基于Ag85B mRNA的荧光定量逆转录PCR快速检测活结核分枝杆菌方法的建立和评价  

Establishment and assessment of real time reverse transcription PCR for rapid detection of viable mycobacterium tuberculosis based on Ag85B mRNA

作　　者：高文凤[1] 罗影殊[2] 黄偌颖[2] 徐园红[3] 钟晶[3] 刘衡川[2] 

机构地区：[1]四川省疾病预防控制中心结核病预防控制所,四川成都610041 [2]四川大学华西公共卫生学院医学检验教研室,四川成都610041 [3]成都市结核病防治院,四川成都610016

出　　处：《中国卫生检验杂志》2013年第18期3477-3480,3491,共5页Chinese Journal of Health Laboratory Technology

摘　　要：目的建立评价荧光定量逆转录PCR快速检测活结核分枝杆菌的方法。方法建立检测Ag85B mRNA的荧光定量逆转录PCR方法,评价其特异性、灵敏度、重复性、区分细菌存活状态及检测临床样本的能力。结果该方法特异性强,仅扩增结核分枝杆菌。最低检出限是50 Copies/反应,菌落灵敏度为1.3×103cfu/ml,重复性好,可有效区分死菌和活菌。以培养法为"金标准",PCR法的灵敏度为100.00%,特异度为84.38%,符合率为93.15%,Kappa值为0.86。结论荧光定量逆转录PCR检测方法简便、快速、可靠,能有效区分结核分枝杆菌的存活状态,具有较高灵敏度和特异性,适用于临床样本的检测,为结核病的早期诊断、疗效评价及药敏情况分析提供了技术支持,有利于结核病的防控。Objective To establish and assess a real time reverse transcript PCR assay for rapid detection of viable mycobacteri- um tuberculosis. Methods The real time reverse transcript PCR was founded for mycobacterium tuberculosis detection targeting on Ag85B mRNA, and the method was assessed regarding its specificity, sensitivity, replicability, differentiation of live and dead mycobacterium tuberculosis and possibility of clinical utilization. Results The establisbed real time reverse transcript PCR could exclusively detect mycobacterium tuberculosis complex apart non tuberculous mycobacteria. The sensitivity was 50 copies/ reaction or 1.3 × 10-3 cfu/ml. This real time RT - PCR could effectively distinguish viable condition of mycobacterium tuberculo- sis. Compared with culture method, the golden criterion, the sensitivity of the method was 100%, the specificity was 84.38% , the consistency was 93.15% and Kappa value was 0.86. Conclusion Based on real time reverse transcript PCR targeting Ag85B mRNA, a rapid, specific and sensitive method was successfully established to detect viable mycobacterium tuberculosis, which could be applied to clinic. The method could serve as a technique support in early detection, efficacy assessment and drug -resistance analysis of TB, being helpful to tuberculosis prevention and control.

关 键 词：结核分枝杆菌 活菌检测 荧光定量逆转录PCR AG85B MRNA 

分 类 号：R378.911[医药卫生—病原生物学]