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

目的　应用PCRSSCP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25株结核分支杆菌敏感分离株用PCRSSCP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、rpsL PCR扩增产物PCR-SSCP分别有28株(87.5%)rpoB基因,19株(59.3%)KatG基因和23株(71.9%)rpsL基因电泳条带与结核分支杆菌标准株H₃₇Rv电泳条带相比有明显差异,而25株结核分支杆菌敏感株的PCR-SSCP条带与结核分支杆菌H₃₇Rv相似,特异性为100%。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,有利于耐多药结核分支杆菌耐药性的快速检测。

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

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.