结核分枝杆菌耐异烟肼katG基因LiPA检测研究

目的利用LiPA技术检测结核分枝杆菌耐异烟肼katG基因，评价LiPA技术临床应用的可行性。方法应用比例法确定80株结核分枝杆菌临床分离株其耐药性，用katG基因PCR扩增产物进行DNA测序，然后用LiPA检测技术检测结核分枝杆菌katG基因的突变，对比3种方法的检测结果，评估LiPA检测技术的准确性。结果在80株结核分枝杆菌中，35株为异烟肼敏感株，45株为异烟肼耐药株。敏感株katG基因扩增阳性率为100％，耐药株为91．1％（41／45）。耐异烟肼茵株katG的缺失率为8．9％。41株扩增阳性耐药株中有26株检测到315位密码子的突变，分别为ACC（60．97％，25／41），ATC（2．44％，1／41），无突变（36．58％，15／41）。LiPA技术检测、药物敏感性试验和基因测序的结果基本一致。结论结核分枝杆菌katG基因突变的LiPA技术敏感性高，特异性强，可用于结核分枝杆菌的快速药物敏感性试验评价。

Line probe assay for detection of katG gene mutations associated with Mycobacterium tuberculosis resistance to INH

Objective To detect the katG gene resistant to isoniazid in Mycobacterium tuberculosis by LiPA and evaluate the feasibility of clinical application of the LiPA technique. Methods There 80 clinical isolates was detected by L-J cultivation to determine drug resistance and the PCR amplified katG genes by DNA sequence.Simultaneously, LiPA was detected the mutation of katG in Mycobacterium, tuberculosis isolates.These three assays were compared to evaluate the accuracy of LiPA technique. Results The 80 clinical isolates including 35 strains sensitive to INH and 45 strains resistant to INH. katG was successfully amplified from 100% of the susceptible strains and 91.1%（41/45） resistant strains. 4 of 45 INH resistant isolates katG was deleted, katG 315 codon mutations were observed in 26 of 41 katG at sites of 315 ACC （Thr,25/41）,ATC （Ile,1/41）.The mechanisms of katG analyzed by LiPA we prepared were identical to katG sequencing. Conclusion The LiPA technique for determination of Mycohaeterium tuberculosis resistant to INH is specific, sensitive and can be used as a routine in clinical laboratory.