用分子生物学技术快速检测耐多药结核分枝杆菌

目的：用PCR—SSCP技术检测耐INH、RFP、SM结核分枝杆菌分离株KatG、rpoB、rpsL基因突变，评价其在快速检测结核分枝杆菌耐药性方面的临床价值。方法：以结核分枝杆菌H；vRv为对照，20株耐多药菌株和20株敏感株，分别用PCR—SSCP方法检测KatG、rpoB、rpsL基因突变，并将SSCP结果与药敏试验结果作对照分析。结果：PCR—SSCP方法检测20株敏感株SSCP带语均与H37RV标准株相同，而20株耐多药结核分枝杆菌KatG、rpoB、rpsL基因突变的阳性率分别为70％，100％和75％。发现20株耐多药菌株中有11株(55％)共INH、RFP、SM3种耐药遗传标志均发生突变，7株(35％)有2种耐药遗传标志突变，2株(10％)只有RFP耐药标志突变。结论：PCR—SSCP方法敏感、特异，可快速检测结核分枝杆菌KatG、rpoB、rpsL耐药基因突变，有利于耐多药结核分枝杆菌耐药性的快速检测。

Rapid Detecting Mycobacterium Tuberculosis of Multiple Drug Resistance by The Molecular Biologic Technique

OBJECTIVE: To evaluate the value of detecting katG, rpoB and rpsL genie mutation in M. tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis. METHODS:Twenty drug-susceptible M. tuberculosis and 20 multidrug-resistant M. tuberculosis (MDRT) which are resistant to rifampin, isoniazid and streptomycin were evaluated by PCR-SSCP, with H37RV reference as control group. RESULTS:Twenty drug-susceptible clinical isolates and M. tuberculosis H37RV strain displayed identical SSCP patterns. Part of multidrug-resistant clinical isolates shown a different SSCP patterns from M. tuberculosis H37RV strain , and a few of MDRT displayed the same patterns.Compared with drug sensitive test, the positive rate to detect katG, rpoB, rpsL genes by PCR-SSCP were 70%, 100%, 75%, respectively.Mutations of three genetic markers of rifampin, isoniazid, and streptomycin resistance were detected in 11 of 20 multidrug-resistant strains. Alterations in two genes were identified in 7 of these isolates. Alteration in one genes were detected in 2 isolates. CONCLUSION: The strains of katG, rpoB, rpsL gene mutations could be readily identified by PCR-SSCP analysis. PCR-SSCP is a simple and reliable method of detecting katG, rpoB, rpsL gene mutation, and it is useful in rapid detecting the multidrug-resistant M. tuberculosis.