肠出血性大肠埃希菌双重实时荧光PCR检测方法的建立

目的建立快速、灵敏、特异、有效的肠出血性大肠埃希菌(EHEC)双重实时荧光PCR检测方法。方法根据GenBank公布的EHEC的stx1和stx2基因序列设计相应的PCR引物和TaqMan探针,探针分别用FAM-BHQ1和HEX-BHQ1标记stx1及stx2,建立双重实时荧光PCR反应体系,并对方法的灵敏度、特异性及其重复性进行分析。结果建立了EHEC双重实时荧光PCR检测方法。结果显示该方法特异性、重复性好、灵敏度高,最低检出限可达102cfu/ml,117.5fg/μl。结论建立的双重实时荧光PCR方法可快速、灵敏、特异、有效地检出EHEC。

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.