猪传染性胃肠炎病毒、猪流行性腹泻病毒和猪轮状病毒A多重荧光定量RT-PCR检测方法的建立  

Development of a multiplex real-time reverse transcriptase PCR assay for simultaneous detection of TGEV, PEDV and PRoVA

作　　者：李军[1] 蔡良语 陈兵[3] 刘建利[3] 孙洁[3] 陈书琨[3] 林庆燕[3] 陶虹[3] 吴绍强[4] 卢体康[3] 陈金顶[1] 秦智锋[3] LI Jun;CAI Liang-yu;CHEN Bing;LIU Jian-li;SUN Jie;CHEN Shu-kun;LIN Qing-yan;TAO Honga;WU Shao-qiang;LU Ti-kang;CHEN Jin-ding;QIN Zhi-feng(South China Agricultural University,Guangzhou 510642,China;Wuhan Polytechnic University,Wuhan 430023,China;Shenzhen Entry-Exit Inspection ＆ Quarantine Bureau,Shenzhen 518045,China;Chinese Academy of Inspection and Quarantine,Beijing 100176,China)

机构地区：[1]华南农业大学,广东广州510642 [2]武汉轻工大学,湖北武汉430023 [3]深圳出入境检验检疫局动植物检验检疫技术中心,广东深圳518045 [4]中国检验检疫科学研究院,北京100176

出　　处：《中国预防兽医学报》2018年第9期801-805,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2016YFD0501103; 2016YFD0500700; 2017YFD0501104; 2017YFD0500600)

摘　　要：为对引起腹泻症状的猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)和猪轮状病毒A(PRoVA) 3种病毒同时快速地鉴别检测,本研究对GenBank中登录的TGEV的S基因、PEDV的S基因、PRo VA的NSP基因进行序列分析,分别设计了针对3种病毒的特异性引物和用3种荧光基团标记的核酸探针,通过条件优化建立了针对这3种病毒的多重荧光定量RT-PCR方法。结果显示,该方法能够特异性检测PEDV、TGEV和PRo VA,而与其它病原无交叉反应;对TGEV、PEDV和PRo VA的最低检测量分别为1.12拷贝/μL、39拷贝/μL和25拷贝/μL;组内和组间变异系数均小于5%;该方法与商品化的试剂盒检测结果符合率达100%。该方法快速、敏感,检测结果准确可靠,为猪病毒性腹泻疾病的流行病学调查和分子生物学快速诊断奠定了基础。In order to simultaneous detection of the transmissible gastroenteritis virus （TGEV）, porcine epidemic diarrhea virus （PEDV） and porcine rotavirus A （PRoVA） which have the similar clinical signs, a multiplex real-time reverse transcriptase PCR （mrRT-PCR） assay was developed with three sets of probes labeled with three different fluorophore and primers designed according to the S gene of TGEV, S gene of PEDV and NSP gene of PRoVA to rapidly distinguish TGEV, PEDV and PRoVA （TGEV/PEDV/PRoVAmrRT-PCR）. The TGEV/PEDV/PRoVA mrRT-PCR assay was specific, and had no false positive and no false negative detection results. The sensitivity of the TGEV/PEDV/PRoVA mrRT-PCR assay was 1.12copies/μL for TGEV, 39copies/lxL for PEDV, and 25copies/μL for PRoVA, respectively. The repeatability of the assay was acceptable with CV ≤5% and the consistency was 100% with commercial kits. The developed TGEV/PEDV/PRoVA mrRT-PCR assay was a promising method for monitor and surveillance of swine viral diarrhea diseases.

关 键 词：猪传染性胃肠炎病毒 猪流行性腹泻病毒 猪轮状病毒A 荧光定量RT-PCR检测 多重检测 

分 类 号：S852.65[农业科学—基础兽医学]