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Real-time Polymerase Chain Reaction for the Food Microbiologist: Technologies, Applications, and Limitations
S COT T E. HANNA, CHRIST OP HER J. CONNOR, AND HUA H. W ANG HRISTOP OPHER ABSTRA CT :R apid detection of pathogenic and spoilage micr oor ganisms is essential for ensur ing the safety and ABSTRACT CT: Rapid microor oorganisms ensuring quality of food. Real-time polymerase chain reaction (PCR) technology has the potential to achieve rapid, sensitive, and specific detection of these micr oor ganisms in food. I n this r eview ,w e discuss r eal-time PCR technologies in microor oorganisms In review eview, we real-time use today , applications of r eal-time PCR in food systems , and some of the associated challenges and limitations . today, real-time systems, limitations. Keywords: real-time PCR, rapid methods, food microbiology

Introduction

Conventional PCR
CR technology has been used to rapidly detect, characterize, and identify a variety of organisms (Campbell and Reece 1996). In a conventional PCR setting, a pair of oligonucleotide primers 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 replication 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 detected within 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; Makino and others 2001; Jensen and Whitfield 2003; Malinen and others 2003). PCR can be a powerful tool for analysis, but it has some shortcomings 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 temperatures, 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 complementing sequences can bind to each other and form primer dimers, which can be detected as a nonspecific amplification product (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, creatVol. 70, Nr. 3, 2005JOURNAL OF FOOD SCIENCE R49 Published on Web 3/17/2005

ontamination by foodborne pathogens is a great threat to human health; the estimated cost of foodborne illness in the United States is between $10 billion and $83 billion annually (USFDA 2001). In addition, food spoilage due to outgrowth of spoilage organisms costs the food industry significant amounts of money through the loss of raw material and finished product, product recalls, loss of sales due to reduced consumer confidence, and potential litigation. While sterilization could eliminate the presence of microorganisms in the final products, extreme processing conditions often cause undesirable physiochemical changes and loss of nutritional values of the food products that lead to consumer unacceptability. Therefore, achieving final product quality assurance through controlling the quality of raw materials and verification of the 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 challenges associated with microbial detection in foods. The initial contamination level in foods is normally low, and sampling with representation 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 limited shelf-life. Therefore, timely detection of these organisms with high degrees of specificity and sensitivity to maintain a safe, wholesome 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.

MS 20040446 Submitted 7/2/04, Revised 9/27/04, Accepted 12/12/04. The authors are with Dept. of Food Science and Technology, The Ohio State Univ., Columbus, Ohio 43210. Direct inquiries to author Wang (E-mail: wang.707@osu.edu).

2005 Institute of Food Technologists

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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). sis can help differentiate various products. The melting point of double-stranded DNA increases with longer length and higher GC content. By analyzing the temperatures at which the DNA strands separate, releasing the dye and therefore reducing the fluorescence, 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, Escherichia coli O157: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 sensitive 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 realtime method (Bagwhat 2004). 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 secondary structure. 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 signal 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 extremely specific assays that other types of probes may not achieve. TaqM an pr obes . The TaqMan real-time PCR assay can overcome aqMan probes obes. some of the pitfalls of SYBR Green I and the lack of flexibility of molecular beacons. The TaqMan-based assay includes a fluorogenic probe (a 3rd oligonucleotide) that binds specifically to the amplicon. 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 fluorescence 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 desired 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 concentration of mismatched products is high, signals generated from them can be detected as well. ies . A fairly new type of chemistry, intended to O ther chemistr chemistries ies. decrease the time needed to generate a fluorescent signal and to improve reliability, is the scorpion probe. This technology incorporates 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 approach. During the PCR process, the primer end begins replication
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Real-time PCR
Overview
Recent advancements in PCR technology include the development of real-time PCR devices and the application of new amplification product-detection chemistries. Real-time PCR adds an optical 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 calculation and plotting, eliminating the need for the post-amplification analysis 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 detection 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 individual 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 sequence, they can offer improved detection specificity. Because the amplification is monitored during the reaction, realtime 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 samples fluorescence will cross the threshold. The Ct produced from a given sample can then be compared with a standard curve, generated from serial dilutions of a known amount of the target DNA, to obtain the initial starting copy number (Ibekwe and others 2002).

