real-time polymerase chain reaction for the food microbiologist

7/27/2019 Real-Time Polymerase Chain Reaction for the Food Microbiologist

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Real-time Polymerase Chain Reactionfor the Food Microbiologist:Technologies, Applications, and LimitationsSSSSSCCCCCOOOOOTTTTTTTTTT E. HE. HE. HE. HE. HANN AANNAANN AANNAANN A, C, C, C, C, CHRISTHRISTHRISTHRISTHRISTOPOPOPOPOPHERHERHERHERHER J. CJ. CJ. CJ. CJ. CONNORONNORONNORONNORONNOR,,,,, ANDANDANDANDAND HHHHHUUUUUAAAAA H.H.H.H.H.WWWWWANGANGANGANGANG

ABSTRAABSTRAABSTRAABSTRAABSTRACTCTCTCTCT: R: R: R: R: Rapid detection of pathogenic and spoilage micrapid detection of pathogenic and spoilage micrapid detection of pathogenic and spoilage micrapid detection of pathogenic and spoilage micrapid detection of pathogenic and spoilage microorooroorooroorganisms is essential for ensurganisms is essential for ensurganisms is essential for ensurganisms is essential for ensurganisms is essential for ensuring the safety anding the safety anding the safety anding the safety anding the safety andquality of food. Real-time polymerase chain reaction (PCR) technology has the potential to achieve rapid, sensitive,quality of food. Real-time polymerase chain reaction (PCR) technology has the potential to achieve rapid, sensitive,quality of food. Real-time polymerase chain reaction (PCR) technology has the potential to achieve rapid, sensitive,quality of food. Real-time polymerase chain reaction (PCR) technology has the potential to achieve rapid, sensitive,quality of food. Real-time polymerase chain reaction (PCR) technology has the potential to achieve rapid, sensitive,and specific detection of these micrand specific detection of these micrand specific detection of these micrand specific detection of these micrand specific detection of these microorooroorooroorganisms in food. Iganisms in food. Iganisms in food. Iganisms in food. Iganisms in food. In this rn this rn this rn this rn this reviewevieweviewevieweview, w, w, w, w, we discuss re discuss re discuss re discuss re discuss real-time PCR technologies ineal-time PCR technologies ineal-time PCR technologies ineal-time PCR technologies ineal-time PCR technologies inuse todayuse todayuse todayuse todayuse today, applications of r, applications of r, applications of r, applications of r, applications of real-time PCR in food systemseal-time PCR in food systemseal-time PCR in food systemseal-time PCR in food systemseal-time PCR in food systems, and some of the associated challenges and limitations, and some of the associated challenges and limitations, and some of the associated challenges and limitations, and some of the associated challenges and limitations, and some of the associated challenges and limitations.....

Keywords: real-time PCR, rapid methods, food microbiologyKeywords: real-time PCR, rapid methods, food microbiologyKeywords: real-time PCR, rapid methods, food microbiologyKeywords: real-time PCR, rapid methods, food microbiologyKeywords: real-time PCR, rapid methods, food microbiology

Introduction

Contamination by foodborne pathogens is a great threat to hu-man health; the estimated cost of foodborne illness in the Unit-ed States is between $10 billion and $83 billion annually (USFDA

2001). In addition, food spoilage due to outgrowth of spoilage or-

ganisms costs the food industry significant amounts of money

through the loss of raw material and finished product, product re-

calls, loss of sales due to reduced consumer confidence, and poten-

tial litigation. While sterilization could eliminate the presence of mi-

croorganisms in the final products, extreme processing conditions

often cause undesirable physiochemical changes and loss of nutri-

tional values of the food products that lead to consumer unaccept-

ability. Therefore, achieving final product quality assurance

through controlling the quality of raw materials and verification ofthe lack of target pathogenic and spoilage organisms in the final

products are still the main choices for the food industry.

When compared with clinical diagnostics, there are several chal-

lenges associated with microbial detection in foods. The initial con-

tamination level in foods is normally low, and sampling with repre-

sentation could be difficult (Jaykus 2003). Foods not only provide

nutrients supporting the growth of microorganisms, but various

ingredients can interfere with the activities of enzymes involved in

detection. In products such as fermented foods, the background

microbial count could be fairly high. Finally, many foods have lim-

ited shelf-life. Therefore, timely detection of these organisms with

high degrees of specificity and sensitivity to maintain a safe, whole-

some food supply is a major task for food microbiologists.

