REAL2TIME QUANTITATIVE RT2PCR ASSAY FOR DETECTION OF IHNV IN FISH

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.