结核分枝杆菌利福平耐药基因突变的研究

Study on Mutation of Rifampin-resistant Genes Mycobacterium tuberculosis

目的评价检测rpoB基因突变在结核分枝杆菌耐利福平(RFP)耐药性测定中的应用价值。方法采用DNA序列分析法和聚合酶链反应-单链构象多态性(PCR-SSCP)法对91株结核分枝杆菌临床分离株(其中药物敏感株35株,耐RFP或含耐RFP耐多药株56株)rpoB基因核心区域的突变情况进行检测。结果以结核分枝杆菌H37RV为对照,所有35株药物敏感株的rpoB基因均无突变,用DNA序列分析方法检测56株耐药株中53株发生突变,敏感性为94·6%(53/56),其中最常见的突变位点是531位丝氨酸和526位组氨酸。PCR-SSCP检测56株耐RFP分离株中,52株rpoB基因SSCP图谱异常,其敏感性为92·9%(52/56)。结论DNA序列分析对判断结核分枝杆菌耐利福平非常有价值。PCR-SSCP可初步筛选结核分枝杆菌RFP耐药性。

Objective To detect rpoB gene mutations in refampin- resistant Mycobacterium tuberculosis using Polymerase Chain reaction- single - strand conformation polymorphism （PCR - SSCP） and DNA sequencing. Method 91 clinical isolates of Mycobacterium tuberculosis （35 drug susceptible, 56 RFP - resistance or multiple drug resistance including rifampin） by DNA sequencing and PCR - SSCP. Results No mutation was found in 35 rif-sensitive strains by DNA sequencing. While 53 of 56 rifampinsensitive strains had mutations rlfampin- resistant determination region of rpoB gene, the mutation rate was 94.6% （53/56）, 531 - Set and 526 - His were the most conmlon positions to be substituted.35 rif- sensitive strains had sanle PCR - SSCP gelprofile with Mycobacterium tuberculasis H37 Rv as control, while 52 of 56 rif- sensitive strains showed different SSCP.The specificity is 92.9%. Conclusion DNA sequencing is valuable in rifampin suscepfibilty test, PCR- SSCP is useful in rifampin resistance strains screening.