PCR-SSCP检测结核分枝杆菌利福平耐药相关基因rpoB突变

Detection of rpoB gene mutations associated with Rifampin in Mycobacterium tuberculosis clinical isolates by PCR-single strand conformational polymorphism method

目的建立聚合酶链反应–单链构象多态性(PCR-SSCP)技术快速检测结核分枝杆菌利福平(RFP)耐药相关基因rpoB突变。方法设计结核分枝杆菌RFP耐药相关rpoB基因PCR引物,建立PCR-SSCP技术检测临床菌株rpoB基因的突变导致的运动变位,同时采用PCR直接测序(PCR-DS)技术检测rpoB基因突变,并对上述方法检测结果进行分析和比较。结果84株临床菌株均含有rpoB基因;PCR-SSCP和PCR-DS检测结果显示,56株RFP敏感菌株中rpoB基因分别有3株和2株检测出突变,检测特异性分别为94.6%(53/56)和96.4%(54/56);28株RFP耐药菌株中rpoB基因分别有27株和28株发生突变,检测灵敏度分别为96.4%和100%。结论本研究建立的PCR-SSCP技术能快速、简便、特异、敏感地检测结核分枝杆菌利福平耐药基因rpoB突变,具有临床应用前景。

Objective To establish PCR-single strand conformational polymorphism（PCR-SSCP） method for rapid detection of rpoB gene mutation associated with rifampin（RFP） resistance in Mycobacterium tuberculosis isolates.Methods The primers were designed for detecting RFP resistance associated rpoB gene in the isolates by PCR-SSCP.PCR-DNA sequencing（PCR-DS） technique was applied to detect the mutations in rpoB gene.All the results from different assays were analyzed and compared statistically.Results All of the 84 clinical isolates had rpoB gene.In the 56 RFP-susceptible isolates,3 and 2 strains had mutation in rpoB genes confirmed by PCR-SSCP and PCR-DS respectively with the specificity rate of 94.6% and 96.4%.The results of PCR-SSCP and PCR-DS also demonstrated that in the 28 RFP-resistant isolates,27 and 28 isolates had the mutation in rpoB gene with the sensitivities of 96.4% and 100% respectively.Conclusion The PCR-SSCP established in this study can be used to rapidly detect RFP-resistance associated rpoB gene mutation in M.tuberculosis with convenience,specificity and sensitivity,which could have a good prospect in clinical application.