结核分枝杆菌异烟肼耐药基因katG点突变的快速检测

摘要

目的探讨快速检测结核分枝杆菌异烟肼(INH)耐药基因型的分子药敏方法.方法用聚合酶链反应-单链构象多态性(PCR-SSCP)检测了30株INH敏感菌株及30株耐药株的katG基因,随后用SSCP方法鉴定扩增产物有无突变,以结核分枝杆菌H37Rv作对照.结果所有INH敏感株均观察到katG基因扩增产物,30株INH耐药株中28株观察到katG基因扩增产物.以H37Rv标准株为对照,30株敏感株SSCP带谱与对照相同;28株INH耐药株中13株与对照相同,15株有不同程度的差异.与药敏试验比较,PCR-SSCP检测INH耐药结核菌的敏感性为57%,特异性为100%.结论多数结核分枝杆菌耐INH是由于katG基因突变所致,用PCR-SSCP筛选突变株可达到快速检测结核分枝杆菌INH耐药基因型的目的.%Objective One of the main mechanisms of isoniazid (INH) resistance is catalase peroxidasekatG gene mutation of Mycobacterium tuberculosis. Polymerase chain reaction (PCR) and singlestrand conforma-tion polymorphism (SSCP) technique is a simple, rapid method for detecting gene mutation. In this study, thekatG gene mutation associated with isoniazid resistance was detected by PCR-SSCP technique. Methods Thirtyisoniazid-susceptible M. tuberculosis clinical isolates and 30 isoniazid-resistant isolates were analysed by PCR-SSCPtechnique, with Mycobacterium tuberculosis H37 Rv reference strains as control group. The detection methodinvolved amplification of the isoniazid resistance region of katG gene by PCR and the identification of mutations ofthe amplification products by SSCP analysis. Results Amplification was observed in all clinical isolates except twoisoniazid-resisant isolates. Thirty isoniazid-susceptible clinical isolates and M. tuberculosis H37Rv displayed identicalSSCP patterns. About twenty-eight isoniazid-resistant clinical isolates, fifteen isolates showed PCR-SSCP patternsdifferent from that of H37Rv, and 13 isolates displayed the same patterns as H37Rv. Around fifty seven percent ofisoniazid-resistant clinical isolates showed mutation or deletion of katG gene by SSCP analysis. Conclusion Thechanges of katG gene are associated with isoniazid resistance. PCR-SSCP technique will be useful to rapidly screengene mutation of isoniazid-resistant M. tuberculosis.