P-000. Rapid Method of Detecting Listeria genus, Salmonella genus, and Campylobacter using Real Time Transcription-Mediated Amplification Assays Targeted to Ribosomal RNA

M. Reshatoff, E. Ong, J. Ritter, J. Garcia, C. Motta, C. Lewis, M. Fullerton, M. Deras, A. Eusebio, K. Pekny, N. Hedrick, S. Lee, S. McDonough, J. J. Hogan;
Gen-Probe Incorporated, San Diego, CA.

Background: Listeria is a common indicator for uncleanliness, while Salmonella and Campylobacter are responsible for major food borne diseases. Current identification by USDA methods can take 48-72 hours. This study describes the performance characteristics of three Gen-Probe research prototype Real Time Transcription-Mediated Amplification (TMA) assays for detecting Listeria, Salmonella, and Campylobacter from bacterial pellets. Studies on poultry rinsates for Campylobacter, and two spiked food matrices for Listeria and Salmonella are presented. Methods: Assay specificity [~1E4 colony forming units (CFU)] and sensitivity (1E5 copies rRNA) were evaluated using lysed bacterial pellets. CFU and rRNA target levels of these bacterial pellets were estimated by plating and by a direct DNA probe assay. Two food matrices, ice cream and ground beef (25 g), were inoculated with either L. monocytogenes or S. Enteritidis (~20 CFU) and processed through a Stomacher® device in broth. Plating and the Real Time TMA monitored a 24-hour time course. The Campylobacter assay was performed directly on aliquots of poultry rinsate without enrichment or pelleting. The Real Time TMA systems utilize two fluorescent probes, one specific for the analyte, one specific for an internal control.. The results were analyzed based on fluorescence emergence curves. Results: The Listeria assay was 100% sensitive to 7 species of Listeria and 100% specific against B. thermosphacta, E. rhusiopathiae, and 8 common food contaminants. The Salmonella assay was 100% sensitive to 22 strains of Salmonella including 6 different subspecies and 100% specific against 22 non-Salmonella organisms. The Campylobacter assay was 100% sensitive to 24 strains of C. jejuni, C. coli, or C. lari and 100% specific against 6 strains of other Campylobacter species and 16 non-Campylobacter common contaminants in poultry. A sensitivity of 5E3 copies rRNA in direct rinsates yielded 20/20 positives that were missed by culture. Conclusion: These rapid Real Time TMA assays can be run in less than four hours, reducing the time needed for testing in food and manufacturing facilities from days to hours.