猪Delta冠状病毒和猪流行性腹泻病毒双重RT-PCR诊断方法的建立及初步应用  

Establishment and preliminary application of a duplex reverse transcription-PCR for the detection of porcine deltacoronavirus and porcine epidemic diarrhea virus

作　　者：韩丽[1,2] 周雍[1,2] 李炳晓 曹贝贝[2] 梁青青[1,2] 张利卫[1,2] 胡慧[1,2] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省动物性食品安全重点实验室,河南郑州450002

出　　处：《中国兽医科学》2017年第5期557-562,共6页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2016YFD0500102);河南省科技开放合作项目(152106000047)

摘　　要：自2014年美国首次报道猪Delta冠状病毒(PDCoV)以来,该病毒相继在我国一些猪场检测到,由于其发病症状和病理剖检与猪流行性腹泻(PED)极其相似,且易出现混合感染,临床很难区分,给养殖业造成严重经济损失,因此建立能够同时检测且能区分这两种病毒感染的诊断方法,对于临床实践具有重要意义。根据GenBank中登录的PDCoV和猪流行性腹泻病毒(PEDV)基因序列,设计了2对分别用于扩增PDCoV N基因和PEDV M基因的目的片段的引物,通过对RT-PCR反应条件进行优化,建立了同时检测PDCoV和PEDV的双重RT-PCR检测方法,并对该方法进行了灵敏性、特异性和重复性研究。结果显示,该双重RT-PCR对PD-CoV和PEDV的最低检测量分别是3.14×10~3 copies/μL和3.88×10~4 copies/μL。该方法仅对PDCoV和PEDV检测为阳性,对猪传染性胃肠炎病毒(TGEV)、博卡病毒(PBoV)、猪繁殖与呼吸综合征病毒(PRRSV)和猪伪狂犬病毒(PRV)等猪常见的感染病毒的扩增均为阴性,说明该方法具有较好的特异性。用建立的双重RT-PCR方法对111份临床样品进行检测,其中PDCoV阳性样品22份,阳性率为19.82%,PEDV阳性样品27份,阳性率为24.32%,PDCoV和PEDV共感染的样品数为10份,阳性率为9.01%。本试验所建立的双重RT-PCR方法能够快速、准确地对PDCoV和PEDV单一或混合感染的临床样品进行快速检测,为临床上对PDCoV和PEDV鉴别诊断和流行病学调查研究提供了新的技术手段。Since the first report of porcine deltacoronavirus（PDCoV） in USA in 2014,it has also been detected in some pig farms in China. Due to the clinical and pathological symptoms are very similar to that of porcine epidemic diarrhea（PED）,it is difficult to distinguish them by clinical diagnosis. Moreover,the co-infection of PDCoV and porcine epidemic diarrhea virus（PEDV） is common clinically,which causes huge economic losses to the swine industry. So it has important significance to establish a rapid diagnostic method for the simultaneous detection of PDCoV and PEDV. According to the GenBank sequences of PDCoV and PEDV,two pairs of primers were designed to amplify the PDCoV N gene and PEDV M gene fragments,respectively. By optimizing the PCR reaction conditions,a duplex RT-PCR detecting method was established for the simultaneous detection of PDCoVand PEDV,and then the specificity,sensitivity and repeatability of this method were analyzed. The results showed that the duplex RT-PCR method had a sensitivity of 3. 14×10^3 copies/μL for PDCoV and 3.88×10^4/μL copies L for PEDV,as well as a good specificity,which was only positive for PDCoV and PEDV,and negative for porcine transmissible gastroenteritis virus（TGEV）,porcine bocavirus（PBoV）,porcine reproductive and respiratory comprehensive syndrome virus（PRRSV） and porcine pseudorabies virus（PRV）.Further more this duplex RT-PCR method was used to test 111 clinical samples that collected from the diarrheic pigs. PDCoVpositive rate was 19.82%（22/111）,PEDV positive rate was 24.32% （27/111）,and PDCoV and PEDV co-infection positive rate was 9.01%（10/111）.Therefore,the duplex RT-PCR method in this experiment was established and can be used for quick and accurate diagnosis of PDCoV and PEDV infection clinically,especially for the PEDV and PDCoV mixed-infection,which provides a new technical method for the clinical identification of these two diseases.

关 键 词：猪Delta冠状病毒(PDCoV) 猪流行性腹泻病毒(PEDV) 双重RT-PCR 

分 类 号：S852.659.6[农业科学—基础兽医学]