禽呼肠孤病毒TaqMan实时荧光RT-PCR方法的建立及病毒组织分布比较研究

发布时间：2019-03-26 21:25

【摘要】：根据本实验室分离禽呼肠孤病毒(Avian reovirus,ARV)毒株和GenBank中已发布ARV标准毒株σC基因序列比对结果,在保守区域设计1对引物和1条特异性TaqMan探针。通过对反应条件和反应体系的优化,建立的Real-time RT-PCR在1×10~1~1×10~(10) copies/μL范围内具有良好的线性关系,灵敏度达到10 copies/μL,是常规PCR方法的100倍。本研究建立的RTPCR与其他禽病病原无交叉反应,且批间与批内重复性试验变异系数分别小于1%和2%,表明该方法具有良好的特异性和重复性。将本实验室分离流行毒株MS01和标准毒株S1133感染SPF鸡后,利用建立的Real-time RT-PCR对ARV在鸡体内组织脏器的分布及病毒载量进行比较研究,结果表明,毒株MS01及S1133对SPF鸡的致死率分别为80%和46.7%,两株病毒均能在肝脏、脾脏、肺脏中有效复制,毒株MS01病毒载量明显高于毒株S1133,但与毒株S1133不同,毒株MS01在肾脏组织中也能有效复制。本研究不仅为禽呼肠孤病毒的诊断提供了技术手段,也有利于进一步探索流行毒株MS01高致病性的分子机制。
[Abstract]:According to the sequence alignment of 蟽 C gene of avian reovirus (Avian reovirus,ARV) strain isolated in our laboratory and ARV standard strain published in GenBank, a pair of primers and a specific TaqMan probe were designed in the conservative region. Through the optimization of the reaction conditions and reaction system, the established Real-time RT-PCR has a good linear relationship in the range of 1 脳 10 ~ 1 ~ 1 脳 10 ~ (10) copies/ 渭 L, and the sensitivity is up to 10 copies/ 渭 L, which is 100 times higher than that of the conventional PCR method. There was no cross-reaction between RTPCR and other pathogens in this study, and the coefficient of variation of inter-assay and intra-assay repeatability tests were less than 1% and 2%, respectively, indicating that the method has good specificity and repeatability. After SPF chickens were infected with MS01 and S1133, the distribution and viral load of ARV in the tissues and organs of the chickens were studied by using the established Real-time RT-PCR. The results showed that the distribution of ARV in the tissues and organs of chickens was compared with that of the standard strain S1133. The fatality rates of MS01 and S1133 strains to SPF chickens were 80% and 46.7%, respectively. The two strains could replicate effectively in liver, spleen and lung. The load of MS01 virus was significantly higher than that of S1133 strain, but different from S1133 strain. The strain MS01 can also replicate effectively in kidney tissue. This study not only provides a technical means for the diagnosis of avian reovirus, but also helps to explore the molecular mechanism of the high pathogenicity of MS01.
【作者单位】：中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室;
【基金】：现代农业产业技术体系建设专项基金(CARS-42-G07)
【分类号】：S852.65

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