Use of Specific CRISPR-2 Spacers for the Detection of the Big Six STEC Serotypes by Real-time PCR

Introduction: Molecular typing of pathogenic Escherichia coli is important for diagnostic microbiology and trace back epidemiology to quickly establish food-clinical linkages and as an indicator of virulence potential. A clustered, regularly interspaced, short palindromic repeat (CRISPR) presents an alternative genetic marker for molecular typing and detection of pathogenic Shiga toxin-producing E. coli (STEC).These regions are conserved among phylogenetically related E. coli, and can be used as genomic signatures for food safety related applications.

Purpose: The purpose of this study was to evaluate the application and utility of CRISPR loci and spacers as potential genetic markers in molecular sub-typing of several STEC strains including the “big six” serotypes O145, O121, O103, O111, O26 and O45 by a real-time PCR assay.

Methods: One hundred different E. coli strains from our collection were tested. The CRISPR-2 regions and spacers of the big six E. coli serotypes were identified and 30 conserved spacers were selected based on BLAST information and http://crispr.u-psud.fr/CRISPRcompar. Real-Time PCR assays were performed in a volume of 25 µl in 96-well plates using the 30 spacer panel.

Results: We have analyzed over 100 strains using the panels and no template control (NTC) was used as a negative control. We were able to distinguish the big six serotypes using the cycle threshold (Ct) value of 30; the Ct value < 30 was considered positive.

Significance: Our data demonstrated CRISPR-2 spacers could be utilized as molecular markers associated with the big six serotypes with proper databasing to reference strains associated particularly with each E. coli serotype. Further studies include expansion of spacer repertoire for broader application to E. coli serotypes beyond the “big six.”