RT-PCR products is
another method of choice, and is frequently applied in the clinical realm, and
more recently when amplicons are obtained from foods implicated in outbreaks
(69, 91).

REAL-TIME DETECTION

Real-time detection refers to the simultaneous detection and confirmation of
amplicon identity as the amplification reaction is progressing, thereby linking
nucleic acid amplification with hybridization. There are currently five main
chemistries used for real-time amplification and detection. One of the earliest
and simplest approaches to real-time PCR, called DNA binding fluorophores,
uses ethidium bromide or SYBR green I compounds that fluoresce when asso-
ciated with double stranded DNA and exposed to a suitable wavelength of
light. These methods tend to lack specificity but this has been addressed
recently by coupling the assay with melting curve analysis. The 5' endonucle-
ase assay (e.g., TaqMan oligoprobes, Applied Biosystems, Foster City, CA),
adjacent linear probes (e.g., HybProbes, Roche Molecular Biochemicals,
Germany) and hairpin oligoprobes (e.g. molecular beacons, Molecular Probes,
Eugene, Oregon) have received considerable attention of late. Self-fluorescing
amplicons (e.g., Sunrise primers, Amplifluor hairpin primers, Intergen Co.,
Purchase, NY) incorporated into the PCR product as the priming continues
have a 3' end complimentary to the target strand and Scorpion primers have 5'
end complementary to the target strand) (reviewed in 72). Recently, investiga-
tors have developed real-time PCR systems for the detection of a wide array of
bacterial pathogens in foods. Prototype real-time RT-PCR amplification tech-
nologies have been developed for the detection of hepatitis A virus (17) and

Acknowledgments 109

noroviruses (79) using the TaqMan format, and the norovirus detection uses
the SYBR Green melting curve format (73). Beuret et al. (10) have used multi-
plex real-time PCR for the simultaneous detection of a panel of enteric viruses.
Research efforts are currently underway to apply these methods to the detec-
tion of viruses in food matrices. Indeed, Myrmel et al. (78) recently reported
the detection of viral contamination in shellfish using a commercial
SYBRGreen PCR kit, while Narayanan et al (79) used their TaqMan assay to
detect noroviruses in shellfish.

CONCLUSIONS

It should be clear from the preceding discussion that the current methodology
for the detection of enteric viral contamination in foods is less than ideal and
that research is necessary to improve these methods. Indeed, these protocols are
applied infrequently and usually only in response to known or suspected food-
borne disease outbreaks. The most important reasons for their limited use
include: (1) the inability of molecular amplification methods to discriminate
between infectious and inactivated virus, (2) the lack of widely accepted, col-
laboratively tested methods, (3) the requirement that most methods be product
specific, meaning that universal approaches do not exist, and (4) the cost and
need for highly trained personnel (83). When taken together, detection limits
ranging from approximately 1-100 infectious units/g food have been obtained
using various RT-PCR methods. The use of internal amplification standards to
simultaneously evaluate RT-PCR inhibition and/or to provide a semiquantita-
tive assay is also frequently done (5, 6, 67, 69, 89, 93, 103).

The failure to discriminate between infectious and inactivated virus is of
critical importance because the inactivated forms of these pathogens pose no
real public health threat. There is also a need to develop more universal sam-
ple extraction methods. For the most part, virus concentration from foods is
likely to remain product dependent but research is needed to develop and
refine the prototype methods into collaboratively tested protocols.
Researchers continue to seek efficacious methods to concentrate the
pathogens from the food matrix with the simultaneous removal of matrix-
associated inhibitors. Even so, the methods will probably never be perfect and
will always require a high degree of sample manipulation by the laboratory
personnel (83). There is hope, however, that over time these rapid methods to
detect human enteric viruses in foods may become more widely available
to the food safety community.

ACKNOWLEDGMENTS

This effort was supported in part by a grant from the USDA National Research
Initiative, Competitive Grants Program: Ensuring Food Safety, 2002-35201-
11610. The use of trade names in this chapter does not imply endorsement by

110 Molecular Approaches for the Detection of Foodborne Viral Pathogens

the North Carolina Agricultural Research Service nor criticism of similar ones
not mentioned.

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108. Wang, X, X. Jiang, H.P. Madore, J. Gray, U. Desselberger, T. Ando, Y Seto,
I. Oishi, IF. Lew, K.Y Green, and M.K. Estes. 1994. Sequence diversity of small,
round-structured viruses in Norwalk virus group. J. Virol. 68:5982-5990.

109. Wiedbrauk, D.L. and A.M. Devron. 1995. Nucleic acid detection methods. In
D.L. Wiedbrauk and D.H. Farkas (eds.), Molecular Methods for Virus Detection,
pp. 1-24. Academic Press Inc., San Diego, CA.

References 117

110. Wilde, X, J. Eiden, and R. Yolken. 1990. Removal of inhibitory substances from
human fecal specimens for detection of group A rotaviruses by reverse transcrip-
tase and polymerase chain reactions. J. Clin. Microbiol. 28:1300-1307.

111. Wilson, J. G. 1997. Inhibition and facilitation of nucleic acid amplification. Appl.
Environ. Microbiol. 63:3741-3751.

112. Wolfaardt, M., M.B. Taylor, H.F. Booysen, L. Engelbrecht, WO. Grabow, and
X. Jiang. 1997. Incidence of human calicivirus and rotavirus infection in patients
with gastroenteritis in South Africa. J. Med. Virol. 51:290-296.

113. Yan, H., F. Yagyu, S. Okitsu, O. Nishio, and H. Ushijima. 2003. Detection of
norovirus (GI, Gil), sapovirus and astrovirus in fecal samples using reverse tran-
scription single-round multiplex PCR. J. Virol. Methods 114:37^44.

CHAPTER 7

Molecular Tools for the Identification

of Foodborne Parasites

Ynes Ortega*, Ph.D.

Center for Food Safety, College of Agriculture and Environmental Sciences, The University

of Georgia, Griffin, GA 30223

Introduction

DNA Extraction Procedures
Protozoal Infections
Cryptosporidium parvum

Parasite Description and Identification

Molecular Detection
Cyclospora cayetanensis

Parasite Description and Identification

Molecular Detection
Giardia intestinalis

Parasite Description and Identification

Molecular Detection
Toxoplasma gondii

Parasite Description and Identification

Molecular Detection
Microsporidia

Parasite Description and Identification

Molecular Detection

Helminth Infections

Viability Assays

Conclusions

References

INTRODUCTION

Parasites have long been associated with food and waterborne outbreaks.
Although parasites have been consistently reported in developing and endemic
countries, the number of parasites present in the food supply of Americans has
multiplied by more than a factor of 8 during the past 1 5 years. This is partly due
to the increase of international travel and population migration. Rapid and
refrigerated food transportation from foreign countries facilitates consumer
contact with emerging parasites. Cultural habits have also changed towards the

Corresponding author. Phone: (770) 233-5587; e-mail: ortega@uga.edu

119

120 Molecular Tools for the Identification of Foodborne Parasites

consumption of (raw or undercooked) fresh produce. These conditions have
increased the probability that parasites infect naive populations and cause gas-
trointestinal illness (91).

