【作者】 王淑敏；

【导师】 贺建华； 顾万军；

【作者基本信息】 湖南农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 重症急性呼吸综合症(Severe Acute Respiratory Syndrome，SARS)的病原是SARS冠状病毒(SARS.CoV)，为了调查野生动物和家畜家禽体内携带的冠状病毒的情况以及其与SARS.CoV的基因序列同源性，本研究根据SPA能与多种动物血清IgG结合的特性，建立了间接PPA-ELISA检测多种动物血清中SARS病毒相关抗体的方法；根据SARS冠状病毒聚合酶基因序列设计引物，建立套式PCR(nest-PCR)检测方法。对11种动物146个样品进行RT-PCR检测，并用PCR产物测序。与Genbank中的SARS相关冠状病毒的聚合酶基因进行序列比较，构建系统发育树，分析不同宿主来源动物冠状病毒与SARS冠状病毒的同源性。两种检测方法的实验结果表明： SPA与多种哺乳动物血清中的IgG有较好的结合能力，本实验确定了PPA-ELISA的抗原-抗体最佳反应时间，HRP-SPA与抗体反应最佳时间及底物溶液的显色时间，在此基础上，建立了间接PPA-ELISA检测多种动物血清中SARS病毒相关抗体的检测程序。 通过对冠状病毒聚合酶基因的扩增和序列分析，结果表明被检测的动物样品中的冠状病毒聚合酶的保守基因序列与SARS冠状病毒聚合酶的同一位置的基因序列同源性为100％～95.8％。 通过对同一动物的PPA-ELISA和nest-PCR检测表明，两种检测的约为符合率60％～70％，两种方法的同时使用对动物冠状病毒的检测有着重要的参考价值。更多还原

【Abstract】 The pathogen of Severe Acute Respiratory Syndrome(SARS) is Corona Virus, For investigating the truth about Corona virus in animals, and find the homologous with SARS. CoV, a essay have been establish that based on the characters that SPA can combine the IgG in the serum of many kinds of animals, indirect PPA-ELISA. To detect antibody of SARS virus in the serum of many animals was established. Two pairs of primer was designed according to the partly polymerase gene of SARS associated corona virus sequences published in Genbank. 146 samples from 11 kinds of animals were detected by nest-PCR., Then PCR product was sequenced. Phylogenetic analysis was done on the amplified region .And the relationship between Corona virus for the animals and SARS. CoV was analyzed.The results indicated that SPA can combine with many mammals IgG in the serum well, and determined the best time of the antigen combined antibody reaction HRP-SPA commeded antibody, At the same time, we made the method of PPA-LEISA which detected the SARS antibody dedicational.The result of amplifying of polymerase gene of SARS associated corona virus and sequence analyze showed that there were high homology between the samples from human and animals, around 100%~95. 8% in nucleotide acid.By detect the sample of the same animals using PPA-ELISA and nest-PCR separated, the accordance rate of the two methods is around 60%~70%, it indicated that it’ s valuable for the detection of Corona virus in animals by using the two assay.更多还原