Although methods such as microbial culturing and biochemical

assays have proven to be useful in quality control, they still cannot

meet all the demands of the food industry because of their intrinsic

limitations. For instance, because normal bacteria generation time is

approximately 20 to 40 min, it can take anywhere from 18 h to several

days for enough microbial multiplication to occur to allow for bacterial

culturing or metabolism-based detection. More sensitive and rapid

detection is desired, and one of the recent attempts to fill this need

has been with polymerase chain reaction (PCR) technology.

Conventional PCR

PCR technology has been used to rapidly detect, characterize,and identify a variety of organisms (Campbell and Reece1996). In a conventional PCR setting, a pair of oligonucleotide prim-

ers complementing to sequences at both ends of the target gene,

DNA from the target organism to act as a template, free nucleotides,

salts, and DNA polymerase are combined in a reaction mixture,

resulting in the replication of the target DNA fragment. This repli-

cation of an individual DNA fragment, known as amplification, can

be achieved within a minute or two. Because the amplification is an

exponential process, after repeated rounds of this amplification

using a thermal cycler, enough copies of the fragment will have

accumulated to be detectable. Therefore, the presence of even 1

copy of the template within the reaction mixture can be detectedwithin a couple of hours.

Some DNA sequences are similar across class or genus lines, and

others are unique to a particular species or strain. Because primers

can be designed to target specific DNA sequences conserved at

these various levels, detection of the presence of a microbial genus,

species, or strain can be achieved by observation of the targeted

PCR products. Since its invention, many studies have shown the

effectiveness of PCR for rapid detection of numerous species of

bacteria (Allmann and others 1995; Kaiser and others 2001; Maki-

no and others 2001; Jensen and Whitfield 2003; Malinen and others

2003).

PCR can be a powerful tool for analysis, but it has some short-

comings that limit its effectiveness in many cases. Nonspecific

amplification is a major problem associated with PCR. Particularly

under low-stringency conditions, such as low reaction tempera-

tures, primers might anneal to regions with minor mismatches and

amplify unrelated PCR products. This can lead to false-positive

results. In addition, hairpin loop formation, in which 2 segments of

a single primer hybridize with each other, can not only prevent the

primers from binding to the desired template but also result in the

formation of an undesired fragment. Also, 2 primers with comple-

menting sequences can bind to each other and form primer

dimers, which can be detected as a nonspecific amplification prod-

uct (Miller and others 1996).

Other PCR limitations have also been documented. For instance,

detection of the target DNA fragment using gel electrophoresis

must be carried out after PCR amplification has taken place, creat-

MS 20040446 Submitted 7/2/04, Revised 9/27/04, Accepted 12/12/04. Theauthors are with Dept. of Food Science and Technology, The Ohio State Univ.,Columbus, Ohio 43210. Direct inquiries to author Wang (E-mail:[email protected]).
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Real-time PCR for the food microbiologist . . .

ing a need for additional labor as well as allowing opportunities for

carry-over contamination of PCR products (Fratamico 2001). And in

rare cases, reagents might cause a contamination problem due to

improper purification during the manufacturing process (Corless

and others 2000).

Real-time PCR

OverviewOverviewOverviewOverviewOverview

Recent advancements in PCR technology include the develop-

ment of real-time PCR devices and the application of new amplifi-

cation product-detection chemistries. Real-time PCR adds an op-

tical module to a standard PCR assay, allowing the capture of

fluorescent signals from labeled PCR products. This method of

detecting the amplicon, the fragment of DNA replicated during the

PCR reaction, is the main difference between conventional and

real-time PCR technologies, as the instrument detects the intensity

of the fluorescent signal during each replication cycle of the PCR

(Mackay 2004). Computer software records and displays the

amount of fluorescence in relative fluorescence units (RFU). The

amplification cycle at which the fluorescence exceeds a defined

threshold level is known as the threshold cycle (Ct) (Corless and

others 2000). Data analysis software enables real-time calculationand plotting, eliminating the need for the post-amplification anal-

ysis of conventional PCR.

