Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes

Abstract

In this report, we present a PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min.

FIG. 1

Multiplex amplification of a dilution series of the Stx double producer EHEC EDL 933 for evaluation of sensitivity of the PCR protocol. (A) stx₁-specific signal in the −d(F)/dT plot of the third channel (LightCycler Red 705); (B) stx₂-specific signal in the −d(F)/dT plot of the second channel (LightCycler Red 640) (the melting points of both stx genes were 72°C); (C) submarine agarose gel of the PCR products. A negative control (lane 1) and 10-fold dilution series of genome equivalents of EHEC EDL 933 (lanes 2 to 5) at 1,000 copies (lane 2), 100 copies (lane 3), 10 copies (lane 4), and 1 copy (lane 5) were tested. Both PCR fragments had the expected size of 418 bp for stxA₁ and 246 bp for stxA₂, calculated from the nucleotide positions of their amplification primers. A 1-kb DNA ladder was used as the DNA size marker (left side of the panel).

FIG. 2

Multiplex PCR of EHEC EDL 933 and three strains harboring stx_2e, the gene of the pig edema disease toxin. (A) stx₁-specific signal in the −d(F)/dT plot of the third channel (LightCycler Red 705); (B) stx₂ specific signal in the −d(F)/dT plot of the second channel (LightCycler Red 640). (the melting points were 72°C for stx₁ and stx₂ and 62.5°C for stx_2e); (C) agarose gel of the respective PCR products. A negative control (lane 1) and EHEC EDL 933 (lane 2), EHEC ED 42 (lane 3), EHEC ED 43 (lane 4), and EHEC ED 68 (lane 5) were tested. The size of the three stxA_2e amplicons was 246 bp, as expected. The positions of a 1-kb DNA ladder are indicated along the left side of the panel.