High-resolution melt curve PCR assay for specific detection of E. coli O157:H7 in beef

Abstract

Among the disease-causing Shiga toxin-producing Escherichia coli (STEC), E. coli O157:H7 is estimated to cause one-third of the total STEC illnesses and the most cases of hemolytic uremic syndrome (HUS) in the U.S. The uidA gene which is present in the majority of E. coli strains, codes for the synthesis of β-d-glucuronidase (GUD). In E. coli O157:H7, the uidA gene has a single point mutation at the +93 position that leads to an alteration in the amino acid sequence encoding the GUD enzyme. The aim of this study was to distinguish E. coli O157:H7 from other E. coli using a high resolution melt curve (HRM) real-time PCR assay. Based on the uidA mutation in E. coli O157:H7, a reliable PCR assay targeting the uidA gene was developed to differentiate E. coli O157:H7 from other E. coli serotypes and the closely related Shigella spp. The specificity of the assay was validated using a set of 128 pure bacterial DNA samples. Following enrichment and immunomagnetic separation (IMS), the assay was further applied to spiked ground beef and beef trim. Isolates of E. coli O157:H7 formed distinctive melt peaks that were easily distinguishable from those of other E. coli serogroups and Shigella isolates in the PCR plot. Therefore, this assay was able to clearly discriminate E. coli O157:H7 strains from other E. coli and Shigella. With a 5–8 h enrichment time, followed by IMS, 10 CFU of E. coli O157:H7 were detectable in 325 g spiked beef samples. The HRM E. coli O157:H7 detection assay standardized in this study will allow for accurate identification of contaminated food samples and help in preventing foodborne outbreaks caused by this pathogen.