基于环介导等温扩增的三种马属动物疫病病原核酸检测方法的建立  

A nucleic acid detection method for three equine pathogens based on loop-mediated isothermal amplification

作　　者：高家乐 钮冰[1] 陈沁[1] 王艳 GAO Jia-le;NIU Bing;CHEN Qin;WANG Yan(School of Life Sciences,Shanghai University,Shanghai 200444,China;Plant and Food Inspection and Quarantine Technology Center,Shanghai Customs,Shanghai 200135,China)

机构地区：[1]上海大学生命科学学院,上海200444 [2]上海海关动植物与食品检验检疫技术中心,上海200135

出　　处：《中国兽医科学》2023年第12期1480-1487,共8页Chinese Veterinary Science

基　　金：上海市科技兴农项目(2020-02-08-00-10-F01470)。

摘　　要：本研究根据GenBank数据库中登录的非洲马瘟病毒、西尼罗病毒和日本脑炎病毒基因序列(GenBank accession No.:NC_006011,No.:NC_009942和No.:NC_001437),构建重组质粒(命名为pUC57-AHSV,pUC57-WNV和pUC57-JEV)并设计合成特异性引物,采用环介导等温扩增方法(LAMP)进行检测,并进行特异性、敏感性和重复性试验。结果显示,该方法能够在1 h内准确检测靶标,与其他8种核酸均无交叉反应,且最低检测限为10 copies。重复性试验显示,Cq值变异率均小于5%。上述结果表明,本研究建立了一种速度快、成本低、特异性好及灵敏度高的LAMP检测方法,可用于马属动物的有效监测和快速检测。In this study,recombinant plasmids(named pUC57-AHSV,pUC57-WNV and pUC57-JEV)were con-structed to mimic the viruses according to the gene sequences of GenBank(GenBank accession No.:NC_006011,No.:NC_009942 and No.:NC_001437).Primers were synthesized and performed by loop-mediated isothermal amplification.The specificity,sensitivity,and reproducibility of this detection method were evaluat-ed.The results show that the method can detect the target specifically in one hour and the lowest detec-tion limit reaches 10 copies.The repeatability verification shows that the coefficient of variation of Cq value is less than 5%.In brief,a fast,low-cost,specific,and sensitive LAMP detection method was es-tablished,which can be used for effective monitoring of equines and rapid detection of pathogens.

关 键 词：非洲马瘟病毒 西尼罗病毒 日本脑炎病毒 环介导等温扩增 核酸检测 

分 类 号：S852.659.4[农业科学—基础兽医学] S852.659.6[农业科学—兽医学]