29G-31 |
Rapid, specific detection of Salmonella Enteritidis (SE) in pooled eggs using real time PCR method |
K. H. SEO1, R. E. Brackett1, I. E. Valentin-Bon1, P. S. Holt2, and K. Lampel1. (1) Center for Food Safety & Applied Nutrition, U.S. Food & Drug Administration, Office of Plant & Dairy Foods & Beverages, 5100 Paint Branch Pkwy., HFS-300, College Park, MD 20740-3835, (2) Airborne Disease Controls, USDA-ARS-Southeast Poultry Research Lab., 950 College Station Rd., Athens, GA 30605-2796 Although contamination of eggs with SE occurs sporadically, most of the cases have involved grade- A table eggs. To help address this problem, many egg producers implemented flock surveillance to detect the presence of SE at their facilities. However, the success of such surveillance would be improved by the availability of a rapid and simple method for detecting SE from poultry samples. We developed a rapid assay for the specific detection of SE in eggs using a novel application of the fluorogenic 5’ nuclease assay (TaqMan). In this assay, a segment of the gene sef14 specific to Salmonella group D strains, such as Salmonella Enteritidis and Salmonella Dublin, was amplified by polymerase chain reaction (PCR). The amplification of the target gene products was monitored in real-time by incorporating fluorescent dye-labeled gene-specific probes in the PCR reaction. This method correctly detected and distinguished SE from nearly 100 of non-group D Salmonella and other non-Salmonella strains. The contents of 3 egg pools of 220 eggs each were artificially contaminated with low concentrations of SE (8, 40, and 400 CFU per bulk pool, respectively) to form individual contaminated bulk egg pools. Twenty pools of approximately 10 eggs each were withdrawn from the bulk egg pool and allowed to incubate at 25C for 4 days. The test portions of 25 ml of the incubated eggs from each egg pool were incubated in tryptic soy broth for 24 h at 35C. The newly developed PCR method yielded a 100% correlation with results obtained using a conventional culture method. However, the PCR method required only 2 days, compared to the 5 days required by the cultural method. The sensitivity of this assay was 1 CFU per 600 g of egg pool. The real-time PCR assay proved to be a rapid, highly sensitive test for detection and quantification of low concentrations of SE in egg samples.
Session 29G, Food Microbiology: General
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