Rapid detection of shiga toxin-producing bacteria in feces by multiple PCR with molecular beacons on the smart cycler

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 Escherichia coli (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 stx1 and stx2, 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 (n 23) from diverse geographic locations showed analytical sensitivities of about 10 genome copies per PCR. Non-STEC strains (n 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 stx1 and/or stx2. 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