摘要
目的应用实时PCR熔解曲线法快速检测MTB对利福平和异烟肼的耐药性，并与比例法检测结果进行比较。方法收集深圳市2007--2009年耐药监测项目、全国耐药基线调查项目和深圳市慢性病防治中心门诊患者临床分离株311株，采用实时PCR熔解曲线法检On,0rpoB基因耐药突变热点区、ahpC启动子区（硝～30及-15～-3位点）、inhA启动子区（-7～-8位点）、inhA94和katG315位耐药突变，并与耐药基因测序及表型药敏试验结果进行比较。以比例法药敏试验结果作为MTB药敏试验的金标准，评价实时PCR熔解曲线法的敏感度、特异度与金标准的符合率。结果实时PCR熔解曲线法2～3h即可完成检测，扩增和结果判定在1个反应管内完成。检测利福平耐药的敏感度为97．8％，特异度为97．1％，准确性为97．4％；检测异烟肼耐药的敏感度为86．6％，特异度为98．7％，准确性为92．6％。对耐多药MTB的检测敏感度为88．6％，特异度为100．O％，重复性为100．0％。结论实时PCR熔解曲线法检测MTB对利福平和异烟肼耐药性，与比例法检测结果比较具有很好的一致性，且具有快速、准确的优点，有较好的临床应用价值。
Objective To evaluate the application of a real-time PCR and melting curve analysis assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis （M. tuberculosis）. Methods A total of 311 clinical isolates of M. tuberculosis obtained from Shenzhen Center for Chronic Disease Control （SZCCC） were included in the study. These isolates were collected originally from national baseline survey on drug-resistant tuberculosis, project for drug resistance surveillance in Shenzhen and clinical patients in SZCCC between 2007 and 2009. rpoB gene resistance-determining region, ahpC promoter （-44 to -30 and -15 to -3 ） , inhA promoter （-17 to -8）, inhA 94 and katG 315 were detected by melting curve analysis after real-time PCR, and the resuhs were compared with that of proportion method and DNA sequencing. The performances of the assay in detecting the resistance of rifampin and isoniazid were compared to that of reference proportion method drug susceptibility test. Results Real-time PCR and melting analysis was a closed-tube assay that could be completed within 2 -3 h. Compared to the results of the proportion method, the sensitivity, specificity and accuracy of the assay for rifampin resistance were 97. 8%, 97. 1% and 97. 4% respectively, and for isoniazid resistance were 86. 6%, 98.7% and 92. 6% respectively. Conclusions Real-time PCR and melting analysis is a rapid, accurate and closed-tube method that can be used as a screening test for raoid identification of Muhidrug-resistant tuberculosis.
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2011年第12期888-893,共6页
Chinese Journal of Tuberculosis and Respiratory Diseases
基金
国家“十一五”科技重大专项课题（2008ZX10003-004）
关键词
分枝杆菌
结核
抗药性
多药
利福平
异烟肼
突变
聚合酶链反应
Mycobacterium tuberculosis
Drug resistance, muhiple
Rifampin
Isoniazid
Mutation
Polymerase chain reaction
