大鲵虹彩病毒TaqMan实时荧光定量PCR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(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。

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.