摘要
针对鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,CyHV-2)DNA解旋酶基因编码区序列设计特异性引物,利用PCR技术扩增出长度为1 446 bp的基因编码区片段,克隆到pMD19T载体上,构建重组质粒。经PCR鉴定与测序分析确认正确后,以10倍梯度稀释重组质粒,作为标准模板进行TaqMan real-time PCR扩增,制作标准曲线,建立了鲤疱疹病毒Ⅱ型的荧光定量PCR检测方法。检测结果显示,标准曲线的相关系数(R2)达到0.999 1,斜率为-3.412;对初始模板定量检测的范围为1×101～1×107copies/μL;特异性试验结果表明,该方法可特异性地检测出鲤疱疹病毒Ⅱ型,而对大鲵虹彩病毒(GSIV)、锦鲤疱疹病毒(KHV)以及空白对照无检测信号。取江苏射阳和宝应两地疑似患病鲫组织核酸作为模板进行荧光定量PCR,结果表明反应体系中的病毒量分别为6.89×104copies/μL和3.02×102copies/μL。本研究建立的鲤疱疹病毒Ⅱ型TaqMan实时荧光定量PCR方法灵敏度高、特异性强,对因鲤疱疹病毒Ⅱ感染引起的养殖鲫造血器官坏死症的诊断与病毒病原定量检测有重要意义。
A 1 446 bp coding region of Cyprinid herpesvirus Ⅱ（CyHV-2）DNA helicase gene was amplified by PCR and cloned into pMD19T vector for the construction of recombinant plasmid.After being identified and confirmed with PCR reaction,10-fold serial dilutions of recombinant plasmid were used as standard templates for TaqMan real time PCR to quantify the virus genomic copy number and generate standard curve.Herein,a TaqMan real-time PCR of detecting CyHV-2 was developed.It had a good linear relationship between the initial templates and Ct values with a detection range from 1×101 copies/μL to 1×107 copies/μL,the correlation coefficient（R2）was 0.999 1,and the slope value of standard curve was-3.412.The detection results showed that the specificity of this assay was high for CyHV-2 without cross-reactions with DNA templates from KHV and GSIV.The diseased crucian carp from Sheyang and Baoying,Jiangsu Province,were detected with the established method and the results showed that the content of CyHV-2 were 6.89×104 copies/μL and 3.02×102 copies/μL,respectively.The real-time PCR assay described here with high sensitivity and accuracy is considered to be a powerful tool for the rapid detection and quantification of CyHV-2 in fish.
出处
《水产学报》
CAS
CSCD
北大核心
2013年第4期607-613,共7页
Journal of Fisheries of China
基金
现代农业产业技术体系建设专项(CARS-46-11)
中国水产科学研究院基本科研业务费(2013A0606)
