摘要
目的 建立耐异烟肼 (isoniazid ,INH )结核分枝杆菌多重聚合酶链反应 单链构象多态性分析 (multiple polymerasechainreaction singlestrandconformationpolymorphism ,multi PCR SSCP)方法 ,快速、特异地同时检出aphC启动子、inhA、katG基因的突变情况 ,用于快速诊断结核分枝杆菌对INH的耐药性。方法 根据结核分枝杆菌的aphC启动子序列、inhA序列、katG序列 ,分别设计出 3对特异性寡聚核苷酸引物 ,采用multi PCR及SSCP技术 ,同时检测对结核分枝杆菌耐INH起作用的这 3个基因的突变情况。结果 对H3 7Rv标准株、临床分离INH敏感株 (2 3株 )及INH耐药株 (3 5株 )分别采用常规PCR和multi PCR同时进行扩增 ,两种扩增方法均能扩增出预期的目的片段 ,且结果符合率达10 0 % ;采用单基因PCR SSCP ,aphC启动子序列突变检出率 17% (6/3 5)、inhA序列突变检出率 2 0 % (7/3 5)、katG序列突变检出率 66% (2 3 /3 5) ;multi PCR SSCP突变检出率 83 % (2 9/3 5)。结论 耐药基因检测指导治疗是一种新探索 ,multi PCR SSCP方法敏感、特异 ,能同时快速有效地检测结核分枝杆菌aphC启动子、inhA、katG 3个INH耐药基因的突变 ,提高检验效率 ,有望成为临床指导用药的好方法。
Objective To develop a new multiple-polymerase chain reaction-single strand conformation polymorphism (multi-PCR-SSCP) system for detecting the aphC promoter, inhA, and katG gene mutations in isoniazid-resistant Mycobacterium tuberculosis isolates in the single reaction, and for the quick diagnosis of isoniazid-resistant Mycobacterium tuberculosis isolates. Methods Three pairs of oligonucleotide primers were designed according to the aphC promoter, inhA, and katG genes of Mycobacterium tuberculosis to examine isoniazid-resistance by multi-PCR-SSCP. Results Isoniazid-sensitivity and resistance were analyzed with general PCR and multi-PCR at the same time, and H 37 Rv was used as a control. These two protocol s amplified the anticipated fragments, the rate of consistency being 100%. By single gene PCR-SSCP, the mutation rates of aphC promoter, inhA, and katG gene were 17%, 20%, and 66%, respectively. The mutation rate detected by multi-PCR-SSCP was 83%. Conclusions Multi-PCR-SSCP is a sensitive and specific method for rapid detection of aphC promoter, inhA, and katG gene mutations in isoniazid-resistant Mycobacterium tuberculosis isolates. Drug-resistant gene detection may be clinically useful in the therapy of tuberculosis.
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2004年第1期23-26,共4页
Chinese Journal of Tuberculosis and Respiratory Diseases
