A simple method for the direct detection of Salmonella and Escherichia coli O157:H7 from raw alfalfa sprouts and spent irrigation water using PCR

摘要

The U.S. Food and Drug Administration recognizes that raw seed sprouts are an important cause of foodborne disease and is now recommending that either spent irrigation water or final product be screened for Salmonella and Escherichia coli O157:H7 as a means of assuring the safety of product intended for consumption. In an effort to streamline such testing efforts, a simple method to preconcentrate pathogens from sprouts and spent irrigation water was investigated to facilitate the direct (without prior cultural enrichment) detection of pathogens using the PCR technique. Alfalfa sprouts and spent irrigation water were seeded with Salmonella enterica serovar Typhimurium and E. coli O157:H7 at 10(-1) to 106 CFU/g or CFU/ml, respectively. Samples were blended (sprouts only) and then centrifuged at high speed to sediment the total bacterial population. The precipitate was processed for DNA isolation, PCR amplification, and amplicon confirmation by Southern hybridization. Mean pathogen recoveries after centrifugation ranged from 96 to 99% for both pathogens in both matrices. Using primers targeting the invA gene for Salmonella Typhimurium and the stx genes of E. coli O157:H7, it was possible to detect both pathogens in alfalfa sprouts at seeding concentrations as low as 10 CFU/g. PCR detection limits for both pathogens from spent irrigation water were 10(-1) CFU/ml, the equivalent of 100 CFU/liter of water. Because spent irrigation water is constitutionally simple, it is particularly well suited for bacterial concentration by simple centrifugation steps. In this study, progress was made toward development of a rapid, inexpensive, and sensitive method for the detection of sprout-associated pathogens that is relevant to current industrial practices and needs.