H3N8亚型马流感病毒 RT-LAM P 检测方法的建立

摘要

为了建立检测 H3N8亚型马流感病毒的RT‐LAMP方法，根据 H3N8亚型马流感病毒 HA基因序列，设计了2对LAM P引物，经过优化反应条件，建立了RT‐LAM P检测 H3N8亚型马流感病毒方法。结果表明，RT‐LAMP方法能够在63℃恒温条件下、75 min内实现目的基因片段的大量特异性扩增，通过荧光显色就可直接用肉眼判断结果。对 H7N7亚型马流感病毒、马动脉炎病毒、马鼻肺炎病毒的核酸无交叉反应，具有较好的特异性；方法的灵敏度比常规RT‐PCR的高10倍；该方法操作简便快速、省时省力，而且灵敏度高、特异性强，对 H3N8亚型马流感病毒的检测具有实际的应用价值。%To develop a rapid ,sensitive ,and specific approach for the detection of H 3N8 subtype equine in‐fluenza virus (EIV) ,an RT‐LAMP assay with four pairs of primers designed according to the conserved sequences of EIV HA gene was established in the present study .With optimized reaction condition ,the vi‐ral RNA was amplified within 75 min at 63 ℃ ,then the positive results of RT‐LAMP assay were judged through visible green .The established RT‐LAMP was highly specific and no cross reaction with other e‐quine viruses such as H7N7 subtype EIV ,EAV ,EHV .The sensitivity was 10 times higher than that of conventional RT‐PCR .The method established provided a promising method for the detection of H 3N8 subtype EIV .