Most parasites are obligate intracellular organisms. In contrast with bac-
teria, parasites are inert and do not multiply in the environment. Any isola-
tion and detection procedures are crucial because an enrichment process for
parasites is not available. Molecular assays overcome these difficulties and
specific limitations per organism will be discussed.

Based on their morphological attributes, parasites are classified into two
groups: protozoa and helminths. The protozoa are single-celled organisms and
the helminths are metazoans with a rudimentary digestive and reproductive
tract. The helminths are grouped as the nematode or roundworms, the cestoda
or tapeworm, and the trematoda or flukes. The primary objectives in develop-
ing diagnostic molecular assays have been focused on protozoa because of the
limitations of conventional parasitological methods to identify them in foods
and the environment.

DNA EXTRACTION PROCEDURES

Many protocols have been described for the isolation of parasite DNA. Some
are protocols prepared at individual laboratories. The trend is now to use
DNA extraction kits, as this will reduce the possible variables among proce-
dures and laboratories. The recovery efficiency of the extraction procedures,
particularly with parasites, is important because of the potentially low num-
ber of parasites in environmental samples and food matrices. The sensitivity
of the PCR or any molecular assay is dependent on the DNA extraction
methodology. Enzyme digestion of the protozoal oocysts has been done using
proteinase K in lysis buffer (10 mg proteinase K/ml, 120 mM NaCl 2 , 10 mM
Tris and 0.1% SDS) followed by phenol chloroform-isoamyl alcohol (25:24:1)
separation, and DNA precipitation using salts such as 0.3 M sodium acetate
with 10 |ig glycogen, a DNA carrier, and 2 volumes of 100% ethanol. This
method extracts DNA efficiently from parasites, but the most important step
in this process is to break open the oocysts to release the protozoal DNA. Not
all protozoal cysts or oocysts will be effectively digested; therefore other
means of oocyst rupture have been described. One of the commonly used
methods for cyst/oocyst breakage is the freeze/thaw method with cycles of
freeze/thaw that vary from 3 to 12 cycles. The freezing is done in dry
ice/ethanol slurry and thawed at 55 °C. This process may change accordingly
to the parasite and the laboratory (114). Oocysts can also be disrupted using
a bath sonicator (101), keeping under consideration the possible denaturation
of the DNA. Based on forensic studies, an extraction free, filter based prepa-
ration of DNA has been used and can successfully rupture the parasite cysts
or oocysts. This method uses an FTA filter, which is impregnated with denat-
urants, chelating agents, and free radical traps. The sample is placed on the
filter and cut with a hole puncher. The membrane is then rinsed and used

Cryptosporidium Parvum 121

directly for PCR amplification (92). The use of this methodology needs to be
evaluated for the processing and evaluation of large sample sizes and the
potential for cross-contamination.

PROTOZOAL INFECTIONS

Protozoan parasites relevant to public health and associated with foodborne
infections include the ciliates (i.e., Balantidium coli), amoeba (i.e., Entamoeba his-
tolytica), flagellates (i.e., Giardia lamblia), and coccidia (i.e., Toxoplasma gondii,
Cryptosporidium parvum and Cyclospora cayetanensis). Taxonomical placement
of some protozoan pathogens has been changing as we learn more about the
genetic makeup of these organisms. Such is the case with the Microsporidia,
which are now considered to be more closely related to fungi than to the proto-
zoa. Cryptosporidium, originally from the phylum Apicomplexa, has more char-
acteristics associated to the gregarines than to coccidia. Its taxonomical
classification is uncertain and as more evidence is published, these parasites may
be reclassified to more appropriate groups.

CRYPTOSPORIDIUM PARVUM

Parasite Description and Identification. Cryptosporidium sp. was first recognized
by Tyzzer in 1907, from the stomach of a mouse (78). It is currently identified
as C muris. Subsequently, other species of Cryptosporidium were described and
renamed. Cryptosporidium species that were morphologically similar were
named Cryptosporidium parvum, but recent molecular analysis have lead to
reclassification into several species: C hominis (C. parvum genotype 1 or the
human genotype) in humans, C andersoni (C muris-like or C muris bovine
genotype) and C bovis {Cryptosporidium bovine genotype B) in calves and adult
cattle, C canis, (C parvum dog genotype) in dogs, and C suis {Cryptosporidium
pig genotype I) in pigs. To date, there are 15 established Cryptosporidium species
in fish, reptiles, birds, and mammals and 8 have been reported in humans
(C hominis, C bovis, C canis, C felis, C meleagridis, C muris, C suis, and
Cryptosporidium cervine genotype). Cryptosporidium has been associated with
gastrointestinal illness in humans and it is acquired by ingestion of fecally
contaminated water or foods. It can also be acquired via person to person.

The life cycle of Cryptosporidium starts when mature and infectious oocysts
are excreted in the feces of an infected host. The oocysts are ingested along with
contaminated water and/or foods and excyst in the gastrointestinal tract.
Sporozoites are released and infect the epithelial cells of the small intestine, par-
ticularly the ileum. Parasites multiply asexually, producing type I and II
meronts containing 8 and 4 merozoites, respectively. Asexual multiplication can
continue or differentiate to produce the sexual stages of the parasites.
Microgametocytes (male) fertilize the macrogametocyte (female) producing the
zygote, which in turn becomes the oocyst. If a thin-walled oocyst is formed, the

122 Molecular Tools for the Identification of Foodborne Parasites

life cycle can initiate again. If a thick-walled oocyst (which is environmentally
resistant) is formed, it is excreted in the feces (78).

Individuals at risk of acquiring cryptosporidiosis include children in daycare
centers, individuals caring for animals, the elderly, immunocompromised indi-
viduals, and travelers. Since Cryptosporidium is highly resistant to common dis-
infectants, including chlorine, water parks and fountains have been implicated
in numerous outbreaks.

A large waterborne outbreak occurred in 1993 in Milwaukee (76, 146),
where more than 400,000 people suffered gastrointestinal illness. Initially,
C parvum was considered to be the agent responsible for this outbreak, how-
ever, molecular analysis of clinical specimens and water samples demonstrated
that it was actually C hominis. Contamination occurred when tap water was
contaminated with sewer water back-flow (76, 146).

