实时定量RT-PCR检测鱼类传染性造血器官坏死病毒方法的建立与应用
REAL2TIME QUANTITATIVE RT2PCR ASSAY FOR DETECTION OF IHNV IN FISH
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摘要: 建立了Taqman实时定量RT-PCR方法检测传染性造血器官坏死病毒(IHNV)。选取IHNV病毒的N蛋白基因保守序列,利用Primer Express 2.0软件设计引物和探针。以梯度稀释的含有IHNV目的扩增片段的质粒作为标准品,进行定量RT-PCR反应以确定检测灵敏度。病毒浓度在5×106—5个拷贝,共7个数量级的范围内,定量RT-PCR反应有"S"型扩增曲线,检测灵敏度为5个拷贝。根据病毒拷贝数与定量反应Ct值的关系,绘制了标准曲线。该方法具有特异性,对鲤春血症病毒(SVCV)、病毒性出血性败血症(VHSV)、传染性胰脏坏死病毒(IPNV)、草鱼呼肠孤病毒(GCRV)、流行性造血器官坏死病毒(EHNV)、EPC细胞系、牙鲆的核酸都没有扩增反应。在50批待检样品中,有3批鱼类感染IHNV,利用标准曲线进行了定量分析。实时定量RT-PCR检测IHNV方法,灵敏度高,特异性好,可以进行定量分析,在鱼病的快速检测上具有重要意义。
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关键词:
- 传染性造血器官坏死病毒(IHNV) /
- 实时定量RT-PCR /
- Taqman探针
Abstract: A real-time quantitative RT-PCR assay was developed for detection of infectious hematopoietic necrosis virus ( IHNV).Primers and probe were designed based on the nucleocapsid gene of IHNV by Primer Express 2. 0 software. The plasmid contain-ing the target sequence was constructed to detect the sensitivity and prepare the standard curve. The real-time RT2PCR assay hada detection limit of 5 copies, with a dynamic range of detection between 5 ×106 —5 copies. The standard curve was preparedbased on the linear relationship between the amount of plasmid DNA and cycle threshold (Ct). The primers and probe were spe-cific for IHNV and did not react with either spring viremia of carp virus (SVCV), viral hemorrhagic septicemia virus (VHSV),infectious pancreatic necrosis ( IPNV), grass carp reovirus (GCRV), epizootic haematopoietic necrosis virus (EHNV), EPC cellline or fish tissue RNA. Collected samples were detected with the real-time RT-PCR assay and three positive samples were usedfor quantitative analysis. The real-time RT2PCR assay that described here with high sensitivity, specificity and accuracy is con2sidered to be a powerful tool for the rapid detection and quantification of IHNV in fish.-
Keywords:
- IHNV /
- Real-time quantitative RT2PCR /
- Taqman probe
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