PNA-PROBE BASED RT- PCR KIT FOR DETECTING THE NV GENE VARIATION OF VIRAL HEMORRHAGIC SEPTICAEMIA VIRUS ISOLATED IN SOUTH KOREA

Mi Young Cho1, Hyun-Ja Han1, Seong Don Hwang2, Jee youn Hwang2, Jaehun Cheong2 and Sung-Hee Jung1
 
1Pathology Research Division, National Institute of Fisheries Science(NIFS)
2Aquatic life Disease Control Division, NIFS, 46083, ROK
3Pusan University, 46083, ROK

The viral hemorrhagic septicemia virus (VHSV) is a viral pathogen to cause severe losses in farming industry of olive flounder. Thirteen VHSV strains are isolated from olive flounders showing VHS symptoms and mortality in Korea, and then molecular variation of DNA and amino acid sequences of the NV genes were compared. Although common mutations were detected at four sites in DNA sequences of NV gene, there was no difference in the amino acid sequences among thirteen VHSV variants. Without common amino acid variation, we found six amino acid variations in mortality-derived VHSV NV proteins. Based on finding of NV-mediated decrease of intracellular ATP level, protein expression of six NV variants in flounder cells decreased more than control NV protein. We have developed a highly sensitive and simple methods using a peptide nucleic acid (PNA) mediated real-time PCR to detect the specific viral DNA of variation sites of NV gene. PNA probes (SeaSun Biomaterials, Korea) for each variation site were designed to maximize their hybridization efficiencies and to minimize any non-target hybridization. The use of real-time PCR and PNA probes have advantages in terms of flexibility because we can easily alter the targeted mutations by adding or removing PNA probes and primers. In this study, PNA based real-time PCR have been successfully applied for the highly sensitive detection of target viral DNA of VHSV mutations of the Korean isolates. The Kit developed in this study is expected to find increasing use in routine disease monitoring and diagnosis in aquaculture and field epidemiology.