新现猪Delta冠状病毒RT-PCR检测方法的建立及其应用  

Establishment and Application of a RT-PCR Assay for Detection of Newly Emerged Porcine Deltacoronavirus

作　　者：张帆帆[1] 宋德平[1] 周信荣[1] 黄冬艳[1] 李安琪[1] 彭棋 陈燕君[1] 吴琼[1] 何后军[1] 唐玉新[1] 

机构地区：[1]江西农业大学动物科学技术学院,南昌330045

出　　处：《中国农业科学》2016年第7期1408-1416,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金面上项目(31372457);江西省科技厅落地计划项目(KJLD13029)

摘　　要：【目的】猪Delta冠状病毒(Porcine Deltacoronavirus,PDCoV)是近年来新发现的可引起猪只,特别是新生仔猪腹泻的冠状病毒,其引发的腹泻具有较高的发病率和死亡率,是造成新生仔猪死亡的重要原因之一。试验拟建立新现PDCoV RT-PCR检测方法,并调查当前江西腹泻猪群中PDCoV的感染情况。【方法】通过对Gen Bank数据库中PDCoV全基因组序列的比对分析,找出保守序列,用Primer 3.0在线软件设计1对扩增PDCoV核衣壳(N)蛋白基因片段的特异性引物,基于该引物建立PDCoV RT-PCR检测方法;用建立的方法检测腹泻猪粪便及肠道样品,挑选阳性扩增产物进行克隆、测序;用本试验获得的PDCoV序列与其他国家或地区的PDCoV序列以及猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)共同构建进化树,分析PDCoV各毒株及其与PEDV和TGEV之间的进化关系;以PEDV、TGEV、猪库布病毒(PKoV)、猪星状病毒(PAstV)、猪繁殖与呼吸综合征病毒(PRRSV)及猪瘟病毒(CSFV)RNA为模板验证引物的特异性;构建PDCoV核衣壳蛋白基因片段重组质粒,并以系列稀释的重组质粒为模板确定所建立的PDCoV RT-PCR方法的敏感性;应用建立的RT-PCR方法调查249份2012—2015年江西省猪群腹泻样品中PDCoV的存在情况,随机抽取一定数量的RT-PCR阳性扩增产物进行克隆、测序进一步验证反应的特异性。【结果】1以腹泻猪粪便样品RNA为模板,应用所设计的特异性引物扩增出了329 bp的单一条带,与预期目的片段大小一致,扩增产物经克隆后测序,将获得的序列与NCBI Gen Bank数据库序列进行比对,比对结果表明试验所获得的序列与数据库中PDCoV序列的同源性高达99.1%,证明所扩增的序列属于PDCoV;2将8条本试验获得的PDCoV N基因片段序列与18株其他国家或地区的PDCoV毒株以及PEDV、TGEV的相应N基因片段序列构建进化树,结果表明26条PDCo V序列属于同一个分支,而PEDV和TGEV则分别属�【Objective】Porcine Deltacoronavirus（PDCoV） is a newly emerged coronavirus, which has caused diarrhea in pigs, especially in newborn piglets leading to high morbidity and mortality, such that it has become one of the most important causes of death in newborn piglets. In this study, we attempted to establish a reverse transcription-polymerase chain reaction（RT-PCR） assay for detection of newly emerged PDCoV in diarrheal pigs, and investigate the infection situation of PDCoV in diarrheal samples of pigs.【Method】Based on the result of multialignment analysis on the published PDCoV genome sequences on Gen Bank database, a pair of primers specific to the conserved nucleocapsid（N） gene of PDCoV was designed by using the online software Primer 3.0, and then a RT-PCR for detection of PDCoV was established by utilizing the designed primers. Diarrheal samples were tested by the established RT-PCR assay, and several positive PCR products were selected for cloning and sequencing. To analyze the phylogenetic relationships between PDCoV and porcine epidemic diarrhea virus（PEDV） as well as transmissible gastroenteritis virus（TGEV）, a phylogenetic tree was constructed based on the partial N gene sequences of PDCoV obtained in this study and 18 reference PDCoV sequences from other countries/areas, and corresponding N gene sequences of representative PEDV and TGEV strains. To determine the specificity of the RT-PCR assay established, RNAs of PEDV, TGEV, porcine kobuvirus（PKoV）, porcine astrovirus（PAstV）, porcine reproductive and respiratory syndrome virus（PRRSV） and classical swine fever virus（CSFV） were tested. To evaluate the detection limit of the assay, a recombinant plasmid containing the N gene fragment was constructed and served as templates with 10-fold serial dilution starting from 1.0×10^6 copies/μL to 1.0×10^1 copies/μL. Clinic diarrheal samples of pigs（n=249） from sows and piglets collected in Jiangxi Province from 2012 to 2015 were tested for the presence of PDCoV by

关 键 词：猪Delta冠状病毒 RT-PCR 腹泻 进化树 应用 

分 类 号：S852.651[农业科学—基础兽医学]