This ability to detect the presence of DNA throughout the entire

replication process, rather than just the end result, is one of the

main advantages of real-time over conventional PCR. Deviations in

amplification efficiency can be easily seen, and quantification is

much more precise (Schmittgen and others 2000). Furthermore,

real-time PCR offers a better platform for multiplexing, the detec-

tion of more than 1 target DNA fragment in a single reaction tube.

Simultaneous monitoring of multiple fluorophores and melting

curve analysisdetermining the temperature at which the indi-

vidual double-stranded DNA amplicons separate, or meltmake

this detection of multiple genes or multiple organisms possible.Finally, because many real-time PCR approaches involve the use of

a 3rd oligonucleotide that must also anneal to the target DNA se-

quence, they can offer improved detection specificity.

Because the amplification is monitored during the reaction, real-

time PCR can be used for quantification of the target as well as

basic detection. The higher the number of copies of the target DNA

that exist in the original sample, the earlier in the reaction that sam-

ples fluorescence will cross the threshold. The Ct produced from a

given sample can then be compared with a standard curve, gener-

ated from serial dilutions of a known amount of the target DNA, to

obtain the initial starting copy number (Ibekwe and others 2002).

Detection chemistriesDetection chemistriesDetection chemistriesDetection chemistriesDetection chemistries

Nucleic acid dyes.Nucleic acid dyes.Nucleic acid dyes.Nucleic acid dyes.Nucle ic acid dyes. Several types of real-time PCR assays have

been used in research and clinical settings. One type of assay uses

a fluorescent dye that binds to nucleic acids and returns a signal to

the optical module on the thermal cycler. One commonly-used flu-

orescent dye is SYBR Green I, which can be used in assays designed

to detect many different targets (Ramos-Payan and others2003).

SYBR Green I assays do not require a specific probe to be devel-

oped, as in some other assays.

SYBR Green I assays can provide useful quantitative informa-

tion, but the dye binds to any double-stranded DNA molecules in

the reaction, including nonspecific PCR products or primer dimers

(Missel and others 2001; Ramos-Payan and others 2003). False pos-

itives can arise if the PCR primers amplify any nonspecific products

other than the targeted sequence, although melting curve analy-

sis can help differentiate various products. The melting point of

double-stranded DNA increases with longer length and higher G-

C content. By analyzing the temperatures at which the DNA strands

separate, releasing the dye and therefore reducing the fluores-

cence, a distinction can be made between the desired amplicon

and any nonspecific products, or between different amplicons in a

multiplexed reaction (Wang and others 2004). For instance, Escher-

ichia coliO157:H7, Listeria monocytogenes, and Salmonella strains

in fresh produce have been detected simultaneously using SYBR

Green I and melting curve analysis (Bagwhat 2003). SYBR Green I

assays ultimately provide a relatively inexpensive and fairly sensi-

tive method for detecting double-stranded DNA (Missel and others

2001; Malinen and others 2003) and can even incorporate existing

primers already in use for conventional PCR assays (Bagwhat 2003,

2004). Recently, SYBR Green I was added to the reaction mix of the

original commercially available BAX test for Salmonella, successfully

transforming this conventional PCR test into a more rapid real-

time method (Bagwhat 2004).

Molecular beacons.Molecular beacons.Molecular beacons.Molecular beacons.Molecular beacons. Another permutation of real-time PCR is the

implementation of molecular beacons, which are single-stranded

oligonucleotide molecules between 25 and 35 bases in length

(Pierce and others 2000). Five to eight bases on the 3 and 5 ends

must complement, forming an intentional hairpin loop secondarystructure. The theory of this chemistry is to incorporate a quencher

dye on the 3 end and a fluorescent dye on the 5 end; when the 2

dyes are near each other, the quencher absorbs or modifies the sig-

nal produced by the fluorophore. When the beacon binds to an

external complementary sequence, the internal hairpin structure

is flattened out and emission from the fluorescent dye is recorded

by the optical module (Pierce and others 2000).

The development of molecular beacons can be more difficult than

other types of probes because even a single base pair mismatch can

prevent detection by the system. If such a mismatch occurs, the

beacon will remain in the more thermodynamically stable hairpin

loop instead of binding to the template (Tyagi and Kramer 1996).

This property of molecular beacons can be used to create extremelyspecific assays that other types of probes may not achieve.

