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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 technique for histoplasma infection based on the principle of loop-mediated isothermal amplification (LAMP). Methods Tissue-specific Histoplasma was used as the research object. According to the species-specific M antigen gene sequence, several specific primers based on LAMP technology were designed and screened, and the reaction conditions were optimized. Rapid molecular identification technology. Results The sensitivity of LAMP technology to detect genomic DNA of S. histolyticus was 5.3pg / reaction (ie, 5.3 × 10-12g, about 160 copies of genomic DNA) at 65 ℃ for 90min. A fungal cross-reaction. Conclusion The Histoplasma molecular diagnostic technique based on the LAMP principle we developed has the advantages of simple operation, high specificity and high sensitivity, and has the potential of clinical application.