实时荧光定量RT-PCR检测鱼类传染性胰脏坏死病病毒方法的建立

Establishment of Real-Time RT-PCR Assay for Detection of IPNV

[目的]建立Taqman探针荧光定量RT-PCR检测传染性胰脏坏死病病毒(IPNV)的方法。[方法]选取IPNV病毒的基因保守序列,利用Primer Express 2.0软件设计引物与探针。以梯度稀释的含有IPNV目的扩增片段的质粒作为标准品,探索定量RT-PCR反应条件。[结果]当标准品浓度在102～106 copies/μl之间时,标准品浓度(X)与Ct的关系为Ct=-3.426 lgX+4.481,相关系数R2为0.999 8。该法对病毒性出血性败血症病毒(VHSV)、传染性造血器官坏死病病毒(IHNV)、鲤春病毒血症病毒(SVC)和流行性造血器官坏死病病毒(EHNV),草鱼呼肠孤病毒(GCRV),大鳞大麻哈鱼胚胎细胞(CHSE-214)和虹鳟的核酸都没有扩增反应。[结论]该检测方法灵敏度高,特异性好,可用于鱼类传染性胰脏坏死病病毒的快速定量检测。

[Objective] The aim was to develop the detection of infectious pancreatic necrosis virus（IPNV）by real-time RT-PCR assay.[Method] Primers and probe were designed using the software Primer Express 2.0.The plasmid containing the target sequence was constructed to detect the sensitivity and prepare the standard curve.The standard curve was prepared based on the linear relationship between the amount of plasmid DNA and cycle threshold（Ct）.[Result] The linear relationship between virus concentration（X） and Ct was Ct=-3.069 6＋42.246（R2=0.999 8）.The method was specific for IPNV and did not react with other comparative fish virus and nucleic acid of rainbow trout.[Conclusion]The determination of IPNV by real-time RT-PCR with high sensitivity and specificity was an effective tool for the rapid detection of IPNV.