环介导等温扩增技术快速检测结核分枝杆菌核酸  

Rapid detection for Mycobacterium tuberculosis by loop-mediated isothermal amplification

作　　者：沈会平[1] 张耀祺[1] 杨坚 石磊[1] 陈涛[3] 李国周 赵红波[5] 莫自耀[5] 

机构地区：[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

出　　处：《临床检验杂志》2011年第5期378-380,共3页Chinese Journal of Clinical Laboratory Science

基　　金：呼吸疾病国家重点实验室开放课题(2007DA780154F0904);广州经济技术开发区科技项目(20090723000210000015)

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100 fg;而实时荧光PCR检测限为1 pg。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。Objective To develop a rapid loop-mediated isothermal amplification （LAMP） method for detecting the nuclear acid of Mycobacterium tuberculosis（MTB）. Methods The repetitive insertion sequence IS6110 was used as target gene to develop an efficient LAMP method by which MTB was specifically detected. MTB in 100 clinical 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 as 100 fg of DNA for each tube, while the detection limit of conventional PCR was 10 pg for each tube. In the 100 clinical sputum samples the positive rate of MTB detected by direct smear, LAMP and real-time PCR was 28%, 39% and 38% , 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 identification of MTB in clinical laboratory.

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 

分 类 号：R378.91[医药卫生—病原生物学]