RNA恒温扩增实时检测技术快速鉴定鸟分枝杆菌  

Analysis and identification for Mycobaterium avium with real-time isothermal transcription-mediated RNA amplification assay

作　　者：李园园[1,2] 陆俊梅[2] 程松[2] 胡忠义[2] 崔振玲[2] 

机构地区：[1]苏州大学医学部基础医学与生物科学学院,江苏苏州215123 [2]同济大学附属上海市肺科医院/上海市结核病(肺)重点实验室,上海200433

出　　处：《临床检验杂志》2013年第9期641-644,共4页Chinese Journal of Clinical Laboratory Science

基　　金：国家"十二五"传染病重大专项课题(2013ZX10003001-003)

摘　　要：目的建立RNA恒温扩增技术快速鉴定鸟分枝杆菌(RIARD-MA)的方法,评估鉴定分枝杆菌临床分离株的应用效果。方法以鸟分枝杆菌(MA)的16S rRNA的特异序列为检测靶标,设计RNA探针和带有T7启动子的逆转录扩增引物,42℃恒温扩增实时检测,对21种分枝杆菌标准株、4株非分枝杆菌和259株分枝杆菌临床分离株进行鉴别检测;同时以PCR测序结果为参考对照。结果 RIARD-MA可以在2 h内完成检测,能准确鉴定出21种分枝杆菌标准株及4株非分枝杆菌检测中的MA,检测灵敏度达103CFU/mL;检测分枝杆菌临床分离株中MA的敏感性、特异性均可达100%。结论 RIARD-MA鉴定MA具有较高的敏感性、特异性,检测快速,有望作为一种新的MA临床分离株鉴定方法。Objective To establish and evaluate a real-time isothermal transcription-mediated RNA amplification assay for identifica- tion of Mycobacterium avium in clinical isolates. Methods RNA probes and specific primers of reverse transcription and amplification for T7 promoter were designed based on the sequence of M. avium 16S rRNA. The isothermal successive cycles of amplification using T7 RNA polymerase at 42 ℃ were performed for real-time detection. In total, 21 reference strains of Mycobacterium, 4 non-mycobacte-rium strains and 259 clinical strains were detected by the established assay to evaluate the specificity and sensitivity, and the results were compared with those of PCR sequencing. Results The procedures of the established assay could be completed within 2 hours. The 21 reference strains of Mycobacterium and 4 non-mycobacterium strains were correctly identified and the detection limit of the assay for M. aium was 10^3 CFU/mL. Both the sensitivity and specificity reached up to 100% in the detection for clinical isolates. Conclu-sion The real-time isothermal transcription-mediated RNA amplification assay established in this study should be sensitive, specific and rapid for the identification of M. avium and may be hopeful for rapid identification of M. avium in clinical isolates.

关 键 词：RNA恒温扩增 鸟分枝杆菌 鉴定 

分 类 号：R378.91[医药卫生—病原生物学] R446.5[医药卫生—基础医学]