探针熔解曲线法快速检测结核分枝杆菌利福平、异烟肼、乙胺丁醇及链霉素耐药突变  

Rapid detection of rifampin-,isoniazid-,ethambutol- and streptomycin-resistant mutations in Mycobacterium tuberculosis by probe melting curve analysis

作　　者：严虹[1] 施旭东[1] 胡春梅[2] 郭晶[2] 张侠[2] 

机构地区：[1]南京市胸科医院检验科,南京210029 [2]南京市胸科医院结核科,南京210029

出　　处：《临床检验杂志》2015年第10期729-733,共5页Chinese Journal of Clinical Laboratory Science

基　　金：南京市医学科技发展重点项目(ZKX12035;ZKX13042);南京市医学科技发展资金(QRX11229)

摘　　要：目的评价探针熔解曲线法检测结核分枝杆菌利福平(RIF)、异烟肼(INH)、乙胺丁醇(EMB)及链霉素(SM)耐药突变的方法学应用价值。方法用PCR探针熔解曲线法检测110株结核分枝杆菌临床分离株RIF、INH、EMB及SM耐药突变情况,以传统的比例法药物敏感性试验检测结果为标准,评价探针熔解曲线法的敏感性、特异性和一致率,并应用测序方法分析结果不一致菌株的耐药突变位点。结果以比例法检测结果为标准,熔解曲线法检测RIF耐药的敏感性为98.51%,特异性为95.35%,诊断符合率为97.27%;检测INH耐药的敏感性为94.12%,特异性为95.24%,诊断符合率为94.55%;检测EMB耐药的敏感性为88.64%(39/44),特异性为75.76%(50/66),诊断符合率为80.91%(89/110);检测SM耐药的敏感性为82.98%,特异性为80.95%,诊断符合率为81.82%。两种检测方法结果不符菌株的测序结果大部分与熔解曲线法的检测结果一致。结论探针熔解曲线分析法检测速度快,敏感性高,特异性强,可用于结核分枝杆菌耐药突变的快速检测。Objective To evaluate the application value of a probe melting curve analysis-based assay for detection of rifampin-,isoniazid-,ethambutol- and streptomycin-resistant mutations in Mycobacterium tuberculosis. Methods rifampin-,isoniazid-,ethambutol- and streptomycin-resistant mutations in 110 Mycobacterium tuberculosis（ MTB） clinical isolates were detected by fluorescent PCR probe melting curve analysis assay and conventional drug susceptibility test（ proportion method）. The sensitivity,specificity and consistency of the new method were analyzed by comparing the results with that of proportion method. The resistance-related mutations in the strains with different results were detected by DNA sequencing. Results Compared to the results of proportion method,the sensitivity,specificity and accuracy of probe melting curve analysis for rifampin resistance were 98. 51%,95. 35% and 97. 27%,for isoniazid resistance were94. 12%,95. 24% and 94. 55%,for ethambutol resistance were 88. 64%,75. 76% and 80. 91%,and for streptomycin resistance were82. 98%,80. 95% and 81. 82%,respectively. We sequenced the strains with different results for the two methods and the results exhibited that most of the strains was consistent with melting curve analysis assay. Conclusion Probe melting curve analysis assay should be a rapid method for identification of multidrug-resistance mutation of MTB with high sensitivity and specificity as a screening test.

关 键 词：结核分枝杆菌 耐药突变 利福平 异烟肼 乙胺丁醇 链霉素 

分 类 号：R446.5[医药卫生—诊断学]