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大鲵虹彩病毒TaqMan实时荧光定量PCR检测方法的建立
引用本文:周 勇,曾令兵,孟 彦,周群兰,张 辉,高正勇,肖 艺,孙建滨.大鲵虹彩病毒TaqMan实时荧光定量PCR检测方法的建立[J].水产学报,2012,36(5):772-778.
作者姓名:周 勇  曾令兵  孟 彦  周群兰  张 辉  高正勇  肖 艺  孙建滨
作者单位:1. 中国水产科学研究院长江水产研究所,湖北武汉,430223
2. 中国水产科学研究院淡水渔业研究中心,江苏无锡,214081
3. 华中农业大学水产学院,湖北武汉,430071
基金项目:中国水产科学研究院淡水渔业研究中心基本科研业务费(2011JBFZ01); 中国水产科学研究院基本科研业务费资助(2012A0505); 公益性行业(农业)科研专项经费(201203086)
摘    要:利用PCR技术扩增出大鲵虹彩病毒(giant salamander iridovirus, GSIV)主要衣壳蛋白(MCP)编码区长度为1 392 bp 的片段, 克隆到 pMD19-T载体上, 构建重组质粒 pMD19-T-MCP。经PCR鉴定确认正确后, 以10倍梯度稀释 pMD19-T-MCP重组质粒, 作为标准模板进行 TaqMan实时荧光定量PCR扩增, 制作标准曲线, 建立了大鲵虹彩病毒的 TaqMan实时荧光定量PCR检测方法。制作的标准曲线有极好的线性关系, 且线性范围宽, 相关系数为0.990 19。组内重复试验的CT值标准偏差为0.52%。检测结果显示, 该方法对大鲵虹彩病毒的检测有高度的特异性, 与锦鲤疱疹病毒、弗氏柠檬酸杆菌、嗜水气单胞菌以及鲤上皮瘤细胞基因组DNA之间均无交叉反应, 特异性好, 检测总DNA灵敏度为10个病毒核酸分子拷贝数, 约1.1×10-3 pg/μL病毒核酸, 较之常规PCR的敏感度高出约1 000倍。研究建立的大鲵虹彩病毒TaqMan实时荧光定量PCR方法灵敏度高、特异性强, 对大鲵虹彩病毒病的快速诊断与病毒病原定量检测有重要意义。

关 键 词:大鲵    虹彩病毒    主要衣壳蛋白    TaqMan  实时荧光定量PCR
收稿时间:1/4/2012 3:51:17 PM
修稿时间:2/15/2012 1:05:58 PM

Establishment of a TaqMan real-time PCR assay for detecting the giant salamander iridovirus
ZHOU Yong,ZENG Ling-bing,MENG Yan,ZHOU Qun-lan,ZHANG Hui,GAO Zheng-yong,XIAO Yi and SUN Jian-bin.Establishment of a TaqMan real-time PCR assay for detecting the giant salamander iridovirus[J].Journal of Fisheries of China,2012,36(5):772-778.
Authors:ZHOU Yong  ZENG Ling-bing  MENG Yan  ZHOU Qun-lan  ZHANG Hui  GAO Zheng-yong  XIAO Yi and SUN Jian-bin
Institution:Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,JiangSu Wuxi,Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,College of Fisheries,Huazhong Agricultural University,HuBei Wuhan,Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,College of Fisheries,Huazhong Agricultural University,HuBei Wuhan
Abstract:A 1 392 bp coding region of giant salamander iridovirus(GSIV)MCP protein was amplified by PCR and cloned into pMD19-T vector for the construction of recombinant plasmid pMD19-T-MCP.After being identified and confirmed by PCR reaction,10-fold serial dilutions of plasmid pMD19-T-MCP were used as standard templates for TaqMan real-time PCR to generate standard curve for quantifying the virus genomic copy number.A good linear correlation was demonstrated in the standard curve for the real-time PCR assay.The coefficients of variance(CV) were 0.52% for intra-assay tests,which indicated good reli-ability.The detection results showed that the specificity of this assay was high for giant salamander iri-dovirus without cross-reactions with DNA templates from KHV,Aeromonas hydrophila,Citrobacter freundii and EPC cells.A minimum of 10 copies of GSIV DNA(1.1×10 3 pg/μL total DNA) could be de-tected,which indicated that the sensitivity of real time PCR is about 1000 times higher than that of the conventional PCR assay.The TaqMan real-time PCR assay established in this study is considered to be a powerful tool for the rapid detection and quantification of GSIV in giant salamander.
Keywords:Andrias davidianus  giant salamander iridovirus  MCP protein  TaqMan real-time PCR
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