IAFP 2009 Program Book and Abstract Book - AB33

Purpose: The main objective of this study was to develop a quantitative real-time PCR-based detection method for the rapid and sensitive detection of toxigenic C. difficile in food animals and retail meat products. Methods: Primers and dual labeled Taqman probes were designed specifically targeting an internal control topoisomerase gene (tpi), and tcdB gene coding for toxin B. Amplification was performed using purified DNA templates of C. difficile isolates with previously characterized toxin profiles. Quantification analysis and sensitivity of the method was measured by performing single- and multiplex reactions to generate standard curves using serially diluted purified plasmids containing each target. Results: We were able to correctly identify and detect C. difficile under multiplex conditions using the tpi and toxB probes. The detection threshold of the multiplex real-time PCR was 10 copies/reaction. We detected high correlation between single and multiplex reactions (r2 >0.96). The real time assay developed in this study had a very high sensitivity of 100% (20/20) for tpi and 91% (10/11) for tcdB genes. The overall specificity was 100% for both genes. The positive predictive power of the assay was 100% for both genes (20/20 and 10/10, respectively) while the negative predictive power was 100% for tpi (6/6) and 94% for tcdB (15/16). Overall, we have developed a method that will allow us to rapidly detect and quantitate C. difficile in different sources. Significance: The ability to quickly identify and assess the toxingenicity C. difficile using the above method is invaluable both for the rapid detection in food animals and retail meats as well as in the diagnosis of CDAD in humans. T3-04 ISO 16140 Validation of a Real-time PCR Method for the Simultaneous Detection of Escherichia coli O157:H7 and Salmonella spp. in Beef in Ten Hours PATRICE CHABLAIN and Sylvie Hallier-Soulier Pall GeneSystems, Bruz, France Introduction: The meat industry routinely releases products having a short shelf life (< 7 days), requiring very rapid analytical methods for safety control before consumers can get them in their plates. Because traditional bacteriological methods and most of the alternative methods cannot deliver analytical results in less than 24 to 48-h , we developed the GeneDisc DUO E. coli O157 and Salmonella spp. allowing the detection of both organisms in less than 10 h for raw ground beef products. The performance of this method had to be demonstrated through official validation schemes before it could be used for routine analysis by the food industry. Purpose: Two innovative multiplex PCR assays were developed and combined for the routine detection of Salmonella spp. and E. coli O157 STEC and the identification of EHEC in meat. This method was validated according to the ISO 16140 standard and was compared to ISO 6579 and ISO 16654 for Salmonella spp. and E. coli O157, respectively. Methods: Briefly, 25 g of food sample were enriched in BPW for 8 h at 41.5°C or for 16 h at 37°C. Bacterial DNA was extracted from the enrichment broth, then analyzed with a PCR-based technology allowing the amplification of specific sequences for (i) Salmonella spp (iroB), (ii) E. coli O157 (rfbE), (iii) stx genes, and (iv) eae gene. When the rfbE PCR and/or STEC PCR was (were) positive, the DNA extract was also further analyzed using a second PCR assay targeting the presence of the flagella H7 (flicH7) and of the main E. coli serovars (O26, O103, O111 and O145) that have been associated with HUS in humans. Results: Accuracies, specificities and sensitivities were evaluated for 70 meat samples. Results obtained using the Standard and GeneSystems methods were shown to be equivalent (α = 0.05). The limits of detection of PCR methods were 0.1 to 1.0 and 0.1 to 1.2 CFU/25 g for Salmonella and E. coli O157:H7, respectively. Finally, 50 target strains and 32 nontarget strains were analysed for the specificity study. The second part of the validation was a ring trial (12 independent laboratories) using ISO 16140 guidelines. Only three discordant assays were noticed amongst a total of 576 analyses. The variability of the PCR and ISO methods were shown to be equivalent. Significance: This multiplex PCR method was AFNOR approved, as it showed results equivalent to the Standard methods. It is an efficient tool, offering fast and reliable results for the routine onsite control of fresh meat products. In addition, the combination of multiparametric assays allowed the discrimination of pathogenic O157 using the detection of stx and eae virulence genes simultaneously to O157. T3-05 Independent Laboratory Evaluation of a Real-time PCR Test for Detection of Listeria spp. in Selected Foods from a Single Primary Enrichment LESLIE K. THOMPSON, Brian Kupski, Jeanette Franklin, Kenya Williams and Stephanie Sowell Silliker, Inc., Food Science Center, South Holland, IL, USA Introduction: Listeria spp. are found frequently in the food-processing environment and have been isolated from a variety of food products. Testing methods are available for the detection Listeria in foods and environmental surfaces, which take 2 days for a presumptive result. R.A.P .I.D.® LT Food Security System for Listeria is a rapid detection method which couples real-time PCR with a short enrichment time. The test method allows for the detection of a presumptive result from a food sample or environmental surface in less than 24 h, utilizing a single primary enrichment followed by detection using real-time PCR. Purpose: The objective of this study was to compare the performance of the test method, using either enrichments either alone or with pooling of multiple sample enrichments into one test sample, to that of established USDA reference methods for deli turkey and stainless steel surfaces. Methods: For deli turkey, twenty 25 g samples inoculated at < 1 CFU/g and 25 uninoculated control samples were analyzed, using the test method with and without pooling and by the appropriate reference method. In addition, a 3-tube MPN analysis was performed in order to determine the level of Listeria present in the deli turkey. For stainless steel coupons, twenty 25 g sponges samples taken from surfaces inoculated at < 1 CFU/g and 25 uninoculated control samples were analyzed, using the test method with and without pooling and by the appropriate reference method. Abstract Book — 33 Technical

IAFP 2009 Program Book and Abstract Book

Table of Contents for the Digital Edition of IAFP 2009 Program Book and Abstract Book