耐多药结核分枝杆菌基因突变在耐药性检测中的应用

Studies of Mycobacterium Tuberculosis Multi-drug Resistant Gene

目的:探讨结核分枝杆菌耐链霉素(SM)和利福平(RFP)的耐药分子机制,应用聚合酶链反应一单链构象多态性(PCR-SSCP)同时检测rpsL基因、rpoB基因突变在结核分枝杆菌耐SM和RFP耐药性测定中的应用价值。方法:采用PCR-SSCP技术对168株结核分枝杆菌临床分离株rpsL基因、rpoB基因突变同时进行检测。即首先用PCR方法同时扩增RFP、SM的rpoB基因和rpsL基因,然后进行SSCP分析。结果:以结核分枝杆菌H37RV为对照,82株药物敏感株的rpsL基因、rpoB基因均未见SSCP图谱异常。特异性为100%。86株同时耐SM和RFP的耐药株中,68株(79.1%)有rpsL基因图谱异常;81株(94.2%)有rpoB基因图谱异常。结论:rpoB基因突变和rpsL基因突变分别是结核分枝杆菌耐RFP和SM的主要分子机制。应用PCR-SSCP技术可同时快速检测结核分枝杆菌SM和RFP耐药性。

Objective To study on the molecular mechanisms of rifampin and streptomycin-resistant Myeobaeterium tuberculosis. Detection rpoB and rpsL gene mutations in rifampin and streptomycin-resistant Mycobaeterium tuberculosis using Polymorphism（PCR-SSCP）. Methods 168 clinical isolates of Mycobacterium tuberculosis of rpoB and rpsI. genes were detected using PCR-SSCP. Results Strain H377 RV was used as control, SSCP pattern of rpoB,rpsL PCR amplification from 82 drug susceptible strains were all the same as control. The specificity was 100%. SSCP pattern of rpsL gene were different from control in 68 drug resistant clinical isolates. SSCP pattern of rpoB gene were different from control in 86 drug resistant clinical isolates. The sensitivity were 79.1 % （rpsL）, 94.2 % （rpoB）, respectively. Conclusions The results confirm that mutations of rpsl. and rpoB genes are the most important mechanisns in streptomycin and rifampin resistant tuberculosis, respectively.