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

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.