结核分枝杆菌耐异烟肼基因突变的快速检测

Rapid detection of gene mutation in isoniazid-resistant M.tuberculosis

目的 :探讨编码过氧化氢 过氧化物酶的katG基因突变与结核分枝杆菌异烟肼 (INH)耐药性的相关关系 .方法 :根据结核分枝杆菌 genebank中katG序列 ,自行设计特异性寡聚核苷酸引物 ,采用聚合酶链反应 单链构象多态性 (poly merasechainreaction singlestrandconformationpolymorphism ,PCR SSCP)分析和直接测序法 (directsequencing,DS)分析结核分枝杆菌中katG基因突变情况 .以H3 7Rv标准株为对照 .结果 :所有 2 3株敏感菌均未有SSCP结果异常 ;35株耐药菌中 ,有 2株 (5 .7% )katG基因扩增阴性 ,且发生在高度耐药菌中 .进一步分析发现 ,SSCP法突变检出 2 3株 (6 5 .7% ) ,测序法突变检出 2 4株 (6 8.6 % ) ,符合率为 95 .8% (2 3/ 2 4 ) .结论 :参照测序法对耐药菌突变序列的分析结果 ,PCR SSCP敏感、特异 ,可快速检测结核分枝杆菌katG耐药基因突变 。

AIM: To investigate the relationship between the mutation of the katG gene and isoniazid resistance in M. Tuberculosis . METHODS: The specific oligonucleotide primers of the katG gene was designed according to the EMBL:X68081 in genebank. KatG gene mutations were detected by PCR SSCP and DS. H 37 Rv standard isolates were used as control. RESULTS: Compared with the control, no difference was found among the 23 sensitive isolates. Among the 35 resistant isolates, 2 (5.7%) lacked katG gene in those highly INH resistant isolates. The mutation of 23 (65.7%) isolates was detected by the PCR SSCP and that of 24 isolates was detected by DS. The coincidence rate for two the techniques was 95.8% (23/24). CONCLUSION: PCR SSCP is a sensitive and specific method for rapid detection of katG gene mutations in M. tuberculosis and its drug resistance.