摘要
目的:建立可同时检测结核分支杆菌rpoB、katG、rpsL、rrs、embB基因突变的单链探针反向杂交试验技术(PCR-LiPA),评价该技术检测结核分支杆菌利福平、异烟肼、链霉素、乙胺丁醇耐药性的应用价值。方法:采集临床标本,按照实验规程培养、鉴定结核分支杆菌,并对分离到的结核分支杆菌进行药敏试验;PCR扩增结核分支杆菌rpoB、katG、rpsL、rrs、embB耐药基因,单链探针反向杂交试验技术检测耐药基因。将PCR-LiPA结果与DNA测序比较验证方法的准确性,将PCR-LiPA结果与药敏结果比较,了解结核分支杆菌耐药性表型与耐药基因突变的相关性。结果:50株结核分支杆菌传统药物敏感性试验结果显示,12株耐INH,7株耐RFP,15株耐SM,2株耐EMB。PCR-LiPA方法分析结果:耐RFP菌株中4株rpoB基因突变;耐INH菌株中5株KatG基因突变;耐SM菌株中7株、SM敏感株1株rpsl基因突变;耐EMB菌株1株、EMB敏感株1株embB基因突变。与DNA测序结果的符合率为100%。结论:用PCR-LiPA可简便、快速、灵敏、特异地检测出一部分结核分支杆菌耐药株的rpoB、katG、rpsL、rrs、embB基因突变,可用于临床结核分支杆菌耐药性的辅助诊断手段。
Objective：To design the line probe assay for the detection of the rpoB,katG,rpsL,rrs,embB genes in drug-resistant Mycobacterium tuberculosis.Methods：The traditional cultivation,species identification and drug-susceptibility testing of 50 M.tuberculosis were performed in the clinical specimens.50 M.tuberculosis species were identified with PCR-LiPA technique.PCR-direct sequencing（PCR-DS） techniques were used as the control.Results：50 M.tuberculosis isolates performed traditional drug-susceptibility showed that there were 7 rifampicin resistant strains,12 Isoniazid resistant strains,15 streptomycin resistant strains and 2 ethambutol resistant strains.50 M.tuberculosis clinical isolates were detected by LiPA.4 rifampicin-resistant M.tuberculosis of 50 showed the mutation in rpoB genes,5 Isoniazid resistant M.tuberculosis of 50 showed the mutation in katG genes,7 streptomycin resistant M.tuberculosis of 50 showed the mutation in rpsL genes,5 ethambutol resistant M.tuberculosis of 50 showed the mutation in embB genes.The results from PCR-LiPA had 100% consistency with PCR-DS.Conclusion：PCR-LiPA might become a rapid,simple,sensitive and specific method to detect rpoB,katG,rpsL,rrs,embB genes mutations in part of drug-resistant M.tuberculosis.It could be used for clinical detection of drug-resistance as an assistant test.
出处
《中国卫生检验杂志》
CAS
2010年第6期1283-1285,共3页
Chinese Journal of Health Laboratory Technology
