结核分支杆菌耐利福平耐药基因的检测

Defection of the Rifampin - Resistant Mycobacterimn Tuberculosis

目的 评估应用聚合酶链反应-单链构象多态性(PCR-SSCP)技术检测rPOB基因突变在结核分支杆菌耐利福平(RFP)耐药性测定中的应用价值。方法 采用PCR-SSCP方法对93株结核分支杆菌临床分离株(其中药物敏感株35株,耐RFP或含耐RFP耐多药株58株)和33例耐RFP肺结核病人痰标本rPOB基因突变进行检测,并对部分常规药敏试验耐药而用PCR-SSCP未检测到rPOB基因突变的PCR扩增产物阴性菌株用双脱氧末端终止法进行测序分析。结果 以结核分支杆菌H_(37)RV为对照,35株药物敏感株的rPOB基因PCR扩增产物258 bp片段SSCP图谱正常;58株耐RFP分离株中,52株rPOB基因SSCP图谱异常,其敏感性为89.7％。SSCP图谱正常的5株耐RFP的PCR产物经测序证实,4株在531位密码子TCG→TTG,1株在526密码子CAC→TAC,l株未改变。33例耐RFP肺结核病人痰标本有30例PCR扩增阳性,23例SSCP图谱与H_(37)RV株有差异,rPOB突变率为76.7％。结论 PCR-SSCP技术可以快速、准确地检测结核分支杆菌rPOB基因突变,该技术有望直接用于临床标本耐药性检测。

Objective Detection of rPOB gene mutations in refampin - resistant Mycobacterium tuberculosis using Polymerase chain reaction - single - strand conformation polymorphiSM (PCR - SSCP). Methods 93 clinical isolates of Mycobacterium tuberculosis (35 drug susceptible, 58 rifampin - resistance or multidrug resistance including rifampin), and 33 sputum specimens from pulmonary tuberculosis were detected using PCR - SSCP. PCR products of isolates that rPOB gene mutations were not found using PCR - SSCP were sequenced using double deoxidization chain terminating. Results SSCP pattern of reference Mycobacterium tuberculosis H^RV as control, SSCP patterns of 52 rifampin resistant clinical isolates were different from the control.The specificity is 89.7% .PCR products of rPOB gene in rifampin resistant isolates that have the same SSCP pattern as the control were sequenced. Sequence analysis showed 5 isolates had rPOB gene mutations and 1 isolate was not found rPOB geng mutations.The two most frequent rPOB gene mutations are Leu - 531 (4 isolates, TCG→TTG) and Tyr-526 (1 isolate, CAC→TAC) .33 sputum specimens from pulmonary tuberculosis were detected using PCR - SSCP, 3 sputum specimens were not detected PCR products as PCR amplification.23 PCR products of sputum specimens had different SSCP patterns from the control.The rPOB mutations rate of sputum specimens was 76.7% using PCR - SSCP. Condutions rPOB gene mutation of Mycobacterium tuberculosis is rapidly and accurately detected using PCR - SSCP. PCR - SSCP is able to apply to detect specimens on drug susceptibility testing.