IAFP 2009 Program Book and Abstract Book - AB115

Purpose: The purpose of this study was to develop a 24 or 48 hour procedure following USP guidelines and testing using the 3M™ Tecra™ Staphylococcus aureus Visual Immunoassay, comparing this to the USP method for the detection of S. aureus in raw and finished dietary supplements. Methods: Twelve different raw and finished dietary supplements were artificially contaminated at a low and zero level with S. aureus ATCC 6538. Each sample was enriched for 24 ± 2 and 48 ± 2, tested for the presence of S. aureus using the 3M™Tecra™ Staphylococcus aureus VIA kit, and compared to the Standard USP detection method for S. aureus. Results: The data suggests that an initial 1:10 dilution of each sample that is incubated for 24 ± 2 h, followed by another 1:10 dilution and an additional incubation, for a total of 48 ± 2 h, showed the best recovery of the inoculated samples. Ten of the samples tested showed exact agreement between the 3M™ Tecra™ Staphylococcus aureus VIA and the cultural method for the detection of S. aureus. Two of the products showed no recovery of the inoculum by either the 3M™ Tecra™ Staphylococcus aureus VIA and the cultural method. Both of these products have known antimicrobial properties. Further dilution may be required. Significance: 3M™ Tecra™ Staphylococcus aureus Visual Immunoassay is an easy to use recommended alternative method for the detection of S. aureus and meets the USP requirements for detection of S. aureus in artificially contaminated dietary supplement products. P2-31 Comparison of Conventional Culture Method and Real-time PCR for Detection of Staphylococcus aureus DSC in Foods JAE-HOON LEE, Ji-Yeon Hyeon, Jeong-Hwan Cheon, Yun-Gyeong Kim, Kwang-Young Song, In-Gyun Hwang, Hyo-Sun Kwak and Kun-Ho Seo Konkuk University, College of Veterinary Medicine, Dept. of Public Health, Gwangjin-gu, Korea, South Introduction: Staphylococcus aureus is one of the most commonly foodborne pathogens, widely distributed on nature and associated to general infection and foodborne outbreaks. Various detection methods have been used for detection of S. aureus from contaminated foods. Therefore validation of various detection methods is required in public health. Purpose: The purpose of this study was to compare the conventional culture method and real-time PCR for detection of S. aureus in artificially inoculated foods. Methods: The bulk food samples (500 g) were artificially inoculated with S. aureus. Inoculation levels were 4 and 18 CFU/500 g in sausage and 14 and 104 CFU/500 ml in milk and 1200 and 1240 CFU/500 g in vegetable salad to generate partial positive samples. The inoculated food samples were then divided into 20 samples (25 g or ml each), and three negative and one positive control were included in each experiment. All samples were added to mTSB (225 ml/sample) with 10% NaCl and incubated at 36°C for 24 h. After the enrichment, broth cultures were streaked onto a selective medium, Baird Parker Medium with egg yolk tellulite, and incubated at 36°C for 24 h. In addition, 1 ml of broth cultures was collected to be tested by real-time PCR using a commercial kit that detects femA gene. Two suspicious colonies from the selective media were picked up and plated on Tryptic Soy Agar with 0.6% yeast extract (TSAYE) and incubated at 36°C for 24 h followed by coagulase confirmation test. Results: In sausage, the number of positive samples detected was similar by culture method and with the real-time PCR, 25/40 (62.5%) and 24/40 (60%), respectively. In milk, the culture method and real-time PCR detected S. aureus equally well, 27/40 (67.5%) and 25/40 (62.5%), respectively. However, in vegetable salad, the numbers of positive samples detected by conventional culture methods were higher than the real-time PCR, 37/40 (92.5%) and 24/40 (60%), respectively. In addition, negative control resulted in false positive by the culture method, but was defined to negative by real-time PCR. Significance: This study shows that real-time PCR is a more sensitive and specific detection method, and the culture method requires higher selectivity to exclude false positive result in food samples with high levels of background flora such as in vegetable salad. P2-32 A toxR-based Loop-mediated Isothermal Amplification Assay for Detecting Vibrio parahaemolyticus DSC SIYI CHEN and Beilei Ge Louisiana State University, Dept. of Food Science, Baton Rouge, LA, USA Introduction: Vibrio parahaemolyticus is an important foodborne pathogen widely present in warm coastal and estuarine waters and shellfish worldwide. A large number of seafood-associated outbreaks due to V. parahaemolyticus have been reported. Sensitive and specific detection methods are needed to better control V. parahaemolyticus infections associated with seafood. Purpose: This study aimed at developing a highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay targeting on the toxR gene of V. parahaemolyticus and comparing it with a previously described LAMP assay targeting on the V. parahaemolyticus thermolabile hemolysin (tlh) gene. Methods: Firstly, a set of four LAMP primers, two outer and two inner, targeting on the V. parahaemolyticus toxR gene were designed and optimized. Secondly, the specificity of the assay was evaluated using 18 V. parahaemolyticus strains and 32 other strains. To test for the assay sensitivity, serial dilutions of a V. parahaemolyticus culture ranging from 1.2 × 106 cell to extinction per reaction were tested. Finally, comparison between the toxR-based LAMP and tlh-based LAMP was conducted for both sensitivity and specificity. Results: Both toxR- and tlh-based LAMP assays were able to specifically detect the 18 Vibrio parahaemolyticus strains with no false positives for other strains. The detection limit observed was 1.2 × 102 cells for the toxR-based LAMP, compared with 1.2 cell for the tlh-based LAMP . Significance: The toxR-based LAMP assay developed in this study for detecting V. parahaemolyticus was sensitive and specific, and holds great potential for future field applications. 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IAFP 2009 Program Book and Abstract Book

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