Two main target antigens of 15-17 and 23 kDa molecular weights have been
used for detection of the humoral immune response of individuals with cryp-
tosporidiosis (102). These antigens have been used in ELISA, western blot, or
multiplex bead assay for the detection of Cryptosporidium antibodies in sera
and oral fluids (85, 99, 148).

Stool specimens are the most common clinical samples examined for diag-
nosis of Cryptosporidium. Oocysts are usually concentrated using ethyl acetate
concentration methodologies (139). The sample is then examined using
microscopy, immunoassays, and molecular techniques. The modified Ziehl-
Neelsen acid-fast stain and modified Kinyoun's acid-fast stain are more com-
monly used in the microscopic identification of Cryptosporidium (87, 103).

Immunofluorescence assays (IFA) are more sensitive and specific than the
modified acid fast stains and are now being used more frequently in clinical lab-
oratories. They are the gold standard when examining new diagnostic assays
(9, 40), but cannot differentiate among the Cryptosporidium species (46). Some of
these commercial kits include the Merifluor Cryptosporidium! Giardia kit
(Meridian Bioscience; Cincinatti, OH), Giardia/ Crypto IF kit (TechLab;
Blacksburg, VA), Monofluo Cryptosporidium kit (Sanofi Diagnostics Pasteur),
Crypto/ 'Giardia Cel kit (TCS Biosciences; Buckingham, UK), and Aqua-Glo G/C
kit (Waterborne; New Orleans, LA). Commercial antigen-capture-based enzyme
immunoassays (EIA) available are the Alexon-Trend ProSpecT Cryptosporidium
Microplate Assay (Alexon-Trend- Seradyn; Ramsey, MN) and Meridian Premier
Cryptosporidium kit. These assays may not react to Cryptosporidium species that
are genetically distant from C parvum, such as C muris, C andersoni, C serpen-
tis and C bailey i (46). Lateral flow immunochromatographic assays have also
been commercialized for use with stool samples (60)

Parasites can be recovered from foods by washing the samples in 0.025M
phosphate buffered saline, pH 7.25 (95). Detergents (1% sodium dodecyl sulfate
and 0.1% Tween 80, or the membrane filter elution buffer from EPA method
1623) and sonication (3-10 min) are also used to facilitate the elution of para-
sites from the food matrices (13, 105). Cryptosporidium can then be concen-
trated by centrifugation and examined by immunofluorescence staining
(13, 95). A sucrose flotation step may be included producing a cleaner sample

Cryptosporidium Parvum 123

but at the cost of losing parasites. Moderate recovery rates of 18.2-25.2% were
reported for a variety of fresh produce. Immunomagnetic separation has been
included in the Cryptosporidium recovery procedures from lettuce, Chinese
leaves, and strawberries to 42% for Cryptosporidium and 67% for Giardia
(105, 106). Cryptosporidium oocysts can be detected by IFA in shellfish gills,
gastric glands, and hemocytes from the hemolymph (32, 49).

Identification of Cryptosporidium oocysts in water samples is achieved
by using IFA after concentration processes (EPA ICR method, EPA
methodl622/1623, United Kingdom SCA method, and United Kingdom regu-
latory method) (72). Oocysts are recovered by filtration of 10-100 L or more
water, concentrated and stained with FITC-labeled Cryptosporidium antibodies.
The ICR or SCA methods use nominal 1 |im 10" cartridge filters and washes
concentrated by flotation (using Percoll, sucrose, or potassium citrate). The
EPA method 1622 and the United Kingdom regulatory method use capsule fil-
ters for filtration followed by immunomagnetic separation (IMS). To determine
oocyst viability, 4', 6-diamidoino-2-phenylindole (DAPI) vital dye is used. The
recovery rates of the EPA method 1622/1623 for Cryptosporidium oocysts are
between 10 and 75% for surface water (67, 115, 137). One of the limitations of
these procedures is the cross-reactivity of the monoclonal antibodies used in the
IMS and IFA kits. Dinoflagellates (120) and algae (111) may not provide accu-
rate detection and quantification of Cryptosporidium oocysts, and may require
confirmation by differential interference contrast microscopy.

Surface water samples may contain Cryptosporidium oocysts, which are a non-
pathogenic species or genotypes for humans. Therefore, identification of oocysts
to the species/genotype level is of significant public health importance. The PCR
has become a useful tool for determining the genotype Cryptosporidium oocysts
found in shellfish (32, 44, 45).

Analysis of environmental samples presents several limitations to these
assays. The number of parasites in foods is usually small. Other structures mor-
phologically similar may nonspecifically react with antibody-based assays for
Cryptosporidium and make them less reliable. In addition, the presence of
Cryptosporidium oocysts, which are not infectious to humans, may be present
and could be confused with those of public health relevance. When examining
environmental samples (soil, water, or foods), density gradients or antibody-
based concentration procedures have been described (i.e., IMS concentration).
Concentrates are then used for direct-fluorescent antibody (DFA) or for PCR
detection.

Molecular Detection. One of the advantages of using molecular assays on envi-
ronmental samples is the capability of identifying low numbers of parasites and
being able to determine their species and genotype. This allows for parasite fin-
gerprinting in waterborne and foodborne outbreaks, particularly when deter-
mining if the parasite is anthroponotic or zoonotic. It also aids in determining
the risk factors associated with transmission of cryptosporidiosis in a particu-
lar setting (4, 5, 43, 66). Subtyping tools have been useful in the investigation of
foodborne and waterborne outbreaks of cryptosporidiosis (43, 71, 124).

124 Molecular Tools for the Identification of Foodborne Parasites

Earlier PCR methods (22, 65, 140) have been used only for the identification
of Cryptosporidium spp. Several PCR-Restriction Fragment Length
Polymorphism (RFLP)-based genotyping tools have been developed for the
detection and differentiation of Cryptosporidium at the species level (6, 62, 70,
74, 86, 120, 146). Most of these techniques are based on the SSU rRNA gene.
However, some of the SSU rRNA-based techniques (62, 70) used conserved
sequences of eukaryotic organisms, which amplify DNA from organisms other
than Cryptosporidium (127). Nucleotide sequencing-based approaches have also
been developed for the differentiation of various Cryptosporidium spp. (82, 84,
125, 126, 136). These techniques use long PCR amplicons, and some amplify
other Apicomplexan parasites and dinoflagellates. Because of this lack of speci-
ficity and sensitivity they cannot be used for diagnostic purposes (125, 126, 136).

Other genotyping techniques are used specifically to differentiate C parvum
from C hominis (15, 83, 97, 118, 141). Out of ten commonly used genotyping
tools for Cryptosporidium species/genotypes, only the SSU rRNA-based PCR
tools can detect all seven Cryptosporidium species/genotypes (57).