TTTTTaqMaqMaqMaqMaqMan pran pran pran pran probesobesobesobesobes..... The TaqMan real-time PCR assay can overcome

some of the pitfalls of SYBR Green I and the lack of flexibility of

molecular beacons. The TaqMan-based assay includes a fluorogen-

ic probe (a 3rd oligonucleotide) that binds specifically to the ampl-

icon. TaqMan probes are designed with the fluorescent reporter

dye on the 5 end and the quenching dye on the 3 end. When the

probe anneals to the amplicon, the 5 exonuclease activity of the

DNA polymerase cleaves the probe. This step frees the 5 reporter

dye, which prevents the quencher dye from masking the fluores-

cence and allows the optical module to record emission (Giuletti and

others 2001).

One advantage of the TaqMan assay is added specificity over

SYBR Green I assays because the probe will only bind to the de-

sired sequence within the amplicon. Unlike molecular beacons,

TaqMan probes can bind to template DNA containing minor base

pair mismatches, although with reduced efficiency. If the concen-

tration of mismatched products is high, signals generated from

them can be detected as well.

OOOOOther chemistrther chemistrther chemistrther chemistrther chemistriesiesiesiesies..... A fairly new type of chemistry, intended to

decrease the time needed to generate a fluorescent signal and to

improve reliability, is the scorpion probe. This technology incorpo-

rates the primer and probe as a single unit, with a quencher and a

fluorophore held in close proximity similar to the molecular beacon.

This also creates a more thermodynamically stable product during

the PCR reaction than with the separate primers and probe ap-

proach. During the PCR process, the primer end begins replication
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of the amplicon; the probe end then attaches to this new amplicon

after separation from the template DNA during the following melt-

ing phase. When the probe hybridizes with the amplicon, the fluo-

rescent marker is separated from the quencher and a signal is gen-

erated (Whitcombe and others 1999).

Yet another type of chemistry, hybridization probes use 2 differ-

ent labeled probes, or a labeled probe and labeled primer, for each

amplicon, one with a donor fluorophore and one with a receptor

fluorophore. These oligonucleotides are designed to lie head-to-tail

when annealed; in this configuration the donor transmits energy to

the receptor and the signal is generated (Giuletti and others 2001).

At melting temperatures, the probes separate from the template,

and the signal is eliminated until the next annealing step (Bernard

and Wittwer 2000). This chemistry holds promise for increasing the

multiplexing capabilities of real-time PCR because in addition to

using differing fluorescent markers, melting point analysis can be

performed to distinguish slightly differing DNA sequences (Bellin

and others 2001).

ApplicationsApplicationsApplicationsApplicationsApplicationsDue to the complexity of ingredients involved in food samples,

the applicability of real-time PCR in microbial detection needs to

be verified, and sample preparation procedures need to be opti-mized for each food commodity. In the past few years, a number of

DNA primers and probes specific for detecting certain foodborne

microorganisms have been developed, and sample preparation

procedures involved in detecting these organisms in certain types

of food have been reported. These studies are essential in evaluat-

ing the feasibility of implementing real-time PCR detection sys-

tems for food industry applications.

Taqman chemistry has been a popular choice in real-time PCR

detection because of its improved specificity, while still maintaining

flexibility in primer and probe design. The prominent foodborne

pathogens belonging to the genus Salmonella have been targets of

real-time PCR studies. UsinginvA gene specific TaqMan primers-

and-probe, Rodriguez-Lazaro and others (2003) reported 100%accuracy in detection of Salmonellae. This method was also more

convenient than traditional culture methods. The foodborne

pathogen L. monocytogeneshas also been a target organism for

real-time PCR detection. Hein and others (2001) developed an as-

say for milk targeting the iap gene ofL. monocytogenesand Listeria

innocua, enabling the specific detection of as few as 6 copies of the

target gene from the 2 organisms.

