Rapid Diagnosis of Necrosis Nervous Virus Infection using Recombinase Polymerase Amplification (RPA)

Abstract

Red Sea bream Iridovirus (RSIV) is one of the most important marine viruses in Asia and infects high demanded fishes. RSIV spreads rapidly in the sea around the world via various routes, such as ocean current, imported fishery products and ships’ ballast water discharged in ports. Therefore, the detection of RSIV is very important for food and economic security worldwide. Also, it is important for preservation of marine life. However, there are not any appropriate diagnostic methods for rapid diagnosis of marine viruses. In