金鱼造血器官坏死病毒实时荧光定量PCR检测方法的建立  

Development of Real-time PCR Assay for the Detection of Goldfish Haematopoietic necrosis Virus(GFHNV)

作　　者：张旻[1] 王姝 王娜[1] 景宏丽[1] 徐立蒲 吴绍 

机构地区：[1]中国检验检疫科学研究院动物检疫研究所,北京100176 [2]北京市水产技术推广站,北京100176

出　　处：《中国动物检疫》2017年第11期99-103,共5页China Animal Health Inspection

基　　金：国家科技支撑计划(2013BAD12B02);北京市观赏鱼产业创新团队(BAIC03-2017)

摘　　要：金鱼造血器官坏死病毒(Goldfish haematopoietic necrosis virus,GFHNV)是一种可感染金鱼等鲤科鱼类,并可导致其死亡的疱疹病毒。为了快速检测病原,本研究根据GFHNV的主要衣壳蛋白(Major captor protein,MCP)序列中的一段保守基因序列,设计引物和Taq Man探针,建立了GFHNV特异性的实时荧光定量PCR检测方法 ;并利用PCR反应扩增出的MCP基因,制备了p UC-GFHNV重组质粒作为阳性对照。经试验,该方法检测限最低可达10个拷贝数的目的基因,而且与锦鲤疱疹病毒(Koi herpesvirus,KHV)、甲鱼虹彩病毒(Softshell turtle iridovirus,STIV)、流行性造血器官坏死病毒(Epizootic haematopoietic necrosis,EHNV)、斑点叉尾鮰病毒(Channel catfish virus,CCV)以及白斑综合症病毒(White spot syndrome virus,WSSV)等5种水生动物病毒无交叉反应。该方法具有简便、快速、敏感、特异等优点,为该病的诊断与病原检测提供了一个重要手段。Goldfish haematopoietic necrosis virus（GFHNV）is a pathogen causing high mortalities of goldfish,which belongs to Cyprinivirus. In order to establish a high efficient and reliable diagnostic method,a real-time PCR assay for detection of GFHNV was developed in this paper. The primers and Taq Man probes were designed according to the conserved regions of major capsid protein（MCP）gene of GFHNV. And the MCP gene was ligated into the p UC57 plasmid vector to prepare of recombinant plasmid p UC-T/GFHNV as positive control. The detection limit of this Realtime PCR assay was 10 copies of viral gene segment（in p UC-GFHNV）,and there was no cross reaction with other 5 aquatic viruses,such as Koi herpesvirus（KHV）,Softshell turtle iridovirus（STIV）,Epizootic haematopoietic necrosis（EHNV）,Channel catfish virus（CCV）and White spot syndrome virus（WSSV）. It＇s showed that this real-time PCR assay was sensitive and specific for detection of GFHNV and provided an important means for the diagnosis and pathogen detection of the disease.

关 键 词：鱼类病毒病 金鱼造血器官坏死病毒 主要衣壳蛋白 实时荧光定量PCR TAQMAN探针 

分 类 号：S941.419[农业科学—水产养殖]