一种基于环介导等温扩增技术原理的组织胞浆菌分子诊断技术  

A loop-mediated isothermal amplification method for molecular detection of Histoplasma capsulatum

作　　者：李娟[1] 陈敏[1] 潘搏[1] 雷文知[1] 方文捷[1] 刘加[1] 洪南[1] 李颖芳[1] 廖万清[1] 潘炜华[1] 

机构地区：[1]上海市医学真菌分子生物学重点实验室,上海长征医院皮肤病与真菌病研究所全军皮肤病重点实验室,第二军医大学附属长征医院皮肤科,上海200003

出　　处：《中国真菌学杂志》2016年第2期79-84,共6页Chinese Journal of Mycology

基　　金：重大传染病防治科技重大专项(2013ZX10004612);国家973项目(2013CB531601,2013CB531606);国家自然科学基金(81201269);上海市科委专项(14DZ2272900)

摘　　要：目的研发一种基于环介导等温扩增(1oop-mediated isothermal amplification,LAMP)技术原理的组织胞浆菌感染的分子诊断技术。方法以组织胞浆菌为研究对象,针对其种特异性的M抗原基因序列,设计并筛选数套基于LAMP技术的特异性引物,同时优化其反应条件,建立一种针对组织胞浆菌感染的快速分子鉴定技术。结果该LAMP技术在65℃、90min反应条件下,对组织胞浆菌基因组DNA的检测敏感度达到5.3pg/反应(即5.3×10-12 g,约160个基因组DNA拷贝),且与其相近种属真菌无交叉反应。结论我们研发的这种基于LAMP技术原理的组织胞浆菌分子诊断技术具有操作简便、特异性及敏感性高等优点,具有临床推广应用的潜力。Objective To develop a molecular diagnostic approach for the diagnosis of Histoplasma capsulatum based on the LAMP technology.Methods To establish and optimize a specific primer set of the LAMP technology targeting the sequence of M Antigen for rapid and molecular detection of Histoplasma capsulatum at species level.Results In our study,no cross-reaction was observed after 90 min of LAMP reaction,including DNA of the phylogenetically related species such as Coccidioides immitis,Sporothrix schenckii,Penicillium marneffei,etc.The limit-of-detection of the LAMP for genomic Histoplasma capsulatum DNA was approximately 5.3×10^-12 g per run（approximately 160 genomic copies）.Conclusion Our proposed LAMP assay could represent a potential molecular detection tool for rapid diagnosis of Histoplasma capsulatum as apoint of care test to be used in clinics in the future.

关 键 词：组织胞浆菌 环介导等温扩增技术 分子诊断 快速检测 

分 类 号：R379.9[医药卫生—病原生物学]