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item Bhagwat, Arvind

Submitted to: International Journal of Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/4/2002
Publication Date: 4/1/2003
Citation: Bhagwat, A.A. 2003. Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR. International Journal of Food Microbiology. 84:217-224.

Interpretive Summary: Detection of human pathogens from fresh produce is a crucial step in implementing food safety. Conventional methods may take up to one week to accurately predict the presence of human pathogens. Detection methods based on DNA analysis (such as polymerase chain reaction, PCR) are rapid, sensitive and highly specific. However, to perform individual tests for a number of different pathogens is time consuming and expensive. Considering the limited shelf-life of produce, rapid methods for pathogen detection are required. Simultaneous detection of more than one pathogen will broaden our ability to screen a large number of samples in a short time. In this study, PCR detection methods for three human pathogens, Escherichia coli O157:H7, Listeria monocytegenes, and Salmonella strains, approved by the Association of Official Analytical Chemists (AOAC), have been modified to enable near-instantaneous detection and quantitative analysis. The three tests can be performed simultaneously using one PCR protocol. Such a protocol will ensure accurate and economic surveillance of human pathogens in American food supply. Both the fresh produce industry and consumers will benefit from the results of this research.

Technical Abstract: A protocol enabling simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains was devised and evaluated using artificially-contaminated fresh produce. Association of Official Analytical Chemists (AOAC)-approved PCR detection methods for three human pathogens were modified to enable simultaneous and real-time detection with high throughput capability. The method includes a melting-curve analysis of PCR products, which serves as confirmatory test. The modified protocol successfully detected all three pathogens when fresh produce was washed with artificially-contaminated water containing E. coli O157:H7 and S. enterica serovar Typhimurium down to the predicted level of 1 to 10 cells/ml and L. monocytogenes at 1000 cells/ml. The ability to monitor several pathogens simultaneously will save time and increase our ability to assure food safety.