• Bergeron, Michel G.
• Picard, Francois J.
• Menard, Christian
• Boissinot, Maurice
• Belanger, Simon D.

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>We have developed a rapid (1-h) real-time fluorescence-based PCR assay with the Smart Cycler thermal cycler (Cepheid, Sunnyvale, Calif.) for the detection of Shiga toxin-producing<jats:italic>Escherichia coli</jats:italic>(STEC), as well as other Shiga toxin-producing bacteria. Based on multiple-sequence alignments, we have designed two pairs of PCR primers that efficiently amplify all variants of the Shiga toxin genes<jats:italic>stx</jats:italic><jats:sub>1</jats:sub>and<jats:italic>stx</jats:italic><jats:sub>2</jats:sub>, respectively. These primer pairs were combined for use in a multiplex assay. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon. Assays performed with purified genomic DNA from a variety of STEC strains (<jats:italic>n</jats:italic>= 23) from diverse geographic locations showed analytical sensitivities of about 10 genome copies per PCR. Non-STEC strains (<jats:italic>n</jats:italic>= 20) were also tested, and no amplification was observed. The PCR results correlated perfectly with the phenotypic characterization of toxin production in both STEC and non-STEC strains, thereby confirming the specificity of the assay. The assay was validated by testing 38 fecal samples obtained from 27 patients. Of these samples, 26 were PCR positive for<jats:italic>stx</jats:italic><jats:sub>1</jats:sub>and/or<jats:italic>stx</jats:italic><jats:sub>2</jats:sub>. Compared with the culture results, both the sensitivity and the negative predictive value were 100%. The specificity was 92%, and the positive predictive value was 96%. Moreover, this assay detected STEC from a sample in which the STEC concentration was at the limit of detection of the conventional culture methods and from a sample in which STEC was not detected by the conventional culture methods. This real-time PCR assay is simple, rapid, sensitive, and specific and allows detection of all Shiga toxin-producing bacteria directly from fecal samples, irrespective of their serotypes.</jats:p>

Topics

• impedance spectroscopy