聚合酶链反应-单链构象多态性技术检测耐多药结核分支杆菌rpoB、KatG、rpsL突变的研究

Rapid detecting of the genomic mutations of the multidrug-resistant Mycobacterium tuberculosis by polymerase chain reaction-single strand conformational polymorphism

目的 应用PCR SSCP技术快速检测耐INH ,RFP ,SM结核分支杆菌分离株KatG、rpoB、rpsL基因突变 ,评价其在检测结核分支杆菌耐药性方面的价值。方法 32株耐INH、RFP、SM结核分支杆菌临床分离株及 2 5株结核分支杆菌敏感分离株用PCR SSCP方法分别检测 ,rpoB、KatG、rpsL基因突变。结果 32株耐多药结核分支杆菌分离株中 ,rpoB、KatG、rpsLPCR扩增产物PCR SSCP分别有 2 8株 (87.5 % )rpoB基因 ,19株 (5 9.3% )KatG基因和 2 3株 (71.9% )rpsL基因电泳条带与结核分支杆菌标准株H3 7Rv电泳条带相比有明显差异 ,而 2 5株结核分支杆菌敏感株的PCR SSCP条带与结核分支杆菌H3 7Rv相似 ,特异性为 10 0 %。结论 PCR SSCP方法敏感、特异 ,可快速检测结核分支杆菌rpoB、KatG、rpsL耐药基因突变 。

Objective RpoB?KatG?rpsL genes mutation analysis of INH?RFP?SM resistance in isolates of M.tuberculosis (MTB) were undertaken by PCR single strand conformation poylymorphism (SSCP),and to evaluating the value of diagnosing resistance of MTB.Methods Detecting of INH?RFP?SM resistance in 32 isolates strains of MTB and drug susceptivity in 25 isolates strains of MTB by PCR SSCP.Results Among 32 multidrug resistant isolates strains of MTB,28?19?23 isolates had very different rpoB?KatG?rpsL gene mutation pattern of PCR SSCP compared with that of the reference strain (H 37 Rv),and drug susceptivity in 25 isolates had the same PCR SSCP pattern.Compared with drug sensitive test,sensitivity to detecting rpoB?KatG?rpsL genes by PCR SSCP was 87.5%,59.3%,71.9% respectively,and specificity was 100%.Conclusion PCR SSCP is a sensitive and specific method for rapid detecting rpoB?KatG?rpsL genes mutations of MTB,and useful in rapid detecting the multidrug resistant MTB.