Submitted to: Veterinary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 28, 2002
Publication Date: May 20, 2003
Citation: SHARMA, V.K., NYSTROM, E.A. DETECTION OF ENTEROHEMORRHAGIC ESCHERICHIA COLI O157:H7 BY USING A MULTIPLEX REAL-TIME PCR ASSAY FOR GENES ENCODING INTIMIN AND SHIGA TOXINS. VETERINARY MICROBIOLOGY. 2003. V. 93. P. 247-260. Interpretive Summary: Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) have emerged in recent years as important foodborne pathogens associated with severe gastrointestinal and systemic diseases in humans. Each year, about 73,000 estimated cases of diarrheal illness due to E. coli O157:H7 occur in the U.S. Most human E. coli O157:H7 infections are caused by consumption of contaminated foods (ground beef, milk, and produce) and water, and cattle are generally considered the major reservoir for E. coli O157:H7 and other STEC that have been isolated from human clinical cases. E. coli O157:H7 and other STEC colonize the intestine of cattle and are secreted into feces of these animals. E. coli O157:H7 can survive in feces for months. Cattle feces, therefore, represents the major source for the transmission of E. coli O157:H7 to other cattle and humans. The objective of this study was to develop real-time PCR (R-PCR) methods for rapid detection and quantitation of E. coli O157:H7 and in cattle feces and intestinal tissues. We developed a specific and sensitive R-PCR assay for detecting very low levels (1 to 10 bacterial cells per gram of feces or tissues) of E. coli O157:H7 in cattle tissues and feces. The meat processing industry, which slaughters more than $50 billion worth of livestock annually, could use this technology to meet current federal regulations that mandate zero tolerance for visible fecal contamination for E. coli O157:H7.
Technical Abstract: A multiplex real-time PCR (R-PCR) assay was designed and evaluated on the ABI 7700 Sequence Detection System (TaqMan) to detect enterohemorrhagic Escherichia coli (EHEC) O157:H7 in pure cultures, feces, and tissues. Three sets of primers and fluorogenic probes were used for amplification and real-time detection of a 106-bp region of the eae gene encoding EHEC O157:H7-specific intimin, and 150-bp and 200-bp segments of genes stx1 and stx2 encoding Shiga toxins 1 and 2, respectively. Analysis of 67 bacterial strains demonstrated that the real-time PCR assay successfully distinguished EHEC O157:H7 serotype from non-O157 serotypes and provided accurate profiling of genes encoding intimin and Shiga toxins. Bacterial strains lacking these genes were not detected with this assay. The detection range of the real-time PCR assay for the three genes was linear over DNA concentrations corresponding to 103 to 108 CFU per ml of EHEC O157:H7. The real-time PCR allowed construction of standard curves that facilitated quantification of EHEC O157:H7 in feces and intestinal tissues. Detection sensitivity of the real-time PCR assay ranged from 104 to 108 CFU per gram of feces or tissues without enrichment. Enrichment of feces in a non-selective broth for 4 and 16 h resulted in the detection of levels (100 to 103 CFU per g of feces) considered sufficient for infection in humans. The real-time PCR assay for eaeO157:H7, stx1, and stx2 proved to be a rapid test for detection of EHEC O157:H7 in complex biological matrices and could also potentially be used for quantification of EHEC O157:H7 in foods or fecal samples.