Microsatellite analysis has been used to characterize diversity between
C parvum or C hominis and their subtypes (19, 20, 35, 142). High-sequence
polymorphism in the gene of 60 kDa glycoprotein precursor has also been used
for subtype analysis (98, 124). Other subtyping tools include sequence analysis
of HSP70 (98, 124), heteroduplex analysis and nucleotide sequencing of the
double-stranded RNA (71, 146), and single-strand conformation polymor-
phism (SSCP)-based analysis of the internal transcribed spacer (ITS-2) (41-42).

The SSU rRNA-based nested PCR-RFLP method has been successfully
used in conjunction with IMS in the detection and differentiation of
Cryptosporidium oocysts present in storm water, raw surface water, and waste-
water (147, 148).

Two SSU rRNA-based PCR-sequencing tools and one other SSU-based
PCR-RFLP tool can differentiate Cryptosporidium oocysts in surface and waste-
water samples (86, 136), suggesting that humans, farm animals, and wildlife con-
tribute to Cryptosporidium oocyst contamination in water. Other genes have also
been used for genotyping of Cryptosporidium. HSP70 and TRAP-C2-genes have
limited use as they do not amplify DNA of Cryptosporidium species distant from
C parvum(51).

A few PCR related techniques have also been used to quantify and deter-
mine viability of Cryptosporidium oocysts. Excy station followed by DNA
extraction and PCR has been developed to detect viable C parvum oocysts
(36, 134). Tissue culture and PCR (26, 67, 108) or reverse transcription-PCR
(RT-PCR) (108, 109) has been used to detect viable Cryptosporidium oocysts.
RT-PCR techniques have been described for the detection of viable oocysts (48,
56, 143), but it may overestimate the viability of oocysts (37). A new integrated
detection assay combining capture of double-stranded RNA with probe-coated
beads, RT-PCR, and lateral flow chromatography has also been developed,
which should shorten detection time (64).

Other molecular tools, such as fluorescence in situ hybridization (FISH), or
colorimetric in situ hybridization of probes to the SSU rRNA have been used in

Cyclospora Cayetanensis 125

the detection or viability evaluation of C parvum oocysts (72, 116). Nucleic acid
sequence-based amplification (NASBA) has been used in the detection of viable
C parvum oocysts (10). More recently, a biosensor technique for the detection
of viable C parvum oocysts has also been described (11), and a microarray
technique based on HSP70 sequence polymorphism has been developed to
differentiate Cryptosporidium genotypes (119).

CYCLOSPORA CAYETANENSIS

Parasite Description and Identification. Cyclospora cayetanensis was initially
identified as a cyanobacteria-like organism. The first reports of gastrointestinal
infections in humans go back to 1988, when it was described as a blue-green
algae or a large Cryptosporidium. It was not until 1992 when Ortega et al.
reported a complete description of this parasite as belonging to the coccidian
(93, 96). Cyclospora has been identified in insectivores, snakes, and rodents. In
1997, three species were identified in nonhuman primates (30, 73). These are
morphologically similar to C cayetanensis, but examination of the 18S rDNA
demonstrated that they are phylogenetically different. A challenge when work-
ing with Cyclospora is that is there is no animal model suitable to propagate it
in laboratory conditions. The same limitation applies to the nonhuman primate
Cyclospora species.

Cyclosporiasis is characterized by prolonged watery diarrhea.
Nondifferentiated oocysts are excreted in the feces of the infected individual
into the environment. Oocysts differentiate after 7-15 days in the environment,
becoming fully sporulated and infectious. When a susceptible individual ingests
oocysts from contaminated water or foods, the oocyst will excyst and release the
sporocysts, which also will undergo excystation (96). Each contains two sporo-
zoites that will infect the epithelial cells of the small intestine (94). Cyclospora
preferentially colonizes the ileum; however, there are few reports suggesting
extraintestinal colonization such as the biliary and respiratory tracts.

Even though there have been few reports of Cyclospora associated with
drinking or swimming in contaminated water, most of the reported outbreaks
in the developed world have been associated with contaminated fresh produce
and berries. Lettuce, basil, and raspberries have been the most frequently impli-
cated products (50). In 2004, the first documented outbreak of cyclosporiasis
was linked to Guatemalan snow peas (113). Some patients reported consump-
tion of untreated water or reconstituted milk.

Diagnosis of Cyclospora in clinical specimens is performed using a modified
acid fast stain, or by direct microscopical examination. A procedure to isolate
Cyclospora oocysts from produce was described by Robertson (107). For mush-
rooms, lettuce, and raspberries, recovery was 12%; while recovery from bean
sprouts was 4% using lectin-coated paramagnetic beads to concentrate the par-
asite. Although the lectin-coated paramagnetic beads did not significantly
improve recovery of the oocysts, it did produce a cleaner and smaller final vol-
ume for easier identification of the protozoan under the microscope.

126 Molecular Tools for the Identification of Foodborne Parasites

Molecular Detection. The first PCR for Cyclospora was developed for clinical
specimens. Fecal samples containing oocysts were disrupted using a bath soni-
cator at 120 W. The product of the nested PCR was a 304 bp amplicon (101).
However, this PCR produced amplicons for Eimeria; a coccidian that infects a
wide range of animals commonly found in environmental samples and is non-
pathogenic to humans. This nested PCR was modified using the same primers
but without the leader sequence. The template was prepared using 6 freeze/thaw
cycles (2 min in liquid nitrogen and heating at 98°C). To address the issue of
PCR inhibitors, Instagene Matrix (Bio-Rad; Hercules, CA) was added during
the DNA extraction. Nonfat milk (50 mg/ml) was used in the PCR reaction to
overcome the effect of inhibitory effects of the food matrix extracts, soil, and
plant matrices. An RFLP PCR was also developed to differentiate between
Eimeria and Cyclospora (58).

An extraction free filter based template preparation was evaluated using
Cyclospora oocysts from fecal and food matrix samples. Pieces of the FTA fil-
ters (Whatman; Florham Park, NJ) were added directly to the PCR mixture
after washing with 10 mM Tris (pH8.0) containing 0.1 mM EDTA and heat
treatment at 56°C. A sensitivity of 10 to 30 Cyclospora oocyst could be detected
in 100 g of fresh raspberries (92).

Quantitative real-time PCR was developed for the identification of
Cyclospora, targeting a 83-bp region of the 18s rRNA gene. This analysis was
based on one Cyclospora isolate. The sensitivity and specificity of the assay
will need to be confirmed when examining a larger number of environmental
samples (132).

