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United States Department of Agriculture

Agricultural Research Service

Title: Simultaneous Detection of Salmonella and Escherichia Coli O157:h7 Using Fluorogenic Pcr and Single Enrichment Broth

Authors
item Sharma, Vijay
item Carlson, Steven

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 11, 2000
Publication Date: N/A

Interpretive Summary: Livestock are an important source for transmission of many foodborne pathogens, such as E. coli O157:H7 and Salmonella, to humans. Facilities engaged in production and processing of livestock for foods are the critical control points for reducing the transmission of these pathogens to humans. Methods with potential for rapid and sensitive detection of multiple bacterial pathogens at these control points are crucial to ensure pathogen-free food to consumer and to monitor the presence of these pathogens in livestock. The objective of our study was to develop and evaluate a single test that rapidly detects O157:H7 and Salmonella in meats and feces. A fluorogenic PCR test was developed that allowed simultaneous detection of very low levels (1 to 10 bacterial cells) of pathogenic Salmonella and E. coli O157:H7 in meats and feces. In addition, the automated PCR amplification and detection capability of this test is conducive for testing large number of samples in a single assay. This method, therefore, should be a significant tool in monitoring the presence of these bacteria in livestock and in foods of animal origin and in facilities used in the production and processing of livestock into foods.

Technical Abstract: Livestock are an important source for many human foodborne pathogens. Facilities engaged in production and processing meat animals for food are critical control points for reducing the transmission of bacterial pathogens to humans. Methods with the potential for rapid and sensitive detection of multiple pathogens are crucial to ensure pathogen-free food to consumers and to monitor the presence of these pathogens in natural reservoirs. A multiplex fluorogenic PCR assay for simultaneous detection of pathogenic Salmonella strains and Escherichia coli O157:H7 was developed and evaluated for detecting very low levels of these pathogens in meat and feces. Two sets of primers were used to amplify a junctional segment of virulence genes sipB and sipC of Salmonella and an intragenic segment of the gene eae of E. coli O157:H7. Fluorogenic reporter probes were included in the PCR assay for automated and specific detection of amplified products. This assay correctly detected all O157:H7 and pathogenic Salmonella strains and did not detect any of the non-Salmonella or non-O157:H7 strains examined. The assay could detect < 10 CFU of Salmonella typhimurium or E. coli O157:H7 per gram of meat or feces artificially inoculated with these pathogens and cultured for 6 to 18 h in a single enrichment broth. Detection of amplification products could be completed in </= 4 h after enrichment. The data indicate that this multiplex fluorogenic PCR assay has the potential for rapid and automated detection of multiple pathogens in a single PCR reaction from complex matrices, such as meat and feces.

Last Modified: 4/20/2014
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