鳜鱼传染性脾肾坏死病毒TaqMan荧光定量PCR检测方法的建立  

Establishment of a TaqMan qPCR assay for detecting the infectious spleen and kidney necrosis virus of Siniperca chuatsi

作　　者：安伟[1] 肖雨[1] 张崇文[1] 张明辉[1] 何正侃[1] 

机构地区：[1]上海市水产研究所上海市水生动物疫病预防控制中心,上海200433

出　　处：《中国预防兽医学报》2014年第3期214-217,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：上海市财政水生动物病害防治专项资金

摘　　要：为了建立检测鳜鱼传染性脾肾坏死病毒(ISKNV)的方法,本研究根据GenBank中ISKNV MCP基因的保守序列设计并合成1对引物和1条TaqMan探针,建立了该病毒的TaqMan荧光定量PCR检测方法。结果表明,以重组质粒标准品构建的标准曲线的相关系数为0.994,具有良好的线性关系,检测下限为20拷贝/μL,是常规PCR的1 000倍;特异性强,只有ISKNV呈阳性,而与流行性造血器官坏死病毒、鲤春病毒血症病毒和草鱼呼肠孤病毒均无交叉反应;重复性好,批内和批间的变异系数均小于0.7%。本研究建立的ISKNV TaqMan荧光定量PCR方法对ISKNV的快速检测及定量检测具有重要意义。In the present study, a TaqMan real-time PCR assay was established for detection of infectious spleen and kidney necrosis virus （ISKNV） with a pair of specific primers and a TaqMan probe designed according to the conserved region in MCP gen of ISKNV. The established assay possessed a fine linear relationship between initial templates and Ct values, and the correlation coefficient of the standard cure was 0.994. The detected limit of method was 20 copies/μL of initial templates, which was 1000-fold more sensitive than that of the conventional PCR. In addition, the method was also highly specific with the flurescent singals only be detected from ISKNV, but not amplification from EHNV, SVCV and GCRV. Moreover, the method was highly reproducible and had a coefficient of variation less than 0.7% for both intra-and inter-assays. The assay established in this study is considered to be an effective tool for the rapid detection and quantification of ISKNV in mandarin fish （Siniperca chuatsi）.

关 键 词：鳜鱼 传染性脾肾坏死病毒 荧光定量PCR反应 

分 类 号：S852.65[农业科学—基础兽医学]