双重实时荧光PCR检测肠出血型大肠埃希菌stx1和stx2基因方法的建立

目的建立一种检测肠出血型大肠埃希菌(EHEC)产毒基因stx1和stx2的TaqMan双重实时荧光PCR法。方法根据GenBank公布的EHECstx1和stx2基因序列进行比对后设计相应的PCR引物和TaqMan探针,探针报告基团分别使用FAM和HEX标记,淬灭基团使用BHQ1标记。建立双重实时荧光PCR反应体系并对反应条件进行优化,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。菌液浓度为102～108cuf/ml时,均可观察到扩增曲线,且扩增曲线平滑;检测30份模拟临床样品,均检测到stx1和stx2基因,且均培养出EHEC,该方法灵敏度为100%;3次重复测定108、105、102cfu/ml3个高、中、低浓度的菌液DNA模板,得到其扩增曲线,计算各浓度的Ct值标准差及变异系数,均小于5%,在误差允许范围内。结论建立的双重实时荧光PCR法可快速、灵敏、特异、有效地检出EHEC。

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.