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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Meat Safety and Quality » Research » Publications at this Location » Publication #373887
Research Project: Mitigation Approaches for Foodborne Pathogens in Cattle and Swine for Use During Production and Processing

Location: Meat Safety and Quality

Title: High-resolution melting real-time PCR assays for detection of Escherichia coli O26 and O111 strains possessing Shiga toxin genes

Author
item SINGH, PRASHANT - Florida State University
item CUBILLOS, GABRIEL - Florida State University
item KIRSHTEYN, GABRIELLE - Florida State University
item Bosilevac, Joseph - Mick

Submitted to: LWT - Food Science and Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/17/2020
Publication Date: 6/23/2020
Citation: Singh, P., Cubillos, G., Kirshteyn, G., Bosilevac, J.M. 2020. High-resolution melting real-time PCR assays for detection of Escherichia coli O26 and O111 strains possessing Shiga toxin genes. LWT - Food Science and Technology. 131:109785. https://doi.org/10.1016/j.lwt.2020.109785.
DOI: https://doi.org/10.1016/j.lwt.2020.109785

Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) are dangerous foodborne pathogens that contaminate beef and fresh produce. For that reason, sensitive and accurate tests are needed to detect them in food. Two types of STEC are O26 and O111, but there are safe non-pathogen E. coli of these types that can trip up current tests. Therefore, this study designed two new tests that can detect STEC-O26 and STEC-O111 and distinguish them from the non-pathogen O26 and O111. The two tests were further shown to work in beef and spinach that was inoculated with different STEC and non-STEC. Use of these two tests improve food safety and reduce response time during foodborne outbreaks.

Technical Abstract: Strains of Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens. These strains are frequently involved in foodborne outbreaks. The aim of this study was to develop high-resolution melting (HRM) real-time PCR assays for the specific detection of O26 and O111. Then apply HRM analysis for differentiation of those that were STEC and potentially pathogenic to humans (virulent) and non-STEC unlikely to be a cause of disease (avirulent) strains of O26 and O111. Serogroup-specific primers targeting the O26 fnl1 and O111 wbdK genes were designed. Two assays were standardized and validated using 87 pure culture bacterial strains. Serogroup specific O26 and O111 primers showed 100% specificity. Potentially virulent strains formed distinct melting profiles and clustered separately in the melting curve plots and were easily distinguishable from avirulent strains. The O26 assay showed 75% specificity and 100% sensitivity, whereas the O111 assay showed 100% specificity and 100% sensitivity for the identification of potentially virulent strains. Both assays were further validated using inoculated beef and spinach samples and the assays were able to detect the pathogen following 15 h enrichment period. The two assays developed in this study can be used to improve food safety and reduce response time during foodborne outbreaks.