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

【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则分别属于不同的分支。试验获得的PDCoV序列与韩国PDCoV毒株KNU14-04亲缘关系最近,同源性高达99.1%;与两株香港毒株HKU 15-44和HKU 15-155亲缘关系相对较远;3本试验建立的RT-PCR方法特异性强,仅能扩增出PDCoV,而对PEDV、TGEV、PKoV、PAstV、PRRSV及CSFV核酸无交叉扩增现象;4所建立的RT-PCR方法灵敏度高,将含扩增片段的重组质粒以1.0×106拷贝/μL为起始浓度依次10倍稀释至1.0×101拷贝/μL作为模板验证方法的灵敏度,结果表明所建立的方法对PDCoV最低可检出量为1.0×103拷贝/μL;5应用建立的RT-PCR方法检测了249份2012—2015年采集自江西省各地区的腹泻母猪粪便及腹泻仔猪粪便/肠道样本,结果显示腹泻样本中PDCoV的检出率为31.33%(78/249),母猪粪便中PDCoV的检出率(27.78%)稍低于仔猪粪便及肠道样品PDCoV的检出率(31.92%),最早可从2012年的样品中检测出PDCoV。【结论】试验成功建立了检测新现PDCoV的RT-PCR方法;应用所建立的RT-PCR方法检测了249份2012—2015年江西地区猪群临床腹泻样品中PDCoV存在情况,结果表明PDCoV是一种普遍存在于腹泻猪群中的病毒;研究建立的RT-PCR方法对猪群PDCoV的临床诊断和流行病学调查等具有应用价值。

【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 the established RT-PCR, and some PDCoV positive amplicons were cloned and then sequenced for the specificity confirmation of the assay.【Result】A single expected DNA fragment band of 329 bp in size was amplified by using the designed primers. Sequences of the amplicons selected were subjected to a BLAST search against the Gen Bank database, and the results indicated that the sequences hit PDCoV with a nucleotide identity as high as 99.1%, demonstrating that the amplified targets were PDCoV. A phylogenetic tree was generated based on eight partial N gene of PDCoV obtained in this study, 18 reference PDCoVs from other countries/areas, and two representative PEDV and two selected TGEV sequences with corresponding fragments. All of the PDCoV strains were classified into a cluster, distinct from PEDV and TGEV strains. The eight PDCoV sequences obtained in this study showed that the highest similarity with a Korea PDCoV strain KNU14-04, but less similarity to two Hong Kong strains HKU 15-44 and HKU 15-155. No cross amplification for PEDV, TGEV, PKoV, PAstV, PRRSV and CSFV was observed, indicating the established RT-PCR assay was highly specific. The detection limit of 1.0×10^3 copies/μL of the assay was determined based on the templates of the recombinant plasmid with 10-fold serial dilution（from 1.0×10^6 to 1.0×10^1 copies/μL）, indicating the assay was sensitive. A total of 249 diarrheal fecal/intestinal samples of sows and piglets collected in Jiangxi Province, China from 2012 to 2015 were examined by the established assay, and the positive rate of PDCoV was 31.3%（78/249）, and the samples from sows showed somewhat lower positive rate（27.8%） when compared with that from piglets（31.9%）. PDCoV could be detected in samples as early as 2012.【Conclusion】In this study, a RT-PCR assay for detection of newly emerged PDCoV in swine was established and evaluated. The results from this study indicated that PDCoV is a frequently detected virus in diarrheal pigs in Jiangxi, China. The RT-PCR assay established in this study is valuable in terms of clinic diagnosis and epidemiological investigation of PDCoV.