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  

DEVELOPMENT AND APPLICATION OF A TAQMAN RT-PCR ASSAY FOR DETECTION OF AVIAN REOVIRUSES IN TISSUES

作　　者：钟丽[1] 孙美玉[1] 高立[1] 刘永振[1] 李凯[1] 祁小乐[1] 高玉龙[1] 王笑梅[1] 

机构地区：[1]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,哈尔滨150001

出　　处：《中国动物传染病学报》2017年第2期56-60,共5页Chinese Journal of Animal Infectious Diseases

基　　金：现代农业产业技术体系建设专项基金(CARS-42-G07)

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。A Real-time PCR method for detection of Avian reoviruses was developed using a pair of primers and specific Taqman probe that were designed according to the σC genes of Avian reovirus isolates and reference strains from GenBank. The assay was optimized to produce linearity from 1×10^1 copies/laL to 1 × 10^10 copies/pL in standard curve. The detection limit of the assay was 10 copies of viral RNA, which showed higher sensitivity than the currently available PCR methods. There was no cross-reaction with other common Avian viruses. Furthermore, 1-day-old SPF chickens were inoculated with the Avian reovirus isolate MS01 and reference strain S 1133. The viral loads in multiple organs were detected using the PCR method developed here. The results showed that the mortality of MS01 and S 1133 was 80% and 46.7% , respectively. The viruses replicated abundantly in livers, spleens and lungs. However, viral loads of the field isolate MS01 were significantly higher than those of the reference S 1133. Besides, MS01 replicated well in kidneys while S 1133 did poorly. The RT-PCR method developed in the present study could be used for diagnosis and molecular pathogenesis mechanism of highly pathogenic Avian reovirus.

关 键 词：禽呼肠孤病毒 σC基因 TAQMAN RT-PCR 组织分布 致病性 

分 类 号：S852.659.4[农业科学—基础兽医学]