一种真鲷虹彩病毒实时荧光定量PCR检测方法的建立  

Development of Real-Time PCR Assay for the Detection of Red Sea Bream Iridovirus (RSIV)

作　　者：张旻[1] 景宏丽[1] 方珍珍[2] 高隆英[3] 江育林[1] 林祥梅[1] 

机构地区：[1]中国检验检疫科学研究院,北京100029 [2]大连海洋大学 [3]深圳出入境检验检疫局

出　　处：《检验检疫学刊》2011年第5期38-41,59,共5页Journal of Inspection and Quarantine

基　　金：国家质量监督检验检疫总局科技项目(2008IK010)和(2006IK013);中国检验检疫科学研究院基础科研业务经费(2008JK004)

摘　　要：真鲷虹彩病毒一直被世界动物卫生组织列入必报疫病病原目录,该病毒与传染性脾肾坏死病毒在基因序列上没有区别。为了快速检测病原,本文根据传染性脾肾坏死病毒主要衣壳蛋白序列中一段基因保守区设计引物和TaqMan探针,建立了真鲷虹彩病毒特异性实时荧光定量PCR检测方法,并利用PCR反应扩增出的衣壳蛋白基因制备了pGEM-T/真鲷虹彩病毒重组质粒作为标准阳性对照。经试验,反应在模板量102-106拷贝浓度范围内有较好的线性关系,检测限最低可达100拷贝数,而且与流行性造血器官坏死病毒、甲鱼虹彩病毒等6种水生动物病毒无交叉反应。结果证明该方法具有简便、快速、敏感、特异等特点,它的建立为该病诊断与病原检测提供了一个重要手段。Red sea bream iridovirus（RSIV） is a pathogen causing high mortalities of red sea bream（Pagrosomus major） and more than 30 other species of culture marine fish belongs mainly to the orders Perciformes and Pleuronectiformes.So the red sea bream iridovirus disease had been in diseases list by OIE（World Organisation for Animal Health）.At genetic level,RSIV cannot been distinguished from Infectious spleen and kidney necrosis virus（ISKNV） which is the representative species of Megalocytivir and both of them could be detected by PCR using same primers.A number of diagnostic methods on detection of RSIV are used in Manual of Diagnostic Tests for Aquatic Animal diseases,such as the histopathological observation of smears or tissue sections,IFAT using a MAb,and polymerase chain reactions（PCR）.In order to establish a high efficient and reliable diagnostic method,a Real-time PCR assay for detection of RSIV was developed in this paper.The primers and TaqMan probes were designed according to the conserved regions of major capsid protein（MCP） gene of ISKNV.And the MCP gene was ligated into the pGEM-T plasmid vector to prepare of recombinant plasmid pGEM-T/RSIV as positive control.The Real-time PCR was carried out with viral DNA template in a 25-μL reaction mixture under the conditions of 45 cycles of denaturation（95 ℃ for 30 s）,annealing（60 ℃ for 30 s）.A good linear correlation was demonstrated in the standard curve for the Real-time PCR assay at the range of 102-106 copies of recombinant plasmid pGEM-T/RSIV template.The slope of standard curve was-3.496,correlation coefficient was 0.991,and efficiency was 93.2%.The detection limit of this Real-time PCR assay was 100 copies of viral gene segment（in pGEM-T/RSIV）,and there was no cross reaction with other 6 aquatic viruses such as Epizootic haematopoietic necrosis（EHNV）,Softshell turtle iridovirus（STIV） and Tiger frog virus（TFV） etc.In contrast,the cell culture method for detection RSIV are time-consuming,and antibody-based ant

关 键 词：鱼类病毒病 真鲷虹彩病毒 主要衣壳蛋白 实时荧光定量PCR TAQMAN探针 

分 类 号：Q784[生物学—分子生物学] S941.419[农业科学—水产养殖]