IAFP 2009 Program Book and Abstract Book - AB116

P2-33 Rapid Detection of Vibrio vulnificus in Oysters with Immunomagnetic Separation Real-time PCR Assay RAVIRAJSINH P. JADEJA, Janet Simonson and Marlene Janes Louisiana State University, Food Science, Baton Rouge, LA, USA Introduction: Vibrio vulnificus is considered one of the deadliest among all human pathogenic vibrios, with about 50% mortality rate. The majority of V. vulnificus infections in the United States are due to consumption of raw molluscan shellfish. The current conventional bacteriological detection methods are very labor intensive and time consuming. Real-time PCR (q-PCR) in combination with immunomagnetic separation (IMS) could be a rapid approach to detect V. vulnificus in complex environmental samples such as oyster meat. Purpose: The aim of this study was to develop and optimize an IMS q-PCR protocol that uses anti-H antibody to rapidly concentrate and quantify V. vulnificus from oyster homogenate. Methods: Monoclonal antibodies (MAbs) were used to prepare two different sets of immunomagnetic separation reagents at a concentration of 5 µg/107 immunomagnetic beads (IMB). Separation of V. vulnificus cells from the spiked oyster homogenate was achieved by incubating 500 µl of oyster homogenate with 107 MAb coated IMB on a shaker at 25°C for 30 min. The eluted V. vulnificus cells were then lysed by heating and assayed by q-PCR, using primers and probes derived from the V. vulnificus structural gene for cytolysin, vvhA. Results: IMB coated with MAbs 3-D-10 and 8-D-4 when coupled with qPCR were able to identify 42% and 37% V. vulnificus cells, respectively, in the oyster homogenate. The minimum detection limits were 1.7 × 103 and 2.3 × 103 cells/ ml of oyster homogenate respectively for the above stated antibodies. Significance: The IMS q-PCR can be employed to identify and quantify V. vulnificus from oysters in < 3 h, eliminating an enrichment step and separating target organisms from PCR inhibitors present in oyster tissue. P2-34 Rapid Capture and Detection of Model Viruses from Large Volumes of Water DSC TRAVIS STEINER and Lawrence Goodridge Colorado State University, Animal Science, Fort Collins, CO, USA Introduction: Foodborne viral infections caused by the noroviruses and Hepatitis A virus are mainly the result of viral contamination during food production due to infected food handlers, or food contact with sewage sludge or polluted water. Current methods to rapidly assess the presence of foodborne viruses in water used for food production are limited. Consequently, there is a need for development of rapid and sensitive methods for the capture and detection of foodborne viruses in large volumes (10 liters or more) of water. Purpose: The objective of this study was to evaluate an integrated sampling and detection method to rapidly assay for foodborne viruses in large volumes of water. Methods: FRNA bacteriophages MS2, GA, SP and Qβ were used in all experiments. These phages were chosen based on their differing isoelectric points, and their acceptance as surrogates of foodborne viruses in the scientific literature. Each phage was individually seeded into 10 liter volumes of tap water at a concentration of 100, 101, 102 and 103 PFU mL-1. One and a half grams of anionic exchange resin (Amberlite® IRA 900) was added to each water sample and kept in suspension by continuous agitation. Aliquots of water were withdrawn at 0 and 60 minute time points and assayed for phage via the double layer plaque assay. Virions bound to the resin beads were detected by isolating viral RNA directly off of the beads, followed by reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR using a SYBR Green assay. Results: The resin captured phages MS2, Qβ and SP with high efficiency (ranging from 86% (phage Qβ, 102 PFU mL-1) to 100% (phages SP Qβ, and MS2 (100 PFU mL-1). The capture efficiency of phage GA was lower and ranged from 55% (100 PFU , mL-1) to 89% (102 PFU mL-1). When RT-PCR and agarose gel electrophoresis was used to detect the phages the detection limits were 2.0 × 102 PFU mL-1 (SP), 4.0 × 101 PFU mL-1 (MS2), 5.5 × 100 PFU mL-1 (GA) and 5.7 × 102 PFU mL-1 (Qβ). Realtime RT-PCR detection limits were 1.0 × 101 PFU mL-1 (SP), 3.0 × 100 PFU mL-1 (MS2), 5.5 × 100 PFU mL-1 (GA) and 1.0 × 100 PFU mL-1 (Qβ ). Significance: These results suggest that anion exchange capture and real-time RT-PCR can be used as a rapid and sensitive assay for detection of foodborne viruses. This method would be especially useful to test water used during the production of foods that are consumed raw, such as produce and certain seafoods. P2-35 Evaluation of Repetitive Extragenic Palindromic Sequence-based PCR Typing of Bacillus Species SARITA RAENGPRADUB and Jacob K. Cannon Food Science Center, Silliker, Inc., South Holland, IL, USA Introduction: The application of molecular subtyping methods to bacterial foodborne pathogens provides the tools to detect and track outbreaks and contamination sources throughout the food system. Pulsed-field gel electrophoresis is often considered the “gold standard” for molecular subtyping, as the method offers a high level of discrimination; however, the drawback is that it is a time-consuming process that requires a high level of skill to perform. Automated subtyping methods for ribotyping and repetitive extragenic palindromic sequence-based PCR (rep-PCR) have emerged as approaches to rapidly discriminate bacterial strains. Purpose: The purpose of this study was to assess the utility of the DiversiLab System™, an automated rep-PCR-based typing system, to effectively differentiate species and strains of the genus Bacillus. Methods: A total of 79 Bacillus strains, representing 19 species, from the Silliker Laboratories Research Culture Collection were used in this study. Isolate DNA was extracted using the UltraClean™ Microbial DNA Isolation Kit, and DNA was amplified using the DiversiLab Bacillus strain typing kit. Rep-PCR products were separated and detected using microfluidics chips and the Bioanalyzer platform. Sample fingerprints were analyzed using the web-based DiversiLab software. 116 — Abstract Book Poster

IAFP 2009 Program Book and Abstract Book

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