Detection chemistries
Nucleic 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 fluorescent dye is SYBR Green I, which can be used in assays designed to detect many different targets (Ramos-Payan and others 2003). SYBR Green I assays do not require a specific probe to be developed, as in some other assays. SYBR Green I assays can provide useful quantitative information, 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 positives can arise if the PCR primers amplify any nonspecific products other than the targeted sequence, although melting curve analyR50

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Real-time PCR for the food microbiologist . . .

of the amplicon; the probe end then attaches to this new amplicon after separation from the template DNA during the following melting phase. When the probe hybridizes with the amplicon, the fluorescent marker is separated from the quencher and a signal is generated (Whitcombe and others 1999). Yet another type of chemistry, hybridization probes use 2 different 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). A primer-and-probe set was developed targeting the shc gene encoding squalene-hopene cyclase, a key enzyme in hopanoid biosynthesis. Hopanoids are involved in maintaining membrane fluidity and stability in extreme environmental conditions. Using this primer-and-probe set, the presence of spoilage Alicyclobacillus spp. can be detected without cross-reactivity with other common foodborne bacteria (Luo and others 2004). Using a primer-and-probe set developed targeting the 16S rRNA-encoding gene, the presence of all 7 species of Alicyclobacillus and a few closely related thermoresistant bacteria can be detected by real-time PCR (Connor and others 2005). In both cases, the presence of less than 100 cells/mL in juice products can be directly detected without enrichment procedures. 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 pathogen detection. As previously noted, the dye can be used in conjunction 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 and Salmonella in sausage using multiplex real-time PCR, with sensitivities of 3 and 4 CFU/g, respectively, following an overnight enrichment 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. coli O157:H7 in milk (McKillip and Drake 2000). Molecular beacon technology has also been used to detect Salmonella in a variety of fresh fruits and vegetables (Liming and Bagwhat 2004), with the ability to detect 1 to 3 CFU/25 g in these products, 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 targeting Salmonella in raw and ready-to-eat meat products provided a detection level of 1 to 10 CFU/g, markedly better than the 103 CFU/g obtained by the traditional 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. coli O157: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 for E. coli O157:H7, Salmonella, L. monocytogenes, and Listeria 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 AFNOR generally regard these as screening tests; presumptive positives must be followed up with conventional detection methods.

Applications
Due 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 optimized 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 evaluating the feasibility of implementing real-time PCR detection systems 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. Using invA gene specific TaqMan primersand-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. monocytogenes has also been a target organism for real-time PCR detection. Hein and others (2001) developed an assay for milk targeting the iap gene of L. monocytogenes and 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 of E. coli O157:H7. A study targeting both the stx1 and stx2 shigatoxin-producing 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 stx2 along 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 cholerae has 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 different 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 enterotoxin yst gene, 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.
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Technical challenges
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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 normally 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 background microflora, need to be identified. DNA primers and probe will be derived based on these conserved sequences. The sequence composition and the relative positions of these oligonucleotides in the genome have to fulfill the technical requirements for the specific detection chemistry, that is, the matching of the melting temperatures of the primers and probe, the lengths of the oligonucleotides 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 Geobacillus and Alicyclobacillus spp., using the same set of primers and probe targeting a 16s rRNA gene sequence (Connor and others 2005). However, detection specificity can be lost when 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 contain relatively high levels of background microflora. Multiplexing is a preferred feature of real-time PCR, but there are 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 oligonucleotides (8 primers and 4 probes) need to be tested for compatibility, and up to 4 PCR reactions need to be optimized for similar amplification efficiencies to produce a reliable multiplexed reaction. Molecular 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 others 2001; Wittwer and others 2001). The problems with optimization and multiple reaction 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 of Cryptosporidium oocysts in fecal samples found that using glass beads to disrupt cells and release the DNA provided 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 levels of Campylobacter jejuni in chicken carcass washes ranged from 104 to 102 CFU/mL, depending on which of the 5 different cell lysis
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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). Using 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 bacterial 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 taken 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 RTPCR 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 detection 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 sensitivity and achieving accurate analysis of food samples. Filtration, immunomagnetic bead capture, and centrifugation are methods that 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

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 culture, 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 particularly useful in finding zero tolerance organisms in food such as E. coli O157: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 detection methods because of its current technical and regulatory limitations. However, with careful assay design and knowledge of these limitations, it can be a powerful tool in food microbiology. Its ability to reduce the time to detect organisms can free up lab personnel 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, freeing 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 improving 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.
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Acknowledgments
The authors thank Drs. Steve Schwartz and Ahmed Yousef for helpful 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 support for author C.J. Connor. Author S.E. Hanna is sponsored by the Dept. of Health Education and Training, Army Medical Dept. Center and School, Fort Sam Houston, Texas.

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Vol. 70, Nr. 3, 2005JOURNAL OF FOOD SCIENCE

R53

R: Concise Reviews in Food Science