Since other nonhuman primate Cyclospora also produced 294 bp amplicon
with the SSU-rRNA nested PCR primers, a multiplex PCR was developed.
Briefly, PCR is performed first using external, nested PCR primers described by
Jinneman, followed with PCR using a series of specific primers that differenti-
ate Cyclospora species and Eimeria species (90).

A restriction fragment length polymorphism (RFLP) assay was developed
using the endonuclease Mnl I. RFLP DNA patterns are different between
Cyclospora and Eimeria (58). To simplify the methodology for use at inspection
sites, an oligo-ligation assay (OLA) was developed. The target amplicon was
detected with an antibody-enzyme conjugate that could be read as a colorimetric
assay (59).

Methodologies that could be used for fingerprinting analysis and genotype
discrimination are not yet available. The intervening transcribed spacer 1(ITS1)
was examined as a potential sequence that will allow discrimination among
Cyclospora genotypes. When compared, the sequences of 5 isolates from a
Cyclospora foodborne outbreak were identical. One of two Guatemalan isolates
and 2 out of 4 Peruvian isolates were also identical (2). Thirty-six Cyclospora
samples were examined using ITS1 specific primers. Sequence homology of
460-465 bp from various isolates varied between oocyst samples from 1 to 6.5%
and between samples and 5.7%. Cryptosporidium ITS is also highly variable
(1.1-1.3% within and 0.8-1.6% between oocyst samples). The lack of animal
models to obtain a clonal population of Cyclospora limits the possibility to

Giardia Intestinalis 127

determine if there are different ITS sequence types within one oocyst or if there
are infections with various Cyclospora strains (88). If intraisolate variation
occurs, as with many coccidian parasites, ITS1 may not be a suitable target for
genotyping of Cyclospora.

GIARDIA INTESTINALIS

Parasite Description and Identification. Giardia was initially described by
Leeuwenhoek in 1681 (1). Giardia infecting humans was renamed in the early
1990s: as G intestinalis, G lamblia, and G duodenalis. The name G lamblia was
well-recognized in the 1970s, but encouraged by other investigators it was
changed to G duodenalis and G intestinalis in the 1990s. Over 40 species names
have been proposed on the basis of hosts of origin. Now, several species are rec-
ognized: G muris from rodents, G agilis from amphibians, G psittaci from
parakeets, G ardae from herons, and G microti from voles and muskrats.
Giardia is the most common cause of waterborne outbreaks; however, there
have also been reports of foodborne outbreaks. In developing countries, chronic
giardiasis has been associated with long-term growth retardation. Giardia can
be asymptomatic or cause intermittent or chronic diarrheal complaints.
Symptomatic giardiasis is characterized by prolonged and intermittent diar-
rhea, anorexia, flatulency, weight loss, and malabsorption. Whether Giardia
lamblia infects humans and other mammals, whether it is a single species, and if
so, whether it is a zoonotic infection has been questioned. Foods implicated
with giardiasis are fresh produce or foods contaminated by food handlers (1).

The infective stage of Giardia is the environmentally resistant cyst and the
vegetative stage is the trophozoite. The trophozoite is pear shaped and has 8 fla-
gella and a ventral adhesive disk. When a susceptible individual ingests con-
taminated water or foods containing the cysts, these cysts will excyst in the
intestine aided by bile salts and gastric acids. The trophozoite will multiply
asexually. Giardia does not invade tissues and propagation occurs on the epithe-
lial surface. The trophozoites attach to the epithelial surface. Some of the
trophozoites will encyst in the jejunum and are passed in the feces.

Giardia is the most commonly isolated gastrointestinal parasite worldwide
and the U.S., (61) with large waterborne outbreaks having been reported. The
cysts are environmentally resistant and survive long periods of time in water at
cold temperatures. Giardia has been detected in 81% of raw water samples and
17% of filtered water samples (68). Children attending day care centers are at
higher risk of acquiring Giardiasis. Waterborne Giardiasis has been associated
with unfiltered water; recreational water, such as swimming pools; water foun-
tains; and travel (78).

Molecular classification tools have been used with various Giardia isolates
and have determined various assemblages or genotypes based on the sequence
comparisons of the SS rRNA, triosephosphate isomerase, and glutamate dehy-
drogenase genes. Genotype A groups 1 and 2 have been isolated from humans
and animals. Genotype B has also being isolated from humans and some

128 Molecular Tools for the Identification of Foodborne Parasites

animals. Assemblages C and D have been isolated from dogs, F from cats,
G from rats, and E from cows, sheep alpaca, goat, and pigs. The significance of
these assemblages in the human infections is being examined (1). In the
Netherlands, patients with assemblage A isolates presented with intermittent
diarrheal complaints, while assemblage B was present in individuals with per-
sistent diarrheal complaints (51).

Diagnosis of Giardia in clinical samples is performed using bright field
microscopy. Immunofluorescent assay and EIA are commercially available.
Merifluor Cryptosporidium! Giardia kit (Meridian Bioscience), Giardia/ Crypto
IF kit (TechLab), Crypto/ Giardia Cel kit (TCS Biosciences), and Aqua-Glo
G/C kit (Waterborne) are some of the commercially available kits. Detection of
Giardia cysts in environmental samples presents a significant challenge.
Recovery of Giardia cysts from surface water to be used by drinking water treat-
ment plants is achieved using the United States EPA Method 1623. Several of
these methods have been adapted for detecting parasites in food matrices.

Molecular Detection. However, the low number of cysts typically present in
these types of samples requires the use of molecular tools. In clinical specimens
Giardia and Cryptosporidium were detected 22 times more often by PCR than
by conventional microscopy (7).

Analysis of nucleotide sequences of glutamate dehydrogenase (GDH), elon-
gation factor la (EFla), SSU rRNA, and triosephosphate isomerase (TPI),
and ADP-ribosylating factor genes can discriminate five to seven defined line-
ages and assemblages of G intestinalis (80, 81, 121). Thus far, the TPI gene has
the highest polymorphism in G intestinalis at both intergenotype as well as
intragenotype levels, and TPI genotyping has proven very useful in epidemio-
logical investigations of human Giardiasis (121, 123).

Molecular characterization of Giardia species in wastewater has been used
for community wide surveillance of human Giardiasis (130, 135). The distribu-
tion of the Giardia species in environmental samples correlates directly with
human, agricultural, and wildlife activities. SSU rRNA-based PCR-RFLP
(130) and beta-giardin-based PCR-RFLP methods were used to identify and
differentiate between Giardia assemblages A and B from water samples.

