猪δ冠状病毒RT-PCR、RT-LAMP及间接ELISA检测方法的建立及应用

发布时间：2018-06-12 22:24

  本文选题：猪δ冠状病毒(PDCoV) + RT-PCR　； 参考：《江西农业大学》2017年硕士论文

【摘要】：猪δ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。猪Delta冠状病毒,属于冠状病毒科(Coronaviridae)冠状病毒属(Coronavirus)。PDCoV在2012年报道后,陆续在美国、中国等出现。目前对于PDCoV的致病和复制机理尚不清楚,仅了解其引起仔猪发病特征与猪流行性腹泻病毒相似。鉴于PDCoV在猪群中的广泛流行性及潜在威胁,本试验建立了针对PDCoV核酸的RT-PCR(Reverse Transcriptional Polymerase Chain Reaction)和反转录环介导等温扩增(Reverse Transcriptional Loop-Mediated Isothermal Amplification,RT-LAMP)的检测方法及针对PDCoV抗体的间接ELISA(Enzyme Linked Immunosorbent Assay)的检测方法。1.PDCoV RT-PCR方法的建立及其流行病学调查比对GenBank中PDCoV全基因序列,根据其保守区域设计一对特异性扩增N基因片段的引物,利用RT-PCR方法扩增获得329 bp的片段,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GenBank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;对建立的RT-PCR方法的特异性、敏感性、稳定性鉴定,结果表明该方法的特异性好、敏感性高、稳定性强;应用建立的RT-PCR方法检测了656份2012—2017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30.79%(202/656),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(30.97%),最早可从2012年的样品中检测出PDCoV。2.PDCoV RT-LAMP检测方法的建立根据比对结果,选取PDCoV N基因全长为目标序列,设计2套RT-LAMP引物。以PDCoV阳性病料提取的总RNA为模板,建立了PDCoV RT-LAMP的检测方法。对反应条件优化后,结果表明该反应的最佳条件是63℃恒温水浴70min,且该方法灵敏度高,特异性好。应用RT-LAMP、RT-PCR和套式RT-PCR对192份临床样品进行检测,结果表明RT-LAMP的灵敏度为RT-PCR的100倍,与套式RT-PCR相当。3.PDCoV N基因的原核表达以PDCoV阳性病料提取的总RNA为模板,扩增PDCoV N基因的全基因序列,扩增片段经双酶切后与表达载体pColdⅠ连接,构建重组表达质粒pCold-PDCoV-N,将重组质粒转入BL21(DE3)感受态细胞,OD600nm值为04-0.6时,冰浴30min后加入终浓度为0.8mM IPTG,15℃诱导表达21 h,破碎菌体得到大小约为41kDa的重组蛋白,且蛋白主要以可溶性的形式存在。使用PDCoV阳性血清作为一抗进行Western Blot试验,结果表明其免疫原性良好。4.PDCoV间接ELISA检测方法的建立以纯化的PDCoV N重组蛋白为包被抗原,采用棋盘滴定确定最佳抗原包被浓度和抗体,对酶标二抗稀释倍数、不同封闭液、封闭时间、一抗作用时间、二抗作用时间和显色时间进行优化建立PDCoV间接ELISA检测方法,结果表明抗原包被量为5μg、3%犊牛血清封闭2 h,检测血清稀释100倍,反应45 min,酶标二抗稀释4000倍,反应为1h为最优条件。建立的ELISA方法阴阳性临界值为0.316,与猪流行性腹泻病毒等6种猪常见病原阳性血清均无交叉反应,具有良好的特异性。批内和批间重复性试验的变异系数均小于10%,重复性和稳定性均较好。应用建立的间接ELISA方法,对收集自江西各地区282份猪血清进行检测,阳性率为12.8%(36/282),表明PDCoV在我省猪群中普遍存在。本实验建立的PDCoV抗体捕获间接ELISA为临床上猪群PDCoV感染监测和流行病学调查提供了实验依据。
[Abstract]:Swine delta coronavirus (Porcine Deltacoronavirus, PDCoV) is a new coronavirus, which has been discovered in recent years, which can cause piglets, especially newborn piglet diarrhea. The diarrhea caused by the pig is one of the most important reasons for the death of newborn piglets. The porcine Delta coronavirus belongs to the coronavirus (Coronaviridae) coronavirus. After reported in 2012, virus genus (Coronavirus).PDCoV appeared in the United States, China and so on. The pathogenesis and replication mechanism of PDCoV are still unclear. It is only known that the pathogenic characteristics of the piglets are similar to those of the porcine epidemic diarrhea virus. In view of the widespread prevalence and potential threat of PDCoV in the pigs, this experiment established the PDCoV nucleic acid. The detection method of RT-PCR (Reverse Transcriptional Polymerase Chain Reaction) and reverse recording loop mediated isothermal amplification (Reverse Transcriptional Loop-Mediated Isothermal Amplification, RT-LAMP) and the establishment of the method for detecting indirect antibodies against the antibodies. The epidemiological survey was compared to the PDCoV whole gene sequence in GenBank, and the primers of a pair of specific amplified N gene fragments were designed according to the conservative region. The fragment of 329 BP was amplified by the RT-PCR method. The sequence of the amplified product was compared with the sequence of the GenBank database and the sequence of the amplified product was compared with the sequence of the GenBank database. The results showed that the sequence obtained by the experiment was 99.1% homologous to the PDCoV sequence in the database, which proved that the amplified sequence was PDCoV, and the specificity, sensitivity and stability of the RT-PCR method established. The results showed that the method had good specificity, high sensitivity and strong stability, and 656 2012 - 20 samples were detected by the application of the established RT-PCR method. 