TaqMan chemistry has also been used for the detection ofE. coli

O157:H7. A study targeting both the stx1 and stx2shigatoxin-pro-

ducing genes was able to detect 10 colony-forming units (CFU)/g in

soil following a 16-h enrichment (Ibekwe and others 2002). Another

assay multiplexed stx1 and stx2along with a uidA sequence unique

to the O157:H7 strain, and achieved 98.6% sensitivity and 100%

specificity for this pathogen (Jinneman and others 2003). Detection

levels in this study were as low as 6 CFU/g. Vibrio choleraehas been

detected in raw oysters, with the detection of 6 to 8 CFU/g (Lyon

2001). This probe was tested on 60 bacterial strains from 21 differ-

ent genera and achieved 100% specificity for V. cholerae. An assay

has also been developed to detect Yersinia enterocolitica in raw meats

and tofu (Vishnubhatla and others 2001). This assay was capable

of detecting 102 CFU/mL in pure culture and 103 CFU/g in ground

pork; conventional culture methods detected only 105 CFU/mL and

106 CFU/g, respectively. Because the target sequence was the en-

terotoxinystgene, the assay could quickly and accurately identify

virulent strains.

Two Taqman real-time PCR assays have also been developed in

our laboratory to detect the presence of spoilage microorganisms.

A primer-and-probe set was developed targeting the shcgene en-

coding squalene-hopene cyclase, a key enzyme in hopanoid bio-

synthesis. Hopanoids are involved in maintaining membrane flu-

idity and stability in extreme environmental conditions. Using this

primer-and-probe set, the presence of spoilageAlicyclobacillusspp.

can be detected without cross-reactivity with other common food-

borne bacteria (Luo and others 2004). Using a primer-and-probe

set developed targeting the 16S rRNA-encoding gene, the presence

of all 7 species ofAlicyclobacillusand a few closely related thermore-

sistant bacteria can be detected by real-time PCR (Connor and oth-

ers 2005). In both cases, the presence of less than 100 cells/mL in

juice products can be directly detected without enrichment proce-

dures. The whole detection process can be completed within 5 h.

The OSU CleanPlant rapid detection system containing multiple

components for spoilage and pathogenic bacteria as well as molds

and yeasts is currently patent pending.

SYBR Green I assays have also been used for foodborne patho-

gen detection. As previously noted, the dye can be used in conjunc-

tion with primers designed for conventional PCR to create efficient

real-time assays (Bagwhat 2003, 2004). Listeria and Salmonella have

also been detected using multiplex real-time PCR at levels of 1 cell

per PCR reaction using an overnight (16 h) enrichment process

(Jothikumar and others 2003). This group also detected Listeria andSalmonella in sausage using multiplex real-time PCR, with sensitiv-

ities of 3 and 4 CFU/g, respectively, following an overnight enrich-

ment step ( Wang and others 2004). These 2 studies used melting

curve analysis to differentiate the 2 species.

The 1st use of molecular beacons in the food microbiology arena

was to detect E. coliO157:H7 in milk (McKillip and Drake 2000).

Molecular beacon technology has also been used to detect Sal-

monella in a variety of fresh fruits and vegetables (Liming and Bag-

what 2004), with the ability to detect 1 to 3 CFU/25 g in these prod-

ucts, and cut the detection time from 3 to 4 d for conventional

methods to 18 h.

Finally, hybridization probes have been used recently in a food

matrix. A real-time PCR study targetingSalmonella in raw andready-to-eat meat products provided a detection level of 1 to 10

CFU/g, markedly better than the 103 CFU/g obtained by the tradi-

tional culture and enzyme immunoassay (EIA) methods currently

used (Ellingson and others 2004). Total time for detection, including

a 6-h enrichment step, was 12 h; the EIA test requires 48 h to

achieve presumptive positive results. Because this study used

hybridization probes, confirmation of results could be achieved

with melting curve analysis.

Several commercially available real-time PCR assays targeting

foodborne pathogen detection have recently obtained Performance

Tested Methods status from the AOAC Research Institute. At the

time of this writing, these include the BAX System assays for L.

monocytogenes, Salmonella, and E. coliO157:H7 (Dupont Qualicon,

Inc., Wilmington, Del., U.S.A.), the Roche Diagnostics Lightcycler

Salmonella Detection Kit (Roche Applied Science, Indianapolis, Ind.,

U.S.A.), and, most recently, the Genevision Rapid Detection Systems

forE. coliO157:H7, Salmonella, L. monocytogenes, andListeria species

(Warnex Diagnostics, Laval, Quebec, Canada) (AOAC Intl. 2004). It is

important to note that each assay is approved only for specific food

matrices and as such may not be appropriate for all products. Also,

regulatory agencies such as USFDA, USDA, and AFNORgenerally re-

gard these as screening tests; presumptive positives must be fol-

lowed up with conventional detection methods.

