新型布尼亚病毒环介导等温扩增可视化检测方法的建立  

Visual detection of SFTSV by loop-mediated isothermal amplification with hydroxynaphthol blue dye

作　　者：吕恒[1] 钟璟浩 张锦海[1] 王平[1] 王长军[1] 

机构地区：[1]南京军区疾病预防控制中心,江苏南京210002

出　　处：《中国病原生物学杂志》2016年第3期225-228,共4页Journal of Pathogen Biology

基　　金：国家重大传染病专项课题(No.20013ZX10004-008;2013ZX10001001-004);全军青年培育项目(No.13QNP051);江苏省科技支撑计划(社会发展)项目(No.BE2012609)

摘　　要：目的应用环介导等温扩增(LAMP)技术建立新型布尼亚病毒(SFTSV)可视化快速检测方法。方法针对新型布尼亚病毒的S片段基因设计LAMP引物建立LAMP方法。反应体系内加入染料羟基萘酚蓝(HNB)作为反应扩增指示剂,根据LAMP浊度仪扩增结果优化反应条件,根据HNB颜色变化进行结果判定,评估LAMP方法的特异性和灵敏性。结果建立的LAMP方法最低检出限为10拷贝/反应管;方法的特异性高,仅SFTSV反应管颜色由紫罗兰变为天蓝色,而汉坦病毒、新疆出血热、Q热、马尔堡、埃博拉病毒及阴性对照反应管均未发生变色反应;LAMP反应的最佳温度为63℃,其扩增效率高于常规PCR,试验过程仅需30min。结论建立的可视化SFTSV LAMP检测方法具有特异性强,灵敏性高,实验设备要求简单等优点,可用于SFTSV的快速检测。Objective A loop-mediated isothermal amplification（LAMP）assay was developed for rapid visual detection of severe fever with thrombocytopenia syndrome bunyavirus（SFTSV）. Methods A set of four specific primers specific to six regions of S gene was designed.A reaction was performed in a single tube with the addition of hydroxynaphthol blue（HNB）dye prior to amplification.A Loopamp Real-time Turbidimeter was used to ascertain amplification conditions.Results were determined based on a change in the color of HNB,and the sensitivity and specificity of the LAMP method were evaluated and verified with agarose gel electrophoresis. Results The optimum temperature was 63 ℃.The optimum concentration of HNB was 150μmol/L.The detection limit of the LAMP assay was 10 copies per tube,which is higher than that of conventional PCR.The LAMP assay had a high level of specificity.Only SFTSV tube changed color.Detection only took 30 minutes. Conclusion Results suggested that the LAMP assay with HNB dye provided a useful tool for the rapid detection of SFTSV.

关 键 词：新型布尼亚病毒(SFTSV) 可视化检测 环介导等温扩增(LAMP) 羟基萘酚蓝(HNB) 

分 类 号：R373[医药卫生—病原生物学]