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

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.