TTTTTechnical challengesechnical challengesechnical challengesechnical challengesechnical challengesSelection of a target gene and development of specific primers

and probes are critical factors to achieve the desired detection spec-


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ificity. Targeting a gene highly conserved among different species

can be used for a broad-based detection strategy, while targeting a

DNA sequence unique to a particular species or strain can produce

a highly specific test. Specific primer and probe development nor-

mally involves 1st identifying a target gene based on the ultimate

goal of the detection and the understanding of the uniqueness of

biological functions of various macromolecules in the organisms.

Then DNA sequences of the target gene from various organisms

will be aligned. Two to three regions that are conserved within the

group of organisms to be detected, yet distinct from the back-

ground microflora, need to be identified. DNA primers and probe

will be derived based on these conserved sequences. The se-

quence composition and the relative positions of these oligonucle-

otides in the genome have to fulfill the technical requirements for

the specific detection chemistry, that is, the matching of the melt-

ing temperatures of the primers and probe, the lengths of the oli-

gonucleotides and the amplification products within the required

range, and so forth. The developed primers and probe further need

to be tested for detection specificity with real microorganisms. In

our lab, we were able to detect closely related thermophilic spoilage

organisms of both GeobacillusandAlicyclobacillusspp., using the

same set of primers and probe targeting a 16s rRNA gene sequence

(Connor and others 2005). However, detection specificity can be lostwhen a highly conserved region is used to design primers and

probes to detect a specific organism. One recent study detecting

environmental molds using species-specific primers and probes

found that a number of the assays also detected closely related

species (Haugland and others 2004). Because the DNA sequences

were not exact matches, the efficiency of the PCR reactions was

greatly diminished and high levels of the alternate organisms were

required to produce these false-positive signals. These examples

point out the importance of carefully selecting the DNA sequences

used to design primers and probes to achieve the desired results.

This can be particularly important in food samples that may con-

tain relatively high levels of background microflora.

Multiplexing is a preferred feature of real-time PCR, but thereare also limits in how many DNA sequences can be analyzed in a

given sample. Multiplexing can be performed with SYBR Green I

technologies using melting point analysis; however, a precise

knowledge of each amplicons melting point is needed, and the

melting points must be far enough apart to be distinguishable.

TaqMan assays rely on different colors of fluorescent markers for

multiplexing, but the colors must be widely separated in the visible

spectrum. Current commercial technologies can differentiate 3 or

4 colors of dye, but from a practical standpoint, up to 12 oligonucle-

otides (8 primers and 4 probes) need to be tested for compatibility,

and up to 4 PCR reactions need to be optimized for similar amplifi-

cation efficiencies to produce a reliable multiplexed reaction. Mo-

lecular beacons, scorpions, and hybridization probes can make use

of both melting curve analysis and different color fluorescence

during the same reaction because unlike the TaqMan probe, these

fluorescent probes are not cleaved (Bellin and others2001; Wittwer

and others 2001). The problems with optimization and multiple re-

action efficiencies, however, still remain.

Another key factor affecting PCR efficiency, both conventional

and real-time, is the DNA extraction technique. A study using

known amounts ofCryptosporidiumoocysts in fecal samples found

that using glass beads to disrupt cells and release the DNA provid-

ed 100% sensitivity, whereas using a freeze-thaw cycle with liquid

nitrogen provided only 83% sensitivity (Lindergard and others

2003). Cheng and Griffiths (2003) reported that the detection lev-

els ofCampylobacter jejuniin chicken carcass washes ranged from

104 to 102 CFU/mL, depending on which of the 5 different cell lysis

methods was used. We have used the commercially available

Qiagen DNeasy Tissue Kit (Qiagen, Valencia, Calif., U.S.A.) for DNA

extraction, and the detection of less than 100 cells/mL sample was

achieved.

A problem inherent to all PCR methods is the presence of factors

that inhibit nucleic acid synthesis by the polymerase enzyme. Such

inhibitory factors can be found in foods, culture media, and various

chemical compounds, including those used to extract DNA (Rossen

and others 1992), emphasizing the importance of careful sample

preparation. Adding substances that block these inhibitors has

been reported to improve detection sensitivities (Grant 2003). Us-

ing a control sample with a known amount of DNA can be used to

measure and adjust for reaction inhibition (Rijpens and Herman

2002).

