PEDV、TGEV二重RT-PCR检测方法的建立与应用  

Establishment and application of PEDV and TGEV dual RT-PCR assay

作　　者：陈希文[1,2] 杨双燕 李莲 尹苗[1] 杨勤 杨凤[1] 罗文涛 马缨[1] 赵洪 CHEN Xiwen;YANG Shuangyan;LI Lian;YIN Miao;YANG Qin;YANG Feng;LUO Wentao;MA Ying;ZHAO Hong(Molecular Diagnostic and Drug Research Center, Mianyang Normal University, Mianyang 621006, China;Fengguangde Laboratory of Sichuan Tieqilishi Group,Mianyang 621006, 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621006 [2]铁骑力士集团冯光德实验室,四川绵阳621006

出　　处：《黑龙江畜牧兽医》2018年第11期138-142,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家生猪产业技术体系建设项目(CARS-36-02A);四川省教育厅科研项目(18ZB0294);绵阳市科技局应用基础项目(16N-ZC-01);绵阳师范学院科技成果培育项目(PY-2016-A02)

摘　　要：为了能快速灵敏地确诊临床腹泻仔猪的病原,试验采用分子生物学方法对基于猪流行性腹泻病毒（PEDV）M基因（KJ778616.1）和猪传染性胃肠炎病毒（TGEV）N基因（GQ374566.1）的二重RTPCR检测方法进行了研究。结果表明：建立的二重RT-PCR检测方法能同时扩增鉴别PEDV和TGEV两个病原;该方法的特异性强,与猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）、猪伪狂犬病毒（PRV）和猪轮状病毒（PoRV）等病毒间不存在交叉反应;敏感性高,PEDV的最低稀释度为1×10^-5,TGEV的最低稀释度为1×10^-4;应用建立的方法对36份临床腹泻仔猪粪便拭子、小肠组织和乳汁等样品进行检测,结果与商品化试剂盒的符合率为100%。说明试验成功建立了能同时检测并鉴别PEDV和TGEV的二重RT-PCR方法,该方法快速、准确、灵敏,在临床发病样品中应用效果良好,适合在疑似PEDV和TGEV发病猪场中推广应用。In order to diagnose the pathogeny of piglet diarrhea quickly and sensitively,the dual RT-PCR detection method based on the M gene（ Gen Bank： KJ778616. 1） of porcine epidemic diarrhea virus（ PEDV） and the N gene（ Gen Bank： GQ374566. 1） of transmissible gastroenteritis virus（ TGEV） was studied by molecular biology. The results showed that the dual RT-PCR detection method could amplify and identify the two pathogens of PEDV and TGEV simultaneously. This method had strong specificity and had no cross reaction with porcine reproductive and respiratory syndrome virus（ PRRSV）,classical swine fever virus（ CSFV）,porcine pseudorabies virus（ PRV） and porcine rotavirus（ PoRV）.The sensitivity of this method was high. When the minimum dilution of PEDV and TGEV were 1 × 10^-5 and 1 × 10^-4,respectively,the virus could also be detected by the PCR. The method was used to detect thirty-six fecal samples,small intestine tissues and milk samples of piglets with clinical diarrhea. The coincidence rate of the detected results with commercial kits was 100%. The results indicated that the dual RT-PCR method which could detect and distinguish PEDV and TGEV at the same time was successfully established. The method was rapid,accurate and sensitive,and had a good effect in clinical samples. It was suitable to be popularized in suspected PEDV and TGEV pig farms.

关 键 词：猪流行性腹泻病毒 猪传染性胃肠炎病毒 二重RT-PCR 鉴别诊断 建立 应用 

分 类 号：S852.659.6[农业科学—基础兽医学]