METHOD VALIDATION FOR THE DETECTION OF CLOSTRIDIUM DIFFICILE A AND B TOXINS IN FOOD AND ANIMAL SPECIMENS BY PCR ASSAY

PROJECT SUMMARY/ABSTRACTClostridium difficile is an important pathogen capable of causing disease in animals and humans. Theorganism possesses genes that encode for the production of toxins. These toxins are the main virulencefactor for C. difficile and are capable of causing pseudomembranous colitis and sometimes death. Thetargeting of these genes for amplification and detection by a real-time PCR assay allows for fasterdiagnosis of disease and treatment.The specific aim of validating a multiplex, real-time PCR assay for the detection of A and B toxins fromClostridium difficile is to improve laboratory detection and turnaround time in samples from infectedanimals and contaminated food/feed. A multiplex, real-time PCR assay is capable of detecting multiplesequences in a limited time by fragment amplification as opposed to traditional culture methods thatrequire the growth and isolation of Clostridium difficile followed by toxin production induction andconfirmation of the presence of A and B toxins. These culture methods can take days to weeks tocomplete while a multiplex PCR assay is performed within hours. The rapid turnaround time of the PCRassay and the ability to batch testing increases the number of assays that can be performed at one time,increasing laboratory surge testing capacity in the face of an outbreak. The PCR assay can also be usedto detect the presence of toxin in multiple matrices so several sample types can be tested at the sametime.Once the assay is validated for use at PADLS New Bolton Center, the longer-term objective is to utilizethe assay for both FDA Vet-LIRN and Food Emergency Response Network (FERN) investigations androutine diagnostic submissions. The currently-available test is not approved for use in all animal speciesand all matrices so a better method of detection is needed.The assay to be validated for this project was previously published by Houser et al 2010. Our project?smatrices will include the sample types most often submitted in routine diagnostics and typical outbreakinvestigations: feces, dog and cat food, livestock feed, and food products routinely consumed byhumans. PCR detection results will be compared against the current gold standard, toxigenic culture,performed on the same samples. Naturally-infected/contaminated samples and spiked samples will beused for the validation.