流行性出血病病毒(EHDV)血清型特异性荧光定量RT-PCR方法的建立及初步应用  

Development and Application of a Real-time Quantitative RT-PCR Method for Detection and Serotyping of the Epizootic Haemorrhagic Disease Virus

作　　者：杨振兴[1] 李占鸿 宋子昂 朱建波[1] 李卓然 李华春[1] 谢佳芮 廖德芳[1] 杨恒[1] YANG Zhenxing;LI Zhanhong;SONG Ziang;ZHU Jianbo;LI Zhuoran;LI Huachun;XIE Jiarui;LIAO Defang;YANG Heng(Yurnan Tropical and Subtropical Animal Virus Disease Laboralory,Yunan Veterinary and Animal Science Institute,Kunming 650224,China;College of Animal Science and Technology,Ywwnan Agricutural University,Kurmning 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,昆明650224 [2]云南农业大学,动物科学技术学院,昆明650201

出　　处：《病毒学报》2020年第5期897-906,共10页Chinese Journal of Virology

基　　金：国家重点研发计划(项目号:2017YFC1200505),题目:蓝舌病等虫媒性动物疫病现场快速检测与实验室鉴定技术研究;国家重点研发计划(项目号:2016YFD0500908),题目:牛羊虫媒病毒病诊断与检测新技术研究;云南省中青年学术和技术带头人后备人才培养项目(项目号: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,本研究旨在建立EHDV的血清型特异性实时荧光定量RT-PCR(qRT-PCR)诊断技术。以决定EHDV血清型的基因节段2(Seg-2)作为靶基因,设计8对特异性扩增引物和TaqMan探针,以不同血清型EHDV代表毒株的cDNA为模板,分析检测方法特异性、敏感性;以不同血清型的EHDV分离毒株及临床血液样本为检测对象,分析该方法的实际检测效果。实验结果显示:建立的EHDV血清型特异性qRT-PCR检测方法具有高度的特异性和灵敏性,可准确鉴定不同血清型EHDV,与蓝舌病病毒、中山病病毒和阿卡斑病毒之间均无交叉反应;qRT-PCR反应的扩增效率均达到90%以上,对不同血清型EHDV核酸检测灵敏度在24拷贝/μL^44拷贝/μL之间。对我国分离的31株EHDV进行血清型qRT-PCR鉴定,结果显示分离的EHDV分别为EHDV-1(6株)、EHDV-5(9株)、EHDV-6(11株)、EHDV-7(2株)与EHDV-10(3株)。对不同血清型EHDV感染动物的血液样本检测结果显示,本方法能在动物感染EHDV早期鉴定出病毒的血清型,与血液样本中分离EHDV毒株的血清型鉴定结果一致。本研究建立的EHDV血清型荧光定量RT-PCR定型方法具特异性强、灵敏度高和耗时少等优点,可用于动物感染EHDV血清型的快速与准确诊断,具有良好的应用与推广价值。The epizootic haemorrhagic disease virus(EHDV)is an arbovirus that endangers ruminants. Nine serotypes of the EHDV have been found worldwide. We wished to establish a real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR) method for EHDV serotypes. Eight pairs of specific primers and TaqMan ? probes were designed to amplify segment 2(which determines the EHDV serotype).We analyzed the specificity and sensitivity of the detection method using the cDNA of different EHDV serotypes. Different serotypes of the EHDV and clinical blood samples were used to evaluate detection. Results showed the high specificity and sensitivity of our serotype-specific detection method,which showed no crossreaction between the bluetongue virus,Chuzan virus or Akabane virus. The amplification efficiency was >90%,and the minimum limit of detection of the nucleic acids of the EHDV was 24 – 44 copies/μL from different serotypes. qRT-PCR amplification of 31 serotypes of EHDV strains isolated from China showed that the strains were EHDV-1(six strains),EHDV-5(nine),EHDV-6(eleven),EHDV-7(two)and EHDV-10(three). Different serotypes of EHDV-infected animals could be identified by this method using blood samples during early infection;those data were in accordance with the results of serotype identification of EHDV strains isolated from blood samples. The qRT-PCR method we developed for EHDV serotypes:showed strong specificity;showed high sensitivity;could be used for rapid and accurate diagnosis of EHDV serotypes for animal samples and could be applied for diagnostic purposes.

关 键 词：流行性出血病病毒(EHDV) 血清型 荧光定量RT-PCR 分子诊断 

分 类 号：S852.65[农业科学—基础兽医学]