小家鼠螺杆菌TaqMan-qPCR检测方法的建立

摘要

目的 建立敏感性高、特异好的小家鼠螺杆菌(Helicobacter muridarum) TaqMan-qPCR检测方法.方法 选择小家鼠螺杆菌保守基因GyrB设计特异性引物进行PCR扩增,并构建质粒标准品GyrB-19T.通过反应体系及条件的优化,建立小家鼠螺杆菌TaqMan-qPCR检测方法,并对GyrB-19T标准品进行定性及定量分析,评估该方法的灵敏度、特异性及重复性.结果 所建立的qPCR检测方法,质粒标准品浓度在2×102～2×108拷贝/μL范围内表现出较好的线性相关性,所得标准曲线的斜率为-3.57,相关系数为0.996,检测灵敏度达到200拷贝/μL.结论 所建立的小家鼠螺杆菌TaqMan-qPCR方法具有特异性好、敏感性高、稳定性强等特点,适用于小家鼠螺杆菌的定量及定性分析.%Objective To establish the sensitive and specific TaqMan-qPCR method for detection of Helicobacter muridarum.Method The specific primers were designed according to the Helicobacter muridarum gyrase B(GyrB) gene,specific fragment was amplified by PCR,and cloned into pMD-19T vector to construct the recombinant plasmid GyrB-19T,which was used as standard DNA of this qPCR method.The qPCR system was optimized and the sensitivity,specificity,repeatability and quantitative range of this method were evaluated.Result The quantitative standard curve from 2 × 108 copies/well to 2 × 102 copies/well of serial diluted plasmid DNAs showed that they had good liner correlation,the slope of the standard curve was-3.57,R2＞0.996,and the lowest detection limit reached 2 × 102 copies/well.Conclusion This qPCR method showed high sensitivity,specificity and stability,which will be utilized for qualitativeand quantitative detection of Helicobacter muridarum.