Development of a duplex real-time fluorescence PCR method for detection of enterohemorrhagic Escherichia coli

OBJECTIVE To establish a duplex real-time fluorescence PCR assay method,which was specific and sensitive for rapidly detecting enterohemorrhagic Escherichia coli.METHODS The primers and Taqman probes of stx1 and stx2 genes of EHEC were designed and synthesized according to gene sequences published by GenBank.Probes were labeled by FAM-BHQ1 and HEX-BHQ1,separately.The duplex real-time fluorescence PCR reaction was developed.The sensitivity,specificity and reproducibility of real-time PCR methods were evaluated.RESULTS The duplex real-time fluorescence PCR method of EHEC was well developed.The detection limit for this method was 102cfu/ml and 117.5fg/μl.The method was with a good sensitivity,specificity and repeatability.CONCLUSION The developed duplex real-time PCR assay can be used as a simple,rapid,sensitive,and effective method for EHEC strain detection.