【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PrimerExpress2.0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。更多还原

【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 containing the target sequence was constructed to detect the sensitivity and prepare the standard curve. The real-time RT-PCR assay had a detection limit of 5 copies, with a dynamic range of detection between 5×106—5 copies. The standard curve was prepared based on the linear relationship between the amount of plasmid DNA and cycle threshold (Ct). The primers and probe were specific 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 cell line or fish tissue RNA. Collected samples were detected with the real-time RT-PCR assay and three positive samples were used for quantitative analysis. The real-time RT-PCR assay that described here with high sensitivity, specificity and accuracy is considered to be a powerful tool for the rapid detection and quantification of IHNV in fish.更多还原