环介导等温扩增技术快速检测结核分枝杆菌核酸.

文章编号:1001-764X(201105-378-03

·研究生园地·环介导等温扩增技术快速检测结核分枝杆菌核酸*

沈会平1,张耀祺1,杨坚2,石磊1,陈涛3,李国周4,赵红波5,莫自耀5(1．华南理工大学轻工与食品学院,广州510640;2．迪澳生物科技有限公司,广州510663;3．广东省结核病防治研究所,广州510630;4．东莞市慢性病防治院,广东东莞

523008;5．广州医学院呼吸疾病国家重点实验室,广州510230

摘要:目的建立一种快速准确的检测结核分枝杆菌(MTB核酸的环介导等温扩增(LAMP方法。方法以重复插入序列IS6110为目的基因,设计LAMP引物,特异检测MTB核酸。用本法与痰涂片抗酸染色镜检法、实时荧光PCR法对100例可疑患者痰标本进行对比检查。结果LAMP法特异性强,仅扩增MTB复合群核酸;灵敏度高,检测限达100fg;而实时荧光PCR 检测限为1pg。对100例疑似结核病患者痰液标本检测,涂片抗酸染色法、LAMP法、实时荧光PCR法的阳性率分别为28%、39%和38%。结论本研究建立的LAMP方法检测MTB核酸特异性强、灵敏度高、时间短且操作简便,有望成为临床快速检测MTB的新方法。

关键词:结核分枝杆菌;环介导等温扩增法;IS6110基因;实时浊度仪

中图分类号:R378．91文献标志码:A

Rapid detection for Mycobacterium tuberculosis by loop-mediated isothermal amplification

SHEN Hui-ping1,ZHANG Yao-qi1,YANG Jian2,SHI Lei1,CHEN Tao3,LI Guo-zhou4,ZHAO Hong-bo5,MO Zi-yao5(1．College of Light Industry and Food Science,South China University of Technology,Guangzhou510640,Guangdong;2．Diao Bio-Technology Co．Ltd,Guangzhou510663,Guangdong;3．Guangdong Research Institute for Mycobacterium tuberculosis Control,Guangzhou510630,

Guangdong;4．Dongguan Hospital for Chronic

Disease,Dongguan523008,Guangdong;5．The State Key Laboratory of Respiratory Diseases,Guangzhou Medical University,Guangzhou510230,Guangdong,China

Abstract:Objective To develop a rapid loop-mediated isothermal

amplification(LAMPmethod for detecting the nuclear acid of Mycobacterium tuberculosis(MTB．Methods The repetitive insertion sequence IS6110was used as target gene to develop an efficient LAMP method by which MTB was specifically detected．MTB in100clinical sputum samples were detected simultaneously by direct smear,LAMP and real-time fluorescent PCR．Results The developed LAMP method showed high specificity for detecting pathogenic strains of MTB complex．The detection limit of LAMP assay was as low as100fg of DNA for each tube,while the detection limit of conventional PCR was10pg for each tube．In the100clinical sputum samples the positive rate of MTB detected by direct smear, LAMP and real-time PCR

was28%,39%and38%,respectively．Conclusion The developed LAMP in this study was effective method with high specificity and sensitivity for detection of MTB,so it is expected to become a valuable tool for rapid detection and i-dentification of MTB in clinical laboratory．

Key words:Mycobacterium tuberculosis;loop-mediated isothermal amplification;IS6110gene;real-time turbidity meter

WHO调查报告,仅2008年全球肺结核人数就增加940万,死亡180万[1-2]。中国是全球22个肺结核高负担国家之一,患者数量位居全球第二。肺结核是由结核分枝杆菌(MTB引起的一种慢性疾病。导致人类感染的结核菌大多数是人型MTB,也有10%左右是由牛型MTB。目前,MTB的检测方法主要有镜检法、培养法和核酸扩增等分子生物学方法[3]。镜检法快速、简单,但是灵敏度低[4];培养法准确,但耗

时长。环介导等温扩增(loop-mediated i-sothermal amplification,LAMP是对靶基因的6个特异部位设定4种引物,利用具有链置换活性的Bst DNA聚合酶在恒温条件下

催化新链合成,从而使靶基因高效扩增的一种核酸扩增技术[5]。本实验用LAMP法