This experiment was conducted to diagnose Peste des Petits Ruminants based on the eukaryotically-expressed PPRV nucleoprotein through an indirect ELISA. The full-length PPRV nucleoprotein gene was obtained from viral ...This experiment was conducted to diagnose Peste des Petits Ruminants based on the eukaryotically-expressed PPRV nucleoprotein through an indirect ELISA. The full-length PPRV nucleoprotein gene was obtained from viral genome RNA by RT-PCR. The amplified fragments were cloned into baculovirus donor vectors pFastHTA of the Bac-to-Bac system. These recombinant plasmids, pFastHTA-PPRV-N, were transformed into DH10Bac host bacteria to obtain recombinant shuttle plasmids, pBacmid-PPRV-N. Recombinant baculovirus, Bacmid-PPRV-N, was generated for expression of the PPRV nucleoprotein by transfecting recombinant pBacmid-PPRV-N with Lipofectamine 2000 into Sf21 insect cells. The efficient expression of PPRV Nucleoprotein by baculovirus in Sf21 cells was verified by SDS-PAGE and Western blot. An indirect ELISA was developed using recombinant PPRV nucleoprotein as the coating antigen. 37 goat sera from an epidemic area in Tibet and 92 goat sera from a non-infected area in Qinghai Province were simultaneously detected by the indirect ELISA, developed here, and the international standard cELISA kit. The sensitivity and specificity of the indirect ELISA was 100% and 96.2% compared with the cELISA kit. The coincidence rate of the two methods was 96.9%. The results demonstrated that the indirect ELISA established in this study works well for diagnosis of PPR.展开更多
小反刍兽疫(Peste des petits ruminants,PPR)是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)感染山羊、绵羊等小反刍动物引起的一种急性、高度接触性传染病,主要临床特征为高热、黏液脓性眼鼻分泌物、口腔炎、急性腹泻和...小反刍兽疫(Peste des petits ruminants,PPR)是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)感染山羊、绵羊等小反刍动物引起的一种急性、高度接触性传染病,主要临床特征为高热、黏液脓性眼鼻分泌物、口腔炎、急性腹泻和死亡等。由于PPR具有较高的发病率和致死率,严重阻碍了养殖业健康发展,并造成农业经济的巨大损失。因此,分析该病的流行特点、临床特征和诊断技术对其防控工作意义重大。文章简述PPR的流行病学及诊断技术的研究进展,以期为该病的防控诊疗提供参考。展开更多
为实现小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)的快速、准确、定量检测,以国内PPRV Clone 9疫苗株N基因为靶位点设计特异性引物及探针,建立了液滴式数字PCR(Droplet digital PCR,ddPCR)方法,并对其特异性、敏感性及重...为实现小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)的快速、准确、定量检测,以国内PPRV Clone 9疫苗株N基因为靶位点设计特异性引物及探针,建立了液滴式数字PCR(Droplet digital PCR,ddPCR)方法,并对其特异性、敏感性及重复性进行评价。将所建立的ddPCR方法与实时荧光定量PCR(Fluorescence quantitative PCR,qPCR)方法的灵敏度进行比较分析并应用于PPRV(Clone 9株)核糖核酸标准物质的定量。结果显示,所建立的ddPCR方法特异性好,仅PPRV检测结果为阳性,其他羊源病毒类生物制品及毒种检测结果均为阴性;敏感性高,基因拷贝数检出限度为4.33拷贝/μL,比qPCR方法的灵敏度(57.3拷贝/μL)高10倍;重复性好,重复性试验的变异系数为1.8%;定量准确,具有一定资质的9家单位各组测量数据组间变异系数小于5%。试验建立的ddPCR方法能够有效地对PPRV核酸进行快速检测,为PPRV的诊断及绝对定量提供了有效方法。展开更多
文摘This experiment was conducted to diagnose Peste des Petits Ruminants based on the eukaryotically-expressed PPRV nucleoprotein through an indirect ELISA. The full-length PPRV nucleoprotein gene was obtained from viral genome RNA by RT-PCR. The amplified fragments were cloned into baculovirus donor vectors pFastHTA of the Bac-to-Bac system. These recombinant plasmids, pFastHTA-PPRV-N, were transformed into DH10Bac host bacteria to obtain recombinant shuttle plasmids, pBacmid-PPRV-N. Recombinant baculovirus, Bacmid-PPRV-N, was generated for expression of the PPRV nucleoprotein by transfecting recombinant pBacmid-PPRV-N with Lipofectamine 2000 into Sf21 insect cells. The efficient expression of PPRV Nucleoprotein by baculovirus in Sf21 cells was verified by SDS-PAGE and Western blot. An indirect ELISA was developed using recombinant PPRV nucleoprotein as the coating antigen. 37 goat sera from an epidemic area in Tibet and 92 goat sera from a non-infected area in Qinghai Province were simultaneously detected by the indirect ELISA, developed here, and the international standard cELISA kit. The sensitivity and specificity of the indirect ELISA was 100% and 96.2% compared with the cELISA kit. The coincidence rate of the two methods was 96.9%. The results demonstrated that the indirect ELISA established in this study works well for diagnosis of PPR.
文摘小反刍兽疫(Peste des petits ruminants,PPR)是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)感染山羊、绵羊等小反刍动物引起的一种急性、高度接触性传染病,主要临床特征为高热、黏液脓性眼鼻分泌物、口腔炎、急性腹泻和死亡等。由于PPR具有较高的发病率和致死率,严重阻碍了养殖业健康发展,并造成农业经济的巨大损失。因此,分析该病的流行特点、临床特征和诊断技术对其防控工作意义重大。文章简述PPR的流行病学及诊断技术的研究进展,以期为该病的防控诊疗提供参考。
