IAFP 2009 Program Book and Abstract Book - AB113

P2-25 Validation of the Reveal® 8-hour and 20-hour Methods for Detection of Escherichia coli O157:H7 in 375-g Beef Samples Susan Alles, Justina Kennedy, Alicia Rider, Michael Wendorf and MARK A. MOZOLA Neogen Corporation, Lansing, MI, USA Introduction: Lateral flow or immunochromatographic assays are routinely used by the beef processing industry in testing for E. coli O157:H7 contamination. A common approach is to composite up to 15-25 g test samples into one 375 g sample for analysis. This allows more testing to be done while controlling testing costs. While several methods, including the Reveal methods, have been extensively validated and have achieved AOAC method status, to this point validation of methods specifically for 375 g samples has been limited. Purpose: The purpose of this study was to validate the Reveal 8-hour and 20-hour methods for use with 375 g ground beef and beef trim samples and to compare performance of the Reveal methods with that of the USDA-FSIS reference culture procedure. Methods: Product was inoculated with low levels of E. coli O157:H7 to yield fractional positive data sets, i.e., a target of 5 to 15 positive samples out of 20 replicates. Beef was inoculated in bulk and then divided into 40 portions, 20 each for the Reveal and reference methods. Enrichment for the reference method was conducted according to the procedure in the FSIS Microbiology Laboratory Guidebook, except that a screening method was not used; all enrichments were plated for confirmation. The Reveal methods were performed according to the manufacturer’s instructions, using either 8-h or 20-h enrichment media as specified. As it was anticipated that an enrichment period longer than 8 h might be required, testing was performed at several time points, including 8, 10, 11 and 12 h. All samples were plated for confirmation. Results: The 20-hour Reveal and reference methods produced the same number of confirmed positives for the ground beef samples. For beef trim, the Reveal method produced more positives than the reference procedure, but this difference was not statistically significant. For the 8-h method, results statistically equivalent to the reference method were achieved after 8, 10 and 11 h of enrichment for ground beef and after 10 or 11 h of enrichment for beef trim, but maximum method sensitivity was only achieved after 12 h of enrichment. For both sample types, the Reveal methods produced more positives than the reference culture procedure, but in neither case were these differences statistically significant. Test specificity for both Reveal methods was 100%. Significance: Results of this study showed that the Reveal 20-h and 8-h methods (with extended enrichment) are highly sensitive and specific when applied to 375 g beef samples. The new procedures have been approved as modifications to AOAC Official Methods 2000.13 and 2000.14. P2-26 A New Immunoassay Method for the Simultaneous Detection of Escherichia coli O26, Escherichia coli O111 and Escherichia coli O157:H7 Cecile Sauvan, Jean-Michel Pradel, Christine Aguilhon and JEAN-LOUIS R. PITTET bioMerieux, R&D, Marcy L’Etoile, France Introduction: Even though enterohemorrhagic E. coli (EHEC) have been demonstrated at the origin of numerous foodborne outbreaks, current detection methods target only E. coli O157:H7. Purpose: The goal of this work was the development and preliminary evaluation of a new immunoassay for the simultaneous detection of 3 important EHEC serogroups, E. coli O26, O111 and O157:H7. Methods: The assay developed associates both monoclonal antibodies and recombinant phage tail proteins for capture and detection of the 3 serogroups. The target strains for the inclusivity study and the natural and artificially contaminated foods tested were culturally enriched for 24 h at 41.5°C in buffered peptone water containing cefixime, tellurite and acriflavine. Positive enrichment broths were immuno-purified using immuno-capture before streaking on the appropriate chromogenic or selective plates, to facilitate identification and confirmation. Results: Inclusivity : the 18 E. coli O26, 6 E. coli O111 and 18 E. coli O157:H7 strains tested were detected with a limit of detection between 5.103 CFU/ml and 5.104 CFU/ml. Exclusivity: no cross reactivity was observed using 30 non-target organisms enriched in buffered peptone water. 300 raw milk cheeses were tested with the new method and a positive result was obtained for approximately 5% of the samples. Confirmations of these samples isolated only E. coli O26 serotype. Further molecular studies were performed to identify any virulence factor. Significance: The new assay is a promising sensitive tool for the simultaneous detection of the three major serogroups E. coli O26, O111 and O157:H7 in food products within 24 h. P2-27 Prevalence of Shiga-toxin Producing Escherichia coli (STEC) in Edible By-products of Cattle Using Multiplex Real-time PCR JAE-HOON LEE, Ji-Yeon Hyeon, Jeong-Hwan Cheon, Kwang-Young Song, Hyo-Sun Kwak, In-Gyun Hwang and Kun-Ho Seo Konkuk University, College of Veterinary Medicine, Dept. of Public Health, Gwangjin-gu, Korea, South Introduction: Shiga Toxin-producing Escherichia coli (STEC) are recognized throughout the world as important zoonotic foodborne pathogens that cause bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). They rarely cause disease in animals, and live in the intestines of healthy ruminants, which are recognized as their main natural reservoir, so they can contaminate meat during slaughtering practices. Purpose: The purposes of this study were to determine the prevalence of STEC and isolate STEC from edible by-products of cattle using multiplex real-time PCR which detects stx1 and stx genes. Methods: A total of 82 samples were collected from omasum (n = 32), abomasum (n = 34), and large intestine (n = 16) purchased from a traditional beef market in Seoul, Korea. All samples (50 g each) were homogenized with mEC (450 ml/ sample) and incubated at 36°C for 24 h. For presumptive screening, the multiplex real-time PCR was performed to detect shiga toxin 1 and 2 (stx1 and stx2) in enriched mEC. Positive samples in the presumptive screening were then streaked on Abstract Book — 113 Poster

IAFP 2009 Program Book and Abstract Book

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