基于PEDV M基因RT-LAMP快速检测方法的建立与应用  

Establishment and application of RT-LAMP rapid detection method for porcine epidemic diarrhea virus

作　　者：陈希文[1,2] 尹苗[1] 汪谦 杨勤 杨凤[1] 李莲[2,3] 罗文涛 周杰珑 马缨 赵洪 CHEN Xi-wen;YIN Miao;WANG Qian;YANG Qin;YANG Feng;LI Lian;LUO Wen-tao;ZHOU Jie-long;MA Ying;ZHAO Hong(Institute of Applied Animal Technology,Mianyang Normal University,Mian yang,Sichuan 621000,China;Fengguangde Laboratory of Si-chuan Tieqilishi Group,Mianyang,Sichuan 621006,China;College of Life Science,Southwest Forestry University,Kunming 650224,China)

机构地区：[1]绵阳师范学院动物应用技术研究所,四川绵阳621000 [2]四川铁骑力士集团冯光德实验室,四川绵阳621006 [3]西南林业大学生命科学学院,云南昆明650224

出　　处：《中国兽医学报》2018年第9期1641-1647,共7页Chinese Journal of Veterinary Science

基　　金：国家生猪产业技术体系建设基金资助项目(CARS-36-02A);绵阳市科技局基金资助项目(16N-ZC-01);绵阳师范学院科技成果培育基金资助项目(PY-2016-A02)

摘　　要：为建立一种快速、灵敏、方便的猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）早期诊断方法,本研究针对PEDV的M基因片段设计了4条引物,对反应体系的时间、温度进行了优化,对试验的特异性和敏感性进行了评估,初步建立了逆转录环介导恒温扩增技术（RT-LAMP）。结果表明,建立的PEDV RT-LAMP方法在65℃恒温下扩增60min,可通过琼脂糖凝胶电泳或SYBR Green I染料肉眼判断结果,灵敏性较普通RT-PCR高100倍。该方法与猪传染性胃肠炎病毒（TGEV）、猪轮状病毒（PoRV）、猪繁殖与呼吸综合征病毒（PRRSV）和猪瘟病毒（CSFV）等无交叉反应,具有良好的特异性。采用该RT-LAMP对100份临床疑似发病粪便拭子、小肠组织、初乳样本进行检测,结果较RT-PCR更灵敏。因此,本研究初步建立了可用于临床PEDV检测的RT-LAMP检测方法,为PEDV的临床快速诊断和综合防控提供了一种新的手段。The aim of this study is to establish a rapid,sensitive,convenient porcine epidemic diarrhea virus（PEDV）diagnostic methods.Four primers based PEDV M gene were designed,and initially established a reverse loop-mediated isothermal amplification（RT-LAMP）,and the specificity and sensitivity were assessed.The results showed that PEDV RT-LAMP method established at65℃isothermal amplification 60 min,can be judged by agarose gel electrophoresis or SYBR Green dye results by the naked eye,the sensitivity is 100 times higher than the ordinary RT-PCR.The method was also specific for PEDV and did not cross react to TGEV,PoRV,PRRSV and CSFV.The RT-LAMP was used to detect 36 clinical samples,and the results were in complete agreement with RT-PCR.Therefore,this study preliminarily established the RT-LAMP detection method which can be used for clinical PEDV detection,and provides a new method for rapid diagnosis and comprehensive prevention and control of PEDV.

关 键 词：猪流行性腹泻病毒(PEDV) 逆转录环介导等温扩增(RT-LAMP) 快速检测 

分 类 号：S852.653[农业科学—基础兽医学]