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

Abstract

A n o n r a d ~o a c t ~v e DNA probe assay was developed to detect and ~d e n t ~f y infect~ous hernatopoiet~c necrosls virus (IHNV) uslng a dot blot format The probe a synthet~c DNA oligonu-cleot~de labeled enzymatlcally w ~t h biotln h y b n d ~z e d spec~f~cally w ~t h nucleocaps~d mRNA extracted from Infected cells early In the vlrus repl~cation cycle A r a p ~d g u a n ~d l n ~u m th~ocyanate based RNA extraction method uslng RNAzol B and rn~crocentrifuge tubes e f f ~c ~e n t l y pioduced h ~g h q u a l ~t y RNA from 3 commonly used f ~s h cell llnes, CHSE-214, CHH-1, and EPC The probe reacted with 6 d ~v e r s e ~solates of IHNV, but d ~d not react \ n t h 2 related rhabdovlruses of fish viral hemorrhagic septlcemla vlrus and H ~r a m e rhabdovlrus The b ~o t ~n y l a t e d probe was sensltlve d e t e c t ~n g plcogram levels of target mRNA Detect~on and ~d e n t i f ~c a t ~o n of IHNV r e q u ~r e d 2 d when cells wele lnoculated at n ~u l t i p l i c ~t ~e s of infect~on (MOI) greater than 2 Flve days were necessary to detect and identify IHNV In cells lnoculated at a MO1 of 0 0002