The phylogenetic distance between G intestinalis assemblages A and B is
greater than typically used to differentiate two protozoan species (79, 81, 121,
131), suggesting that G intestinalis may be a species complex. Phenotypic dif-
ferences have also been observed. Assemblage B is more likely found in patients
with persistent diarrhea, whereas intermittent diarrhea is mostly observed with
assemblage A (51).

TOXOPLASMA GONDII

Parasite Description and Identification. Toxoplasma is a coccidian parasite that
can cause severe complications for individuals with the infection. It can be
asymptomatic or can cause abortion in humans if an acute infection develops

Toxoplasma Gondii 129

during pregnancy. Healthy individuals may develop encephalitis or be asymp-
tomatic. Toxoplasma can be found worldwide and serologically it can be identi-
fied in as high as 85% of the population in some European countries.
Toxoplasma is responsible for 20.7% of foodborne deaths due to known infec-
tious agents. Waterborne outbreaks in Canada and Brazil (12, 54) have been
reported as well.

Infection occurs when water or foods are contaminated with cat feces con-
taining Toxoplasma oocysts. The oocysts excyst and the sporozoites migrate and
preferentially localize in muscle and the brain. The parasites will encyst and
form cysts. These contain bradyzoites, which are slow multiplying parasites.
Once they become active they are called tachyzoites and multiply quickly. The
parasite can cross the placenta to infect the fetal tissues (29).

A large variety of animals can acquire toxoplasmosis, but only felines
(domestic and wild) are the definitive hosts. When infected tissues are ingested,
the parasites will be released from the tissues and develop to the asexual and
sexual stages. Once fertilization occurs, the oocysts are formed and then
excreted in the environment. The oocysts are highly resistant even to desiccation
and survive on dry surfaces for weeks or even months. Toxoplasma causes fatal
meningoencephalitis in a variety of marine mammals. Shellfish has been stud-
ied during the past several years as indicators of or vectors for transmission of
protozoal agents (29). Shellfish can concentrate large volumes of water and it
has been demonstrated experimentally that viable Toxoplasma oocysts can also
be concentrated. Toxoplasma has been classified into three different lineages I,
II, and III (53). Toxoplasma belonging to type I lineage are highly virulent in
laboratory animals, whereas the type II and III lineages are nonvirulent. In
humans, the type II lineage predominates among Toxoplasma associated with
infections in AIDS and non-AIDS immunocompromised patients (75-80%), as
well as congenital Toxoplasma infections (encephalitis, pneumonitis, or dissem-
inated infections). It appears that the type I lineage is more prevalent in con-
genital Toxoplasma infections in Spain (38). Ocular toxoplasmosis is a common
sequela of congenital toxoplasmosis, but can be dormant for years and emerge
at adulthood; causing severe retinochoriditis. PCR analysis of clinical samples
from patients with these conditions determined that most Toxoplasma isolates
were type I, type IV, or novel types (69). Outbreaks in Canada and Brazil char-
acterized by severe ocular toxoplasmosis were caused by Toxoplasma type I
strains (16).

Toxoplasma infections can be diagnosed by serological assays examining the
antibody response towards the infection. Identification of Toxoplasma oocysts
can be identified in the environment using conventional microscopy; however,
one must consider the limitation to this approach: (1) the small number of par-
asites present in environmental samples; and (2) the oocysts are indistinguish-
able morphologically from other coccidians. Toxoplasma oocysts have been
isolated from mussels, which serve as paratenic hosts assimilating and concen-
trating oocysts. Toxoplasma oocysts can be identified from water samples using
the current USEPA method for concentration of Cryptosporidium (54).
Centrifugation and flocculation procedures using aluminum sulfate and ferric

130 Molecular Tools for the Identification of Foodborne Parasites

sulfate can also concentrate Toxoplasma oocysts. Sporulated oocysts were
recovered more efficiently using aluminum sulfate and unsporulated oocysts
could be better recovered using ferric sulfate (63). A TaqMan PCR assay was
developed to detect the ssrRNA. Infectious Toxoplasma oocysts were detected
up to 21 days in mussels as confirmed using the mouse bioassay (8).

Molecular Detection. Most PCR assays used for Toxoplasma identification use
primers targeting the Bl gene. It is a 3 5 -fold repetitive gene that is highly spe-
cific and conserved among strains of Toxoplasma (18). A PCR-enzyme
immunoassay oligoprobe was developed to detect Toxoplasma oocysts. The
PCR was directed towards the amplification of the Bl gene. Avidin coated
plates were used to capture the biotin-labeled PCR amplicons, and an internal,
FITC labeled oligoprobe was allowed to hybridized to the denatured and bound
amplicon. The bound FITC-tagged oligoprobe was detected using anti-FITC
antibodies tagged with horse radish peroxidase. This assay could detect 50
oocysts in a clean preparation (114). Other assays have focused on the sensitiv-
ity of the PCR assay. Jalal designed primers for Bl gene PCR amplification
with a sensitivity of 2 parasites/sample (55).

The freeze thaw procedure in Tris-EDTA buffer and proteinase K digestion
has been used to break open Toxoplasma oocysts. This is followed by DNA
extraction using the QIAamp DNA minikit. Again, the Bl gene was used in a
real-time PCR using an Icycler device (Bio Rad). Sensitivity of the real-time
PCR using experimentally spiked deionized and public drinking water samples
was of 1 and 10 oocysts. However, the sensitivity of the PCR was reduced for
raw surface waters as only 20%, and 50% were positive by real-time PCR for
samples spiked with 100 and 1000 oocysts, respectively (133).

Oocyst heating at 100°C for 40 min in TE buffer followed with 9 freeze-thaw
cycles and proteinase K digestion at 56°C overnight has been used to break
open the oocysts and provide a template for PCR. DNA was subsequently
extracted from the broken oocysts using the phenol: chloroform: isoamyl alco-
hol procedure. DNA amplification using thel8S-rRNA gene designed by
McPherson and Gajadhar (77) had a theoretical detection limit of 0.1 oocyst if
the preparation included oocyst concentration using aluminum sulfate floccu-
lation (63). TaqMan PCR assays were done using Bl and ssrRNA genes to
study experimentally inoculated mussels (8). Real-time PCR was more sensitive
to nested PCR using Bl and bradyzoites specific genes. LC-PCR also had the
advantage to quantify parasites present in serum and peripheral blood mononu-
clear cells (23). Other targets were used for DNA amplification. These included
a 529 bp sequence present at 300 copies in the parasite genome. Using this tar-
get sequence, real-time PCR had a tenfold higher sensitivity compared to PCR
targeting the 35 copy Bl gene (52). Mobile genetic elements (MGE), which has
100-500 copies/cell was also used for Toxoplasma identification followed by
RFLP analysis (128). Other single copy genes SAG1-4 and GRA4 genes have
been used as targets for Toxoplasma characterization and identification.

