实时荧光PCR分子信标检测耐利福平结核分枝杆菌rpoB基因  

Real-time fluorescence PCR-molecular beacon assay for detecting mutations in rpoB genes of rifampin-resistant Mycobacterium tuberculosis

作　　者：孙桂芝[1] 高铁杰 钟镐镐[3] 康丽军[1] 张治国 衡万杰[3] 吴秉铨[3] 刘威[1] 

机构地区：[1]北京胸科医院检验科,100095 [2]北京昌平结核病防治所 [3]北京大学医学部病理学系

出　　处：《中华检验医学杂志》2008年第4期429-432,共4页Chinese Journal of Laboratory Medicine

摘　　要：目的 应用实时荧光PCR分子信标技术，建立快速检测临床标本中结核分枝杆菌利福平rpoB相关耐药突变点方法，探讨其缩短耐药实验报告时间的临床应用价值。方法以分枝杆菌药物敏感性实验绝对浓度法为标准，12株非结核分枝杆菌、4株非分枝杆菌作对照，对174例结核患者临床分离株应用实时荧光PCR分子信标方法，检测利福平rpoB核心区域的耐药突变点并将结果与直接测序进行比较。结果（1）实时荧光PCR分子信标方法：82例结核分枝杆菌利福平敏感菌株中，3例发生rpoB基因突变，特异度为96．3％；92例结核分枝杆菌利福平耐药菌株中，82例检出耐药突变，敏感度为89．1％；准确性为92．5％。（2）DNA直接测序分析：82例结核分枝杆菌利福平敏感株中，1例发生rpoB基因突变，特异度为98．8％；92例结核分枝杆菌利福平耐药菌株中，83例发生rpoB基因突变，敏感度为90．2％；准确性为94．2％。检测174株结核分枝杆菌临床分离菌株，与实时荧光PCR分子信标方法检测一致性为98．3％（171／174）。结论 实时荧光PCR分子信标方法检测耐利福平结核分枝杆菌rpoB基因突变点可作为结核患者快速耐药检测的初筛方法之一。Objective To establish a rapid method to detect mutations in rpoB genes of rifampinresistant Myeobacterium tuberculosis in clinical specimens using Real-time fluorescence PCR molecular beacon assay. Methods 174 strains of Myeobacterium tuberculosis clinical isolates were analyzed using real-time fluorescence PCR molecular beacon assay followed with DNA sequencing while 12 strains of NTM and 4 strains of bacteria other than Mycobacterium tuberculosis were used as the contrast. Results Eightytwo 89. 1 of 92 rifampin （ BIF）-resistant strains and 3 of 82 RIF-sensitive strains were found to harbor mutation in the rpoB gene using real-time fluorescence PCR-molecular beacon assay. The specificity, sensitivity, and accuracy of this assay were 96. 3% ,89. 1%, and 92. 5% ,respectively. Eithty-three of 92 RIF-resistant strains and 1 of 82 RIF-sensitive strains were found to harbor mutation in the rpoB gene using the direct DNA sequencing. The specificity, sensitivity, and accuracy of the direct DNA sequencing were 98. 8,90. 2%, and 94. 2% ,respectively. As compared with real-time PCR molecular beacon assay, 171 of 174（98. 3% ） strains of myeobacterium tuberculosis clinical isolates had the same results. Conclusion Real-time fluorescence PCR-molecular beacon assay can be used as a rapid screen method to detect RIF-resistant isolates.

关 键 词：聚合酶链反应 分枝杆菌 结核 细菌蛋白质类 突变 利福平 抗药性 细菌 

分 类 号：R686[医药卫生—骨科学]