Polymerase chain reactions also cannot distinguish between live

and dead cells. DNA can be rather resistant to degradation and may

be present for some time after the death of its host cell. This can be

a problem in a food matrix, where processing may destroy a bacte-

rial cell but leave its DNA relatively intact. Reverse-transcription

PCR (RT-PCR), which detects RNA rather than DNA, can overcome

this problem, but handling RNA is inherently more difficult than

DNA. RNase, the enzyme that digests RNA, is ubiquitous in the

environment, making RNA very short-lived unless great care is tak-en when extracting and handling it. Also, an extra step is needed to

convert the RNA to DNAthe reverse transcriptionbecause DNA

is the template needed for a PCR reaction. These factors make RT-

PCR somewhat impractical for a commercial food application.

Finally, food samples tend to be less homogeneous and contain

lower levels of target organisms than clinical samples, making

proper sampling essential. Rather than pushing the technical de-

tection limit of real-time PCRthat is, the ability to detect 1 cell in

1 mL or 1 g of sampleit is more meaningful to incorporate a short

enrichment procedure or to concentrate the target organism from

a relatively large sample size, thereby improving detection sensitiv-

ity and achieving accurate analysis of food samples. Filtration,

immunomagnetic bead capture, and centrifugation are methodsthat have been used to concentrate bacteria in diluted samples

and separate them from the food matrix (Fratamico 2001). As noted

in several of the previous examples, however, enrichment periods

and post-enrichment procedures for PCR analysis can be much

shorter than those needed for conventional methods because the

purpose here is just to have enough cells to ensure that at least 1

DNA template will be included in the PCR reaction (Jothikumar

and others 2003; Ellingson and others 2004; Liming and Bagwhat

2004).

Conclusions

S

tudies such as these point out the promise that real-time PCR

holds for the detection of foodborne pathogens and spoilage

microbes. An organism that previously took days or weeks to cul-

ture, isolate, and identify might now be detected in a matter of

hours. Reactions can be multiplexed so that several targets can be

detected in the same reaction, further reducing the time and labor

needed. The ability to run 96 or more samples simultaneously and

to see results without any post-amplification processing also

makes real-time PCR much more user-friendly than standard PCR.

With relatively brief enrichment, bacteria have been detected

down to a single CFU/g. This tremendous sensitivity can be partic-

ularly useful in finding zero tolerance organisms in food such as

E. coliO157:H7 and L. monocytogenes. And if there is a question as

to the viability of cells detected, real-time PCR can be used as a

rapid screening test, followed up by confirmation with conventional

methods for presumptive positives.


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Real-time PCR still cannot completely replace conventional de-

tection methods because of its current technical and regulatory lim-

itations. However, with careful assay design and knowledge of these

limitations, it can be a powerful tool in food microbiology. Its abil-

ity to reduce the time to detect organisms can free up lab person-

nel to perform other tasks, increasing the throughput of laboratory

testing and the efficiency of quality assurance programs. Also, the

rapid screening of samples can allow earlier release of product, free-

ing valuable warehouse space and allowing food to be marketed

earlier in its shelf-life.

In conclusion, despite some limitations, real-time PCR shows

great promise as a tool for the food microbiologist to use in improv-

ing quality assurance and food safety. It can play a valuable role in

the rapid detection of pathogenic and spoilage organisms, and its

usefulness should only increase as the technology continues to

mature.

AcknowledgmentsThe authors thank Drs. Steve Schwartz and Ahmed Yousef for help-

ful discussions. The authors also acknowledge the Ohio Agricultural

Research and Development Center for providing partial funding for

the iCycler. Related research projects on real-time PCR detection

applications are sponsored by the Ohio State Univ. start-up fund,the Center for Innovative Food Technology, and the Center for

Advanced Food Processing and Packaging for author H.H. Wang.

The Wilbur A. Gould Departmental Fellowship offered partial sup-

port for author C.J. Connor. Author S.E. Hanna is sponsored by the

Dept. of Health Education and Training, Army Medical Dept. Cen-

ter and School, Fort Sam Houston, Texas.

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