Characterization of Toxoplasma isolates was achieved using PCR-amplified
products digested with 13 restriction enzymes and determined the genetic

MlCROSPORIDIA 131

relationship among Toxoplasma isolates and other coccidia (17). RFLP-PCR,
random-amplified polymorphic DNA (RAPD), PCR, and sequencing has
allowed for genotyping analysis (3, 14, 47). Sequencing of DNA polymerase
and the gra6 genes have also been used to determine strain types (33).

MlCROSPORIDIA

Parasite Description and Identification. Microsporidia belongs to the phylum
Microspora. It contains more than 1000 species and infects a wide range of
hosts. Five genera have been implicated in human illness: Encephalitozoon,
Enterocitozoon, Septata, Pleistophora, and Vitaforma. Originally called
S. intestinalis, it has been reclassified as Encephalitozoon and Nosema cornea as
Vitaforma cornea. E. bieneusi causes persistant diarrhea in the immunocompro-
mised, where it is found frequently in the feces.

Microsporidia are obligate intracellular organisms that form highly resistant
spores of pirifiorm or ovid shape ranging from 1 to 2 |im in diameter. Spores
are ingested along with contaminated water or foods, or inhaled. The spore
extrudes its polar filament and injects the sporoplasm (infectious spore mate-
rial). The sporoplasm undergoes merogony forming multiple primordial forms.
Sporogony follows, forming dividing sporonts, which form the sporoblast. They
then develop into mature spores. Spores are excreted along with the feces, urine,
or respiratory secretions. E. bieneusi has been reported in mixed infections with
Cryptosporidium.

Microsporidial spores can be identified microscopically using Calcofluor
white, or modified trichrome with Chromotrope 2R stains. The use of other
stains has been reported. Identification of the spores is difficult because of the
small size of the spores, which require a well-trained microscopist. Molecular
assays have played a significant role, particularly in the identification of particu-
lar Microsporidia (78). The various species of Microsporidia can be acquired via
various routes. The species acquired by ingestion of contaminated foods or water
are E. bieneusi and E. intestinalis. The latter can be successfully grown in vitro.

Molecular Detection. Primers were designed to amplify the SSU rRNA gene of
E. bieneusi in fecal and biopsy samples, either by PCR or in situ hybridization
(21). PCR protocols call for rupture of Microsporidia spores using glass beads
and overnight digestion with proteinase K to release the template. This is fol-
lowed by DNA extraction using commercial kits and PCR amplification using
SSU rRNA gene (24). PCR protocols seem to work with fresh as well as for-
malized specimens. Other PCR primers have been developed for detecting the
various Microsporidia species.

To confirm the identity of the amplified PCR products from the SSU rRNA
gene, restriction endoucleases Haelll and Pst I have been used to distinguish
between E. bieneusi and E. intestinalis (34); however, these restriction enzymes
do not differentiate E. intestinalis from E. cuniculi. Primers were also designed
to amplify conserved segments of the SSU rRNA of these two Microsporidia.

132 Molecular Tools for the Identification of Foodborne Parasites

Results were confirmed by standard staining methods and immunofluorescence
assay specific for E. intestinalis (89). Orlandi evaluated the filter based protocol
described for Cyclospora and Cryptosporidium in PCR detection of E. intesti-
nalis. The PCR assay could identify as few as 10-50 E. intestinalis spores (92).
PCR amplification followed by Hinfl endonuclease restriction could identify E.
intestinalis from clinical specimens (100). Examination of a fragment of the ITS
region suggests the presence of genetically distinct strains of E. bieneusi (104).
Detection of Microsporidia spores in environmental samples was evaluated
using immunomagnetic separation followed by PCR and with the concentration
of the Microsporidia spores by immunomagnetic separation, PCR could detect
as few as 10 spores per 100 L of tap water (117). Real-time quantitative PCR
has been developed using the polar tube protein gene 2 of E. intestinalis, which
could be used to quantify the number of spores produced in vitro, or to deter-
mine the effect of inactivation procedures (138). Real-time PCR using com-
mercial DNA isolation kits and an automated MagNA Pure LC instrument
could identify microsporidia. The sensitivity of the PCR was 100-10,000
spores/ml of feces (145). PCR followed by sequencing of the ITS fragment of
various Microsporidia isolates from human and animal origin has demon-
strated high variability among isolates, and that few of the isolates from animal
origin may be of public health relevance (122).

HELMINTH INFECTIONS

There are three groups of helminthic parasites and each group is significantly
relevant to public health. Cestodes or flatworms associated with foodborne
outbreaks include Taenia solium and T. saginata, Diphylobrotrium latum,
Echinococcus granulosus, and E. multilocularis. Identification of the parasites
can be done by observation of the larval (or cystic) stages of the parasite in
meat. Nematodes or round worms have also been identified in meats as larval
forms (Trichinella spiralis, Anisakis) and the eggs can be present in fresh pro-
duce or in contaminated water (Ascaris lumbricoides, Toxocara canis, Capillaria,
Gnatostoma, and Angiostrongilus). Flukes or trematodes can also be acquired
by ingestion of the cystic forms in fish, crabs, or shellfish (Paragonimus wester-
mani, Heterophyes, and Nanophyetus). In most instances, these parasites are
ingested when foods are eaten fresh or raw (not frozen). Another fluke (Fasciola
or Fasciolopsis) can be acquired by ingestion of raw vegetables containing the
metacercaria stages. These parasites can be isolated from meats and produce
(39, 75, 1 12, 129, 144). Molecular assays for detection of these parasites are cur-
rently not done as routine procedure; however, laboratories that do molecular
epidemiology have developed assays to describe the genotypes most commonly
isolated in certain animal species. Most of these parasites have been reported in
developing areas and countries, where Good Agricultural Practices (GAPs) are
not established. Some of these parasites can be acquired by ingestion of game
meats, raw fish, or shellfish. Most of these parasites can be inactivated when
frozen.

Conclusions 133

VIABILITY ASSAYS

The viability of cysts or oocysts has been examined by using vital dyes. In vitro
cultivation has been successful for some genotypes and species of
Cryptosporidium and Toxoplasma. Most of the microsporidian spores can be
propagated in vitro except for E. bieneusi, which is the most commonly identi-
fied microsporidia in humans and associated with gastrointestinal illness
(25, 27, 28). To date, there is no effective in vitro cultivation assay for Cyclospora.
Giardia can be excysted and grown in TYI-S-33 media; however, a large number
of cysts are required for successful propagation. To date, some Giardia assem-
blages or genotypes cannot be propagated in vitro. Animal models that could be
used to determine infectivity and viability are limited to Cryptosporidium and
Toxoplasma (108). Cryptosporidium can be propagated using neonate calves or
mice (110). However, C hominis, which is anthroponotic, does not infect these
animals. Gnotobiotic pigs have been used to propagate C hominis, but they are
not a practical animal model for inactivation and viability studies. No animal
model is currently available for Cyclospora (31), nor has the disease been repro-
duced with this agent in healthy, human volunteers. Toxoplasma tachyzoites can
be propagated using the MRC-5 cell line and most other fibroblast cell lines. It
can infect cats, mice, and chickens. Whether this infectivity is selective to certain
genotypes is currently not known.

As evident from this discussion, there are several viability assays available
for some but not all foodborne parasites. However, how practical and cost-
effective are these assays in assessing effectiveness of certain processes for elim-
inating these pathogens, or reducing their load in food? Obviously, there is a
need for developing methods to discern the effectiveness of certain food
processes for eradicating or reducing these parasites. Reverse transcriptase PCR
developed as "real-time" detection of these foodborne parasites might prove to
be an important tool, in the future, for this endeavor.

CONCLUSIONS

Parasites and the intestinal diseases they cause are more frequently being asso-
ciated with consumption of raw vegetables, fruits, and nonfiltered water. These
outbreaks are most often associated with produce imported from areas, where
these parasites are endemic. Importation of foods is now necessary in order to
satisfy the consumer demands for certain commodities, especially fresh fruits
and vegetables. Incentives for importation of foods include the cost of produc-
tion for particular crops throughout the year. The fast transportation of fresh
produce from the farm to the consumers has favored the survival of these
pathogens on these commodities. Due to advances in medicine, the U.S. demog-
raphy has also changed. The elderly, immunocompromised individuals and
children are at higher risks of acquiring and having a more severe illness. Most
foodborne outbreaks are considered to be bacterial or viral. To determine the
etiology of an outbreak may take many days, by which time samples of the

134 Molecular Tools for the Identification of Foodborne Parasites

implicated product may not be available for investigation. Because of this fac-
tor, food scientists and the medical community need to be aware that parasites
are significant agents for foodborne outbreaks and routine examination of clin-
ical samples may miss the identification of parasites, particularly with
Cyclospora and Cryptosporidium. Molecular assays have been very helpful in
foodborne outbreak investigations, but they have also demonstrated the current
limitations in food parasitology. Parasite isolation and recovery procedures are
crucial to have a very accurate and specific diagnosis. This is particularly impor-
tant, since parasites are generally inert in the environment and enrichment pro-
cedures necessary for bacterial contaminants are not an option in the detection
of these pathogens in foods. With further development and refinement, PCR
will prove to be an important tool in surveillance of foods for these emerging
human parasites.

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148. Zu, S.X., GD. Fang, R. Fayer, and R.L. Guerrant. 1992. Cryptosporidiosis:
Pathogenesis and immunology. Parasitol. Today 8:24-27.

144

Molecular Tools for the Identification of Foodborne Parasites

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INDEX

16SrDNA, 11-12
Amplicons, non-specific, 28-29
Commercial PCR

Campylobacter, 63-64

Escherichia coli, 0157:H7, 63-64
pathogenic, 63-64
Listeria monocytogenes, 63-64

Salmonella, 63-64, 65
Controls, 28, 32, 46, 59-60
DNA databases, 1 1

NCBI, 14-19

searches, 14-19
False-negatives, PCR inhibitors, 34-35
False-positives

live vs. dead cells, 33-34

non-specific amplicons, 29

primer-dimers, 29

viable but nonculturable (VBNC), 34
Interpretation of PCR results, 28-3 1
Laboratory

equipment, 57-58

personnel, 55

reagents and disposables, 58-59, 60

set-up, 52-55

standard operating procedures, 59-60

standardization, 62
Micro arrays, 12
Multiplex PCR, 10, 70, 84
Multiplex PCR, 70, 84
Nucleic acid extraction

parasites, 120-121

viruses, 99-100
Nucleic acid sequence-based amplification

(NASBA), viruses, 102, 107
parvum, 124-125
PCR contamination, 52-55
PCR design, 12-19
PCR detection methods, 6-8

agarose gels, 6

ELISA, 6-8, 56

fluorescence, 8-10, 56-57
PCR detection of

Bacillus cereus, 71

Campylobacter, 63-64, 71-72, 85

Clostridium perfringens, 72-73

Cryptosporidium parvum, 123-124, 144

Cyclospora cayetanensis, 126-127, 144

Escherichia coli 0157:H7, 63-64, 85

Escherichia coli, pathogenic, 63-64,

73-76, 84-85

Giardia intestinalis, 128, 145

hepatitis A virus, 102, 105-106

Listeria monocytogenes, 63-64, 76-77,

Micro sporidia, 131-132, 144

noroviruses, 100-101, 103-104

Salmonella, 63-64, 85, 77-79, 84-85

Shigella, 79-80

Staphylococcus aureus, 80-81

Toxoplasma gondii, 130-131, 145

Vibrio, pathogenic, 81-83, 85

Yersinia enter ocolitica, 83, 85
PCR inhibitors, 34-35, 46^7
PCR theory, 2-4
Quality control, 59-60
Real-time PCR

Campylobacter, 63-64, 85

concept, 8-10

Escherichia coli 0157:H7, 63-64, 85
pathogenic, 63-64, 85

false-positives, 10

hepatitis A virus, 108-109

Listeria monocytogenes, 63-64, 85

noroviruses, 108-109

Salmonella, 63-64, 85

TaqMan, 8

Vibrio, pathogenic, 85,

Yersinia enter ocolitica, 85
Reverse-transcriptase PCR, detection of

bacteria, 33-34, 84-85

viruses, 101-102
Sample preparation, 41^49

concentrating microbes, 47-49, 93-99,

122-123,

dairy, 43, 96-99

enrichment, 47^9

fruits and produce, 45, 96-99

meat, 43^4

seafood, 44-45, 94-96
Sensitivity

false-negatives, 31

limit of detection, 34-35, 47^9, 70

PCR inhibitors, 34-35, 46-47

false-positives, 31
Terminal Restriction Fragment Length

Polymorphisms, 10-12

147

148

Index

Thermocycler

conventional, 4, 57-58
gradient, 5

hot-air capillary, 4-5, 57-58
real-time PCR, 5-6, 57-58

Thermocyclers, 4-6, 57-58

Trouble- shoo ting

false-negatives, 34-35, 46-47
false-positives, 32-33, 45

real-time PCR, 10, 29

sensitivity, 47-49
Validation of PCR, 31, 60-62
Viability

bacteria, 33-34

parasites, 133
Virus, concentration, 93-99

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