APPLICATION OF THE REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION FOR DETECTION AND IDENTIFICATION OF THE INFECTIOUS PANCREATIC NECROSIS VIRUS OF RAINBOW TROUT (ONCORHYNCHUS MYKISS)

Abstract

Purpose: To develop a diagnostic test system for detection and identification of infectious pancreatic necrosis virus of trout based on the reverse transcriptase polymerase chain reaction (RT-PCR). Materials and methods: Viruses: infectious pancreatic necrosis virus, reference strain – Аb (8.0 lg TCID/cm3); Ukrainian isolates – VF-11(6.8 lg TCID/ cm3), VF-08 (6.2 lg TCID/cm3), viral hemorrhagic septicemia virus of trout (VHSV), strain «DH4p101» [AY546581] (8.0 lg TCID/cm3). Finite cell line of fish: FHM (fathead minnow peduncle) and RTG (rainbow trout gonads). For detection and identification of infectious pancreatic necrosis virus of trout, two pairs of oligonucleotide primers were used. For search and analysis of gene homology of VP2 and NS proteins of infectious pancreatic necrosis virus, we used the BLAST 2.0 software of the National Center for Biotechnological Information (NCBI), USA. Constructing and matching of the primers were performed with the use of the «Vector NTI Advanced v.11» software package (Invitrogen, USA). Findings: For selecting effecting primers for PCR, we performed comparative study of nucleotide sequences of IPNV isolate genome of different genotypes. As a result, for the work we synthesized oligonucleotide primers, which flank VP2 protein gene sequence. The size of the amplified DNA fragment is 630 p.n. Additionally, we used oligonucleotide primers specific for the nucleoprotein gene (NS). The size of the amplified DNA fragment is 204 p.n. When checking the specificity of the polymerase chain reaction with matched primers as a matrix, we used RNA samples of the reference IPNV strains and isolates isolated from rainbow trout fingerlings from different fish farms of Ukraine. Conclusions: The developed diagnostic test system based on the reverse transcriptase chain reaction (RT-PCR) is highly specific and sensitive for detection and identification of IPNV virus in the materials of various origins.