17 years from the diarrhoea sow feces and diarrhoea piglet feces / intestinal samples collected from various regions of Jiangxi Province, the results showed that the detection rate of PDCoV in diarrhea samples was 30.79% (202/656), and the detection rate of PDCoV in sow feces (27.78%) was slightly lower than that of piglet feces and intestinal samples (30.97%), and the earliest can be detected from the samples in 2012. The 2.PDCoV RT-LAMP detection method was established according to the comparison results. The total length of the PDCoV N gene was selected as the target sequence and 2 sets of RT-LAMP primers were designed. The detection method of PDCoV RT-LAMP was established with the total RNA of the PDCoV positive disease material as the template. The optimum conditions for the reaction showed that the best condition of the reaction was at 63 C at constant temperature water bath 70min, and the formula was used. The sensitivity of the method was high and the specificity was good. 192 clinical samples were detected with RT-LAMP, RT-PCR and nested RT-PCR. The results showed that the sensitivity of RT-LAMP was 100 times of RT-PCR. The prokaryotic expression of the.3.PDCoV N gene with the set of RT-PCR was the template for the total RNA of the PDCoV positive disease material, and the whole gene sequence of the PDCoV N gene was amplified and the amplified fragment was amplified. After double enzyme digestion, the recombinant plasmid pCold-PDCoV-N was constructed with the expression vector pCold I, and the recombinant plasmid was transferred into the BL21 (DE3) receptive cell. When the OD600nm value was 04-0.6, the final concentration was 0.8mM IPTG after the ice bath 30min, and the 21 h was expressed at 15 degrees C, and the broken strain obtained the recombinant protein about the 41kDa size, and the protein was mainly in the form of soluble form. PDCoV positive serum was used as an anti Western Blot test. The results showed that the positive immunogenicity of.4.PDCoV indirect ELISA detection method was established with the purified PDCoV N recombinant protein as the envelope antigen, the best antigen concentration and antibody were determined by the chessboard titration, the dilution multiple of enzyme standard two, the closed liquid, and the closure were closed. Time, anti action time, two anti action time and color time were optimized to establish PDCoV indirect ELISA detection method. The results showed that the antigen envelope was 5 mu g, 3% calf serum closed 2 h, serum dilution 100 times, reaction 45 min, enzyme standard two diluted 4000 times, and the reaction was the best condition. The critical value of ELISA method was 0.. The critical value of yin and Yang was 0.. 316, there was no cross reaction in the positive sera of 6 pigs, such as porcine epidemic diarrhea virus, which had good specificity. The coefficient of variation in both batch and interbatch repeatability tests was less than 10%, and the repeatability and stability were better. The indirect ELISA method was used to detect 282 pig serum collected from various regions of Jiangxi. The positive rate was 1. 2.8% (36/282) indicates that PDCoV is common in the pig population of our province. The indirect ELISA captured by PDCoV antibody in this experiment provides an experimental basis for the monitoring and epidemiological investigation of swine PDCoV infection in clinical pigs.
【学位授予单位】：江西农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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