应用探针熔解分析法检测结核分枝杆菌rpoB基因突变

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.

Abstract：

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.

Analyzing the mutations of rpoB gene in Mycobacterium tuberculosis clinical isolates by probe melting analysis assay

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.