【作者】 钟涛；

【导师】 师东方；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 本研究用经Ni2+-NTA金属螯合层析柱纯化后获得纯化的重组N蛋白作为抗原检测试剂,抗重组N蛋白的单克隆抗体作为抗体诊断试剂,用非离子去污剂暴露出TGEV的核衣壳蛋白,利用竞争原理针对粪便中的TGEV建立了一种快速的间接竞争ELISA检测方法。并对方法中封闭液的选择及工作条件进行了确定,结果表明0.5%聚乙烯醇在37℃封闭2h效果最理想。该方法对感染TGEV的20头份仔猪的粪便样品进行检测,并用统计学方法对仔猪粪便样品的检测数据进行了分析,确定了阳性值判定标准为:在阴性对照(N)OD492nm>0.9情况下,抑制率大于14.5%为阳性。特异性实验结果表明,建立的间接竞争ELISA对猪轮状病毒、猪流行性腹泻病毒等肠道病毒的检测均为阴性反应。以TGEV TH-98株的强毒株人工口服感染未进初乳的初生仔猪,24h后收集仔猪粪便样品,用间接竞争ELISA和RT-PCR方法分别对粪便进行病原体检测,结果表明,在仔猪感染62h以后,用间接竞争ELISA方法可检测到在肠道内扩增的TGEV;RT-PCR方法在感染后57h即可检测到TGEV。RT-PCR方法比间接竞争ELISA要早近5h检测到TGEV。对63h以后的粪便作10倍稀释时,间接竞争ELISA方法可检测到粪便中的TGEV,而RT-PCR方法对粪便作20倍、40倍稀释时,仍可检出TGEV。表明RT-PCR方法较间接竞争ELISA方法敏感。更多还原

【Abstract】 The virions were lysed by nonionic detergent and the nucleoprotein was exposed. The selection of blocking solution and other paremeters were optimized, the analyzation shows that blocking for 2h at 37℃with 0.5% PVP induces the best result. Indirect competitive ELISA method was established with the recombinant nucleoprotein(rNP)of transmissible gastroenteritis virus(TGEV)and the monoclonal antibody against TGEV N protein to detect intestinal contents of 20 piglets infected with TGEV. The inhibition ratio of the samples which were more than 14.5% was regarded as positive value under the condition that N OD492nm >0.9 by statistical analysis. The specific assay was carried out by indirect competitive ELISA method and the results showed that the detection of porcine rotavirus (PRV) and porcine epidemic diarrhea virus (PEDV) were negative.Colostrum-deprived piglets were orally infected with TGEV TH-98 virulent strain, and the faecal samples were collected since 24 hours post-infection. Indirect competitive ELISA and RT-PCR were carried out for the detection of TGEV. Enteric proliferated TGEV were detected by indirect competitive ELISA in faecal samples obtained since 62 hours post-infection and also detected by RT-PCR in faecel samples taken since 57 hours post-infection. TGEV was detected by RT-PCR 5h earlier than by indirect competitive ELISA. TGEV can be detected by RT-PCR and indirect competitive ELISA in 10-fold diluted faecal samples taken ever since 63h post-infection. However, TGEV in 20-fold and 40-fold diluted faecel samples could only be detectied by RT-PCR. Results shows that RT-PCR is more sensitive than indirect competitive ELISA.更多还原