大鲵虹彩病毒TaqManreal·timePCR检测方法的建立

Establishment of a TaqMan real-time PCR assay for detecting the giant salamander iridovirus

利用PCR技术扩增出大鲵虹彩病毒（Giantsalamanderiridovirus，GSIV）主要衣壳蛋白（MCP）编码区长度为1392bp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0．99019。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鱼上皮瘤细胞基因组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^-3g／μL病毒核酸，较之常规PCR的敏感度高出约1000倍。本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。

A 1392 bp coding region of Andrias davidianus（GSIV） MCP protein was amplified by PCR and cloned into pMD19-T vector for the construction of recombinant plasmid pMD19-T-MCP. After identified and confirmed by PCR reac- tion, 10-fold serial dilutions of plasmid pMD19-T-MCP were used as standard templates for TaqMan real time PCR to gen- erate 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 a good reliability. The detection results showed that the specificity of this assay was high for A. davidianus with- out cross-reactions with DNA templates from KHV, Aeromonas hyclrophila, Citrobacterfreundii and EPC cells. A minimum of 10 copies of GSIV DNA （ - 1.1 × 10-3pg/μL total DNA） could be detected, 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 A. davidianus.