[1]安 伟,肖 雨*,张崇文,等.鳜鱼传染性脾肾坏死病毒TaqMan荧光定量PCR检测方法的建立[J].中国预防兽医学报,2014,(03):214-217.[doi:10.3969/j.issn.1008-0589.2014.03.11]
 AN Wei,XIAO Yu*,ZHANG Chong-wen,et al. Establishment of a TaqMan qPCR assay for detecting the infectious spleen and kidney necrosis virus of Siniperca chuatsi[J].Chinese journal of preventive veterinary medicine,2014,(03):214-217.[doi:10.3969/j.issn.1008-0589.2014.03.11]
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鳜鱼传染性脾肾坏死病毒TaqMan荧光定量PCR检测方法的建立
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2014年03期
页码:
214-217
栏目:
诊断和检测技术
出版日期:
2014-03-25

文章信息/Info

Title:
 Establishment of a TaqMan qPCR assay for detecting the infectious spleen and kidney necrosis virus of Siniperca chuatsi
文章编号:
1008-0589(2014)03-0214-04
作者:
  安 伟肖 雨*张崇文张明辉何正侃
 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
Author(s):
 AN Wei XIAO Yu* ZHANG Chong-wen ZHANG Ming-hui HE Zheng-kan
 Shanghai Aquatic Animal Epidemic Disease Prevention and Control Center, Shanghai Fisheries Research Institute, Shanghai 200433, China
关键词:
 鳜鱼传染性脾肾坏死病毒荧光定量PCR反应
Keywords:
  Siniperca chuatsi  infectious spleen and kidney necrosis virus  qPCR
分类号:
S852.65
DOI:
10.3969/j.issn.1008-0589.2014.03.11
文献标志码:
A
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。
Abstract:
 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).

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期:2013-07-09
基金项目:上海市财政水生动物病害防治专项资金
作者简介:安 伟(1984-),男,河北唐山人,助理工程师,主要从事鱼类病害防治技术研究.
*通信作者:E-mail:envijoy@126.com
更新日期/Last Update: 2014-03-27