新血清型流行性出血病病毒荧光定量qRT-PCR与RT-PCR检测方法的建立及应用  

Development and application of a real-time quantitative RT-PCR and RT-PCR method for detection a novel serotype of epizootic haemorrhagic disease virus

作　　者：杨振兴[1] 李占鸿 宋子昂 李卓然 李华春[1] 朱建波[1] 廖德芳[1] 杨恒[1] YANG Zhen-xing;LI Zhan-hong;SONG Zi-ang;LI Zhuo-ran;LI Hua-chun;ZHU Jian-bo;LIAO De-fang;YANG Heng(Yunnan Tropical and Subtropical Animal Virus Disease Laboratory,Yunnan Academy of Animal Husbandry and Veterinary Sciences,Kunming 650224,China;College of Animal Science and Technology,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南省畜牧兽医科学院,云南省热带亚热带动物病毒病重点实验室,云南昆明650224 [2]云南农业大学动物科学技术学院,云南昆明650201

出　　处：《中国兽医科学》2021年第4期446-454,共9页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2017YFC1200505);国家公益性行业(农业)科研专项(201303035);云南省中青年学术和技术带头人后备人才培养项目(2017HB055)。

摘　　要：流行性出血病病毒(EHDV)是一种严重危害反刍动物的虫媒病毒。目前世界范围已发现9种血清型的EHDV,2018年笔者在云南芒市新分离到1株新血清型EHDV毒株(YNDH/V079/2018),但尚缺乏该血清型的特异性检测方法。本研究建立了新血清型EHDV核酸检测的实时荧光定量RT-PCR(qRT-PCR)和RT-PCR方法。试验结果显示,建立的两种方法均具有良好的特异性与灵敏度,与另外7种血清型EHDV、蓝舌病病毒(BTV)、中山病病毒(CHUV)、阿卡斑病毒(AKAV)和阴性对照均无交叉反应。对病毒核酸的检测下限分别达到10 copies/μL(qRT-PCR)与102copies/μL(RT-PCR)。临床样品检测显示,qRT-PCR能在动物感染EHDV早期检测鉴定出病毒,更适合应用于检测病毒载量较低的血液样品。RT-PCR的扩增产物可以胶回收测序,进一步了解待测病毒的遗传信息,两种方法辅助使用,为新血清型EHDV的早期快速诊断、流行病学调查和试验研究等提供了有效的技术方法。The epizootic haemorrhagic disease virus(EHDV) is an arbovirus that endangers ruminants.So far,nine serotypes of EHDV have been found worldwide.In 2018 a novel serotype of EHDV,named YNDH/V079/2018,was isolated from a sentinel calf in Mangshi County,Yunnan Province,China.In order to provide a method for identification the new serotype of EHDV,real-time quantitative RT-PCR and one-step RT-PCR were established.The results showed that the two methods had good specificity and sensitivity:there was no cross reaction with other 7 serotypes of EHDV,Bluetongue virus(BTV),Chuzan virus(CHUV),Akabane virus(AKAV) and negative control.The detection limit of viral nucleic acid was 10 copies/μL(qRT-PCR) and 102 copies/μL(RT-PCR),respectively.The detection of clinical samples show that qRT-PCR could detect and identify the new serotype EHDV than RT-PCR early in animal blood,which was more suitable for detection of blood samples with low viral load.The products of the RT-PCR can be recovered and sequenced,so the method can be used to further understand the genetic information of the virus.The two detection methods provide effective technical means for rapid diagnosis,epidemiological investigation and experimental research of the new serotype EHDV.

关 键 词：流行性出血病病毒 新血清型 荧光定量qRT-PCR RT-PCR 血清型鉴定 

分 类 号：S852.659.4[农业科学—基础兽医学]