结核分支杆菌耐利福平耐药基因检测研究

目的评价应用DNA序列分析方法和聚合酶链反应-单链构象多态性(PCR-SSCP)技术检测rPOB基因突变在结核分支杆菌耐利福平(RFP)耐药性测定中的应用价值.方法采用DNA序列分析法和PCR-SSCP法对80株结核分支杆菌临床分离株(其中药物敏感株32株,耐RFP或含耐RFP耐多药株48株)rpoB基因核心区域的突变情况进行检测.结果以结核分支杆菌H37RV为对照,所有32株药物敏感株的rPOB基因均无突变.用DNA序列分析方法检测48株耐药株中44株发生突变,敏感性为91.7%(44/48),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48株耐RFP分离株中,45株rPOB基因SSCP图谱异常,其敏感性为93.1%(45/48).结论DNA序列分析可指导临床用药,PCR-SSCP可快速检测结核分支杆菌RFP耐药性.

Defection rifampin-resistant Mycobacterium tuberculosis

Objective: Detection rPOB geng mutations in refampin-resistant Mycobacterium tuberculosis using Polymerase Chain reaction-single-strand conformation polymorphi RFP(PCR-SSCP) and DNA sequencing. Methods:80 clinical isolates of Mycobacterium tuberculosis (32 drug susceptible,48 rifampin-resistance or multidrug resistance including rifampin) by DNA sequencing and PCR-SSCE.Results: No mutation was found in 32 rif-rensitive strains by DNA sequencing. While 44 of 48 rif-rensitive strains had mutations rifampin resistant determination region of rpoB gene, the mutation rate was 91.7%(44/48),531-Ser and 526-His were the most common positions to be substituted.32 rif-rensitive strains had same PCR-SSCP gelprofile with Mycobacterium tuberculosis H37Rv as control, while 45 of 48 rif-rensitive strains showed different SSCP. The specificity is 93.1%. Conclusion:DNA sequencing is significant in drug choosing for tuberculosis treatment. Rifampin-resistant of Mycobacterium tuberculosis is rapidly and accurately detected using PCR-SSCP.