新现猪Delta冠状病毒RT-PCR检测方法的建立及其应用

发布时间：2018-07-31 07:54

【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。
[Abstract]:[Objective] the swine Delta coronavirus (Porcine Deltacoronavirus, PDCoV) is a newly discovered coronavirus which can cause pigs, especially the newborn piglet diarrhea. The diarrhea caused by the swine has a high incidence and mortality, which is one of the important causes of the death of newborn piglets. The experiment is to establish a new method of detection of PDCoV RT-PCR. And investigate the infection of PDCoV in the current Jiangxi diarrhea pigs. [Methods] through the comparison and analysis of the whole genome sequence of PDCoV in the Gen Bank database, the conservative sequence was found, and the specific Primer gene fragment of the amplified PDCoV Nucleocapsid (N) protein gene fragment was designed by Primer 3 online software, and the PDCoV RT-PCR detection method was established based on the primer. The established method was used to detect the feces and intestinal samples of diarrhea pigs and select positive amplification products for cloning and sequencing. The PDCoV sequence obtained in this test and the PDCoV sequence of other countries or regions, and porcine epidemic diarrhea virus (PEDV) and porcine infectious gastroenteritis virus (TGEV) were used together to construct the evolutionary tree, and the PDCoV strains and their PEDV and TG were analyzed. The evolutionary relationship between EV; PEDV, TGEV, PKoV, PAstV, porcine reproductive and respiratory syndrome virus (PRRSV) and swine fever virus (CSFV) RNA as a template to verify the specificity of the primers; construct the recombinant plasmid of the PDCoV nucleocapsid protein gene fragment and determine the PDCoV RT by a series of diluted recombinant plasmids. The sensitivity of the -PCR method was used to investigate the existence of PDCoV in 249 samples of swine diarrhea in Jiangxi province from 2012 to 2015, and a certain number of RT-PCR positive amplification products were randomly selected to clone them, and the specificity of the reaction was further verified. [results] 1, the RNA of fecal samples from diarrhea pigs was used as a template, and the application was designed. Specific primers amplified a single band of 329 BP, consistent with the expected target fragment size. The amplified products were cloned and sequenced. The sequences obtained were compared with the NCBI Gen Bank database sequence. The comparison results showed that the sequence obtained by the test was 99.1% homology to the PDCoV sequence in the database, proving that the amplified sequence was P. DCoV; 2 the sequence of PDCoV N gene fragments obtained from 8 experiments and the PDCoV strains of 18 other countries or regions and the sequence of corresponding N gene fragments of PEDV and TGEV were constructed. The results showed that 26 PDCo V sequences belonged to the same branch, while PEDV and TGEV were in different branches. The phylogenetic relationship of strain KNU14-04 is up to 99.1% recently; the relationship with two strains of Hongkong strain HKU 15-44 and HKU 15-155 is relatively distant; 3 the RT-PCR method established in this experiment is specific and can only amplify PDCoV, while PEDV, TGEV, PKoV, PAstV, PRRSV and CSFV nucleic acids are not intersected, and 4 established RT-PCR methods are highly sensitive and will be expanded. The amplified recombinant plasmid was diluted to 1 x 106 copies / L as the initial concentration to 1 x 101 copies / mu L as a template verification method. The results showed that the minimum detectable amount of the proposed method was 1 x 103 copies / mu L, and 5 used RT-PCR method was used to detect 249 copies of Jiangxi province from Jiangxi province from 2012 to 2015. The results showed that the detection rate of PDCoV in diarrhoea samples was 31.33% (78/249), and the detection rate of PDCoV in sow feces (27.78%) was slightly lower than that of piglet feces and intestinal samples (31.92%), and the earliest possible test of PDCoV. [Conclusion] from samples in 2012 was successfully established. The RT-PCR method of new PDCoV was detected, and the RT-PCR method was used to detect the presence of PDCoV in 249 samples of clinical diarrhea samples from Jiangxi region from 2012 to 2015. The results showed that PDCoV was a virus commonly found in the diarrhea pigs, and the established RT-PCR method was used for the clinical diagnosis and epidemiological investigation of pig group PDCoV. It is of applied value.
【作者单位】： 江西农业大学动物科学技术学院;
【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)
【分类号】：S852.651

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