TaqMan-based Multiplex Real-time PCR Assays for the Detection and Quantification of the Six Major Non-O157 Shiga Toxin-producing Escherichia coli in Cattle Feces

Introduction: Shiga toxin-producing Escherichia coli (STEC) are responsible for severe foodborne illnesses in humans.  Non-O157 STEC, particularly O26, O103, O111, O45, O121 and O145, are gaining attention in recent years as they are responsible for more than 70% of human STEC infections. Cattle harbor non-O157 STEC in the gut and shed in the feces.  Molecular detection methods, particularly real-time PCR, for non-O157 E. coli detection in cattle feces have not yet been developed and validated.

Purpose: To develop multiplex real-time PCR assays to detect and quantify the six major non-O157 STEC, O26, O103, O111, O45, O121 and O145, in cattle feces. 

Methods: Two sets of serogroup-specific assays targeting O antigen genes, O26, O103, O111 (assay 1) and O45, O121, O145 (assay 2) were developed.  Specificity of the assays was assessed by testing a panel of positive and negative E. coli control strains.  Sensitivity of the assays was determined with 10-fold serial dilutions of pure cultures and culture-spiked fecal samples.  Spiked fecal samples were enriched in E. coli broth for 6 h at 40°C.  Fecal DNA extracted before and after the enrichment were used as templates for real-time PCR.  Assays were compared to conventional PCR and culture-based method of detection by analyzing 225 cattle fecal samples.

Results: The assays were specific for the target genes and no cross-reactions were observed.  The detection limits of the assays were 10³ CFU/ml, 10⁴ CFU/g and 10² CFU/g for pooled pure cultures, before and after enrichment of fecal samples spiked with the six non-O157 STEC, respectively.  Real-time PCR assays (171/225) detected a higher number of positive samples than conventional PCR (55/225) and culture-based (110/225) methods.

Significance: The multiplex real time PCR assays developed are sensitive diagnostic tools for the detection and quantification of the six non-O157 E. coli serogroups in cattle feces.