结核分枝杆菌katG、rpoB、rpsL耐药基因快速检测方法的建立

Study on rapid detection of katG,rpoB,rpsL genomic mutations of Mycobacterium tuberculosis

目的:建立一套应用multi-PCR-SSCP(multiple-Polymerase Chain Reaction-Single Strand Cinformation Poly-morphism)快速检测结核分枝杆菌耐药基因突变的方法,评价该方法的临床应用价值。方法:分别用常规PCR和multi-PCR-SSCP检测58株结核分枝杆菌临床分离株katG、rpoB、rpsL基因突变并与常规药敏试验检测结果进行对比。结果:经常规药敏试验检测,58株结核分枝杆菌临床分离株中有41株耐药,其中,耐异烟肼(INH)35株;耐利福平(RFP)31株;耐链霉素(SM)31株。单基因PCR-SSCP与multi-PCR-SSCP分析结果一致,检出katG、rpoB、rpsL基因的突变率分别为65.7%(23/35)、84%(26/31)、71%(22/31),17株药物敏感株中有1株rpoB基因SSCP电泳条带与结核杆菌标准株结果有差异。结论:multi-PCR-SSCP方法敏感、特异,可快速检测结核分枝杆菌katG、rpoB、rpsL耐药基因的突变,将该方法与药敏试验联合应用可互相弥补,成为临床指导用药的好方法。

Objective： To develop a new multi-PCR-SSCP system for detecting the drug-resistant gene mutation in Mycobacterium tuberculosis（MTB）,and evaluate the clinic application of multi-PCR-SSCP.Methods：58 clinical isolated strains were detected by traditional PCR,multi-PCR-SSCP technique and drug susceptibility test.Results：Results of drug susceptibility tests were multidrug-resistant isolated strains had 41strains,among drug-resistant isolated strains of INH,RFP,SM were 35,31,31 strains;multi-PCR-SSCP had the complete same results compared with traditional PCR-SSCP that were the gene mutation rate of katG,rpoB,rpsL was 65.7%（23/35）,84%（26/31）,71%（22/31）.And 17 drug sensitive strains only one strain′s rpoB gene had the different multi-PCR-SSCP pattern compared with that of the reference strain（H37Rv）.Conclusion：Multi-PCR-SSCP is a sensitive and specific method for rapid detecting the genes mutations of MTB,and useful in rapid detecting the multidrug-resistant MTB.Multi-PCR-SSCP combined with traditional drug susceptibility test will improve clinical management of tuberculosis patients.