Detection of stx1 and stx2 genes of enterohemorrhagic Escherichia coli by a real-time fluorescence polymerase chain method

Objective To establish a duplex real-time fluorescence TaqMan PCR method for the detection of stx1 and stx2 of enterohemorrhagic Escherichia coli (EHEC). Methods The primers and TaqMan probes for stx1 and stx2 genes of EHEC were blasted and designed according to the gene sequences published by GenBank. The probes were labeled with reporter dye FAM or HEX. Quencher dye was BHQ1. The duplex real-time fluorescence PCR reaction was set up and conditions were modified. The sensitivity,specificity and reproducibility of the real-time PCR methods were evaluated. Results The duplex real-time fluorescence PCR method for EHEC was developed. Smooth amplification curves were observed when the strain concentrations were 102～108 cuf/ml. Both stx1 and stx2 genes were found and EHEC strains were obtained by the culture method in 30 clinical samples. The method sensitivity was 100%. DNA templates from three high, middle and low concentrations of 108,105,102 cfu/ml were tested three times. The amplification curves were obtained. The standard deviation and coefficient of variation of Ct from different concentrations were less than 5% which was within acceptable error. Conclusion The developed duplex real-time fluorescence PCR method could rapidly, sensitivity, effectively and exactly detect EHEC.