聚合酶链反应-单链构象多态性技术快速筛选结核杆菌的利福平耐药性

Rapid detection of rifampin resistant M. tuberculosis by PCR and single-strand conformation polymorphism analysis

目的 :探讨利用分子生物学方法直接快速检测结核分枝杆菌利福平 (RFP)药物敏感性的可行性。方法 :应用聚合酶链反应 单链构象多态性 (PCR SSCP)技术分别检测了 4 0株RFP药物敏感及耐药的结核分枝杆菌临床分离株。先用PCR扩增rpoB基因 ,随后用SSCP方法鉴定其扩增产物有无突变 ,并与药敏试验结果作对照分析。 结果 :所有临床分离株均观察到rpoB基因PCR扩增产物。 4 0株RFP敏感的临床分离株SSCP带谱与结核分枝杆菌H3 7RV标准株相同 ,4 0株RFP耐药株中37株检测到突变图谱。与Bactec结果对照 ,用PCR SSCP技术检测结核分枝杆菌RFP耐药性的敏感性为 93% ,特异性为10 0 %。结论 :结核分枝杆菌耐RFP是其rpoB基因突变所致。PCR SSCP技术可用于结核分枝杆菌RFP药物敏感性的直接快速检测。

Objective: To evaluate the applicability of molecular biology technique for the detection of activity of rifampin against M. tuberculosis. Methods: Eighty clinical isolates of M. tuberculosis , including 40 rifampin susceptible strains and 40 rifampin resistant strains, were detected by PCR SSCP, with M. tuberculosis H 37 RV reference strains as control. The rifampin resistant region rpoB was amplified by PCR, and the mutations of amplification products were identified by SSCP analysis. Results: The 40 rifampin susceptible strains and M. tuberculosis H 37 RV displayed identical SSCP patterns. Thirty seven of 40 rifampin resistant strains showed SSCP patterns different from H 37 RV, and 3 strains had the same patterns as H 37 RV. Compared with BACTEC culture system, the sensitivity and specificity of PCR SSCP were 93% and 100%, respectively. Conclusion: PCR SSCP appears to be a promising procedure for the rapid detection of rifampin resistant M. tuberculosis.