耐异烟肼结核分枝杆菌katG基因突变快速筛选的研究

Rapid screening of katG gene mutation in isoniazid-resistant Mycobacterium tuberculosis

目的 :建立结核分枝杆菌 (MTB) kat G基因点突变的筛选方法 ,了解结核分枝杆菌耐异烟肼 (INH)的状况。方法 :采用聚合酶链反应 -限制性片段长度多态性分析 (PCR- RFL P)技术 ,对 8株耐 INH的临床 MTB分离株、16株对 INH敏感的临床分离株及 MTB标准株 H3 7Rv的 kat G基因 ,进行聚合酶链 (PCR)反应扩增 kat G基因片段 ,再用 Msp I内切酶分别对 PCR产物进行消化反应。如果有 315位点 AGC突变为 ACC,则将增加一个切点。结果 :检测到 8株耐药株中有 7株增加了一个 Msp I内切酶切点 ,耐药株中 kat G基因点突变检出率为 87.5 % (7/ 8) ;16株敏感株和标准株均无额外的 Msp I内切酶切点。未发现 kat G基因序列的缺失。结论 :kat G基因点突变是 MTB耐 INH的重要机理之一 ;用 PCR- RFLP检测耐 INH的 MTB的 kat G基因点突变具有快速、简便、敏感性高、特异性强的特点。

Objective: To evaluate the relationship between katG gene mutation and isoniazid (INH) resistance and to develop a rapid screening method of point mutation in the katG gene associated with MTB resistance. Methods : Twenty four clinical isolates of MTB with 8 INH resigtance isolates and 16 INH sensitive isolates were analyzed by PCR RFLP, with the H 37 Rv reference strain as the control. Results: G→C point mutations were detected in 7 of 8 isoniazid resistant strains and no gene mutation was shown in 16 isoniazid sensitive isolates. The sensitivity and specificity were 87.5% and 100% respectively. No katG gene sequence deletion was observed in any specimen. Conclusion: Our results suggest katG gene mutation is one of the most important mechanisms of INH resistant TB. PCR RFLP may be useful in detection of katG gene mutation.