三重实时荧光RT-PCR检测三种鱼类弹状病毒的研究

Triplex real-time RT-PCR assay for simultaneous detection of three fish Rhabdoviridae viruses

鱼传染性造血器官坏死病病毒(Infectious hematopoietic necrosis virus IHNV)、鱼病毒性出血性败血症病毒(Viral hemorrhagic septicemia virus VHSV)和鲤春病毒血症病毒(Spring viraemia of carp virus SVCV)是同属弹状病毒科(Rhabdoviridae)的3种鱼类病毒,也是影响水产养殖业的3种重要病毒。通过对这3种病毒基因序列的分析,在病毒基因的高保守区域,设计了3条分别针对这3种病毒的Taqman MGB探针,并选择3种无相互干扰的荧光基团进行标记。以此为基础建立针对该3种病毒的三重实时荧光RT-PCR的检测方法。通过反应条件的优化,该三重实时荧光RT-PCR最低可检测至102拷贝/反应的病毒量。同时,该方法还对病毒混合感染EPC细胞(Epithelioma papulosum cyprini)和攻毒斑马鱼进行了检测。实验结果表明,该方法是一种特异、灵敏、高效的病毒检测方法。

Infectious hematopoietic necrosis virus （IHNV）,Viral hemorrhagic septicemia virus （VHSV） and Spring viraemia of carp virus （SVCV） are the three fish Rhabdoviridae viruses,which could cause serious losses of the aquiculture. Three taqman MGB probes,which could differentiate and detect the three viruses respectively without interference,were designed from highly conserved regions,and labeled with three dyes. Triplex real-time RT-PCR assay for IHNV,VHSV and SVCV was developed based on this. The assay was optimized and the lower limit of detection for three viruses was determined to be 102 viral genome copies per reaction. The co-infection Epithelioma papulosum cyprinid （EPC） cells and zebra fishes infected by the viruses respectively were detected by the assay. The results show that the assay is specific,sensitive and efficient for virus detection.