Multiplex Real-Time PCR Assays for Screening of Shiga Toxin 1 and 2 Genes, Including All Known Subtypes, and Escherichia coli O26-, O111-, and O157-Specific Genes in Beef and Sprout Enrichment Cultures

Abstract

Shiga toxin family members have recently been classified using a new nomenclature into three Stxl subtypes (Stxla, Stxlc, and Stxld) and seven Stx2 subtypes (Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g). To develop screening methods for Stx genes, including all of these subtype genes, and Escherichia coli O26-, O111-, and O157-specific genes in laboratory investigations of Shiga toxin-producing E. coli (STEC) foodborne cases, we developed multiplex real-time PCR assays and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates, recombinant plasmids, and food enrichment cultures and by performing STEC spiking experiments with beef and sprout enrichment cultures. In addition, we evaluated the relationship between the recovery rates of the target strains by direct plating and immunomagnetic separation and the cycle threshold (CT) values of the real-time PCR assays for the Stx subtypes and STEC O26, O111, and O157 serogroups. All three stx1- and seven stx2-subtype genes were detected by real-time PCR with high sensitivity and specificity, and the quantitative accuracy of this assay was confirmed using control plasmids and STEC spiking experiments. The results of the STEC spiking experiments suggest that it is not routinely possible to isolate STEC from enrichment cultures with real-time PCR CT values greater than 30 by direct plating on MacConkey agar, although highly selective media and immunomagnetic beads were able to isolate the inoculated strains from the enrichment cultures. These data suggest that CT values obtained from the highly quantitative real-time PCR assays developed in this study provide useful information to develop effective isolation strategies for STEC from food samples. The real-time PCR assays developed here are expected to aid in investigations of infections or outbreaks caused by STEC harboring any of the stx-subtype genes in the new Stx nomenclature, as well as STEC O26, O111, and O157.