新发PDCoV和TGEV双重RT-PCR检测方法的建立及初步应用  

Establishment and Preliminary Application of Duplex RT-PCR for Detection of Emerging Porcine deltacoronavirus and Porcine transmissible gastroenteritis virus

作　　者：刘玲玲[1] 曹贝贝[1] 张利卫[1] 韩丽[1] 韦学雷 兰培英[1] 胡慧[1] 王亚宾[1] LIU Ling-Ling CAO Bei-Bei ZHANG Li-Wei HAN Li WEI Xue-Lei LAN Pei-YingL HU Hui WANG Ya-Bin(Key Laboratory for Animal-derived Food Safety of Henan Province, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, Chin)

机构地区：[1]河南农业大学牧医工程学院/河南省动物性食品安全重点实验室,郑州450002

出　　处：《农业生物技术学报》2016年第12期1955-1963,共9页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展计划(973)项目(No.2016YFD0500102);河南省科技开放合作项目(No.152106000047)

摘　　要：猪Delta冠状病毒(Porcine deltacoronavirus,PDCoV)是一种新发现的冠状病毒,临床上主要引起感染仔猪出现水样腹泻、呕吐和脱水等症状,和猪传染性胃肠炎病毒(Porcine transmissible gastroenteritis virus,TGEV)感染引起的临床症状极为类似,且临床上PDCoV和TGEV混合感染时有发生,单靠临床诊断很难区分两种疾病,因此建立同时检测且区分PDCoV和TGEV的检测方法具有重要的临床意义。本研究根据Gen Bank中收录的PDCoV N基因和TGEV N基因序列设计了2对引物,在同一反应体系中同时扩增PDCoV的N基因片段和TGEV的N基因片段,通过对RT-PCR反应条件的优化,建立了同时检测PDCoV和TGEV的双重RT-PCR方法,并对该方法进行了特异性、灵敏性和重复性研究。结果显示,所建立的双重RT-PCR对PDCoV和TGEV的最低检测量分别是3.14×102copies/μL和3.68×103copies/μL,利用该双重RT-PCR方法对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)、猪博卡病毒(Porcine bocavirus,PBo V)、猪繁殖与呼吸综合征病毒(Porcine respiratory and reproductive syndrome virus,PRRSV)和猪伪狂犬病毒(P.pseudorabies virus,PRV)的扩增结果均为阴性,显示出较好的特异性。为了解所建立的双重RT-PCR方法的临床应用效果,对252份临床样品进行了检测。结果表明,双重RT-PCR检出PDCoV阳性样品31份(阳性率为12.30%),TGEV阳性样品12份(阳性率为4.76%),同时感染PDCoV和TGEV的阳性样品2份(阳性率为0.79%),且检测结果与单一PDCoV和TGEV RT-PCR相符。因此,本研究所建立的双重RT-PCR方法可用于PDCoV和TGEV的临床检测,为进一步研究PDCoV和TGEV的临床鉴别诊断和流行病学调查提供了理论依据和技术支持。Porcine deltacoronavirus（PDCoV） is a newly discovered coronavirus. It mainly infects piglets and causes severe enteritis accompanied by diarrhea and vomiting and other symptoms. The symptoms are symptomatically indistinguishable from those that caused by Porcine transmissible gastroenteritis virus（TGEV）.In clinic the co-infection of PDCoV and TGEV is common, which causes huge economic losses to the swine industry. In order to control and prevent effectively these diseases, development of rapid detection methods of PDCoV and TGEV is the primary task. Thus, according to the analysis of PDCoV and TGEV N gene sequences in Gen Bank, two pairs of primers targeting the N gene of PDCoV and TGEV were designed respectively. By optimizing the PCR reaction conditions, a duplex RT-PCR detection method was established for the simultaneous detection of PDCoV and TGEV, and the specificity, sensitivity and repeatability of the method were studied. The results showed that the duplex RT-PCR had high sensitivity and specificity with a limited detection of 3.14×102copies/μL for PDCoV and 3.68×103copies/μL for TGEV, respectively, and no specific amplifications from other virus, such as Porcine epidemic diarrhea virus（PEDV）, Porcine bocavirus（PBo V）, Porcine respiratory and reproductive syndrome virus（PRRSV） or Porcine pseudorabies virus（PRV）. 252 clinical samples tested by the duplex RT- PCR showed that the positive rates of PDCoV and TGEV were12.30% and 4.76%, respectively, co-infection rate of PDCoV and TGEV was 0.79%. The date showed that the duplex RT-PCR method established in this study could be used for the clinical detection of PDCoV and TGEV,which provides the theoretical basis information and technical support for further applied to clinical differential diagnosis and epidemiological investigation of TGEV and PDCoV.

关 键 词：猪Delta冠状病毒(PDCoV) 猪传染性胃肠炎病毒(TGEV) 双重RT-PCR 

分 类 号：S855.3[农业科学—临床兽医学]