Development of a biotinylated DNA probe for detection and identification of infectious hematopoietic necrosis virus

Abstract

A nonradioactive DNA probe assay was developed to detect and identify infectious hematopoietic necrosis virus (IHNV) using a dot blot format, The probe, a synthetic DNA oligonucleotide labeled enzymatically with biotin, hybridized specifically with nucleocapsid mRNA extracted from infected cells early in the virus replication cycle. A rapid, guanidinium thiocyanate based, RNA extraction method using RNAzol B and microcentrifuge tubes efficiently produced high quality RNA from 3 commonly used fish cell lines, CHSE-241, CHH-1, and EPC. The probe reacted with 6 diverse isolates of IHNV, but did not react with 2 related rhabdoviruses of fish, viral hemorrhagic septicemia virus and Hirame rhabdovirus. The biotinylated probe was sensitive, detecting picogram levels of target mRNA. Detection and identification of IHNV required 2 d when cells were inoculated at multiplicities of infection (MOI) greater than 2. Five days were necessary to detect and identify IHNV in cells inoculated at a MOI of 0.0002.