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United States Department of Agriculture

Agricultural Research Service

Title: Semi-Automated Fluorogenic Pcr Assays (Taqman) for Rapid Detection of Escherichia Coli O157:h7 and Other Shiga Toxigenic E. Coli

Authors
item Sharma, Vijay
item Nystrom, Evelyn
item Casey, Thomas

Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 13, 1999
Publication Date: N/A

Interpretive Summary: Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) are important agents of severe diarrheal disease in infected animals (calves and swine) and humans. These bacteria are commonly found in the intestines of cattle and are excreted in feces. Foods, such as beef and dairy products, that become contaminated with feces of these animals represent the potential source for the transmission of these bacteria to humans. The objective of our study was to develop and evaluate methods for rapid screening of cattle feces and beef to detect the presence of very low numbers of these bacteria. We have developed fluorescent PCR tests that can detect less than or equal to 10 bacterial cells in beef or feces within 8 to 10 hours. These tests have the potential for complete automation that would allow rapid screening of hundreds of fecal and food samples. These tests will be useful to cattle producers and veterinarians for rapid diagnosis of diarrheal illness caused by E. coli O157:H7 and other STEC and in monitoring cattle herds for the carriage of these bacteria. Moreover, these rapid diagnostic tests will be useful to meat processors to monitor the presence of these disease-causing bacteria on animal carcasses and meat products.

Technical Abstract: Semiautomated detection of Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a Shiga toxin-producing E. coli (STEC), was achieved in feces and foods of bovine origin using fluorogenic polymerase chain reaction (PCR). These PCR assays were designed to amplify 80, 120, and 150 bp regions of virulence genes stx1, stx2, and eaeA, respectively, using specific primers. The fluorogenic probes were used for specific detection of amplified products of the stx1 and stx2 genes of STEC, and the eaeA gene of EHEC O157:H7. For multiplex PCR assay, the three sets of primers and fluorogenic probes were included in one reaction to simultaneously amplify and detect any of the three targeted virulence genes. In non-multiplex PCR assay, each of the three virulence genes was amplified and detected in independent reactions. The specificity of these assays was evaluated using suspensions of STEC and other bacterial species lacking stx1, stx2, and eaeA. The multiplex assay detected all STEC harboring any combination of three virulence genes. Three non-multiplex PCR reactions identified types of Shiga toxin genes carried by a STEC and identified STEC as either EHEC O157:H7 or non-O157:H7 STEC. Sensitivity limits of these assays in beef and feces inoculated with EHEC O157:H7 were 5.8 to 580 cfu and 1.2 to 1200 cfu, respectively. The automated PCR amplification-detection capability of these assays is conducive for rapid screening of large number of samples for detecting STEC and for specific detection of EHEC O157:H7 within 8 to 10 h.

Last Modified: 11/25/2014
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