CAJ | 학술논문

目的研究结核分枝杆菌利福平耐药突变实时PCR检测试剂盒(探针熔解分析)的临床应用价值.方法用37株非结核分枝杆菌考察该方法的特异性,用菌株H37Rv考察其检测限,并检测962份结核分枝杆菌培养标本的rpoB基因利福平耐药决定区突变,检测结果经测序验证.结果 37株非结核分枝杆菌中仅有3株出现耐药突变峰,检测限考察结果表明该方法每反应可重复检出30个菌.用该试剂盒检测962份标本,检出突变株186份,野生株751份,扩增失败标本25份.选取2009年11月以后的标本中试剂盒检出为突变的标本112份,并数字表法随机选取200份试剂盒检出为阴性的标本,进行测序验证,除5份测序失败其余107份标本的DNA序列分析结果与试剂盒检测结果一致.结论该试剂盒准确快速,方便易行,是检测结核分枝杆菌rpoB基因突变的有力工具.
목적 연구결핵분지간균리복평내약돌변실시PCR검측시제합(탐침용해분석)적림상응용개치.방법 용37주비결핵분지간균고찰해방법적특이성,용균주H37Rv고찰기검측한,병검측962빈결핵분지간균배양표본적rpoB기인리복평내약결정구돌변,검측결과경측서험증.결과 37주비결핵분지간균중부유3주출현내약돌변봉,검측한고찰결과표명해방법매반응가중복검출30개균.용해시제합검측962빈표본,검출돌변주186빈,야생주751빈,확증실패표본25빈.선취2009년11월이후적표본중시제합검출위돌변적표본112빈,병수자표법수궤선취200빈시제합검출위음성적표본,진행측서험증,제5빈측서실패기여107빈표본적DNA서렬분석결과여시제합검측결과일치.결론 해시제합준학쾌속,방편역행,시검측결핵분지간균rpoB기인돌변적유력공구.
Objective To evaluate the clinical performance of a probe melting analysis ( PMA)-based real-time PCR detection kit in rapid detection of rifampin-resistant mutations in Mycobacterium tuberculosis (MTB). Methods The specificity of the assay was evaluated by detecting 37 non-tuberculous mycobacteria (NTM) ,and the detection limit of the method was evaluated by genomic DNA of a standard strain H37Rv. Finally, 962 clinical isolates were analyzed with the PMA assay by detecting mutations in rifampin resistance-determining region (RRDR) of rpoB gene, and results were verified with DNA sequencing. Results Among 37 NTM strains,three strains showed drug resistant mutation signals. The PMA method could detect down to 30 bacteria per reaction. Sample analysis showed that 186 of 962 isolates were mutants,751 isolates were wild type and 25 isolates failed to give amplification signals. Among the mutant samples detected, 112 samples from November 2009 to April 2010 were further analyzed by sequencing, as well as 200 wild-type samples. The results showed a complete agreement with the PMA assay except for 5 samples failed in sequence analysis. Conclusion The PMA assay is rapid, accurate and easy-to-use, and thus can be used for detection of rifampin-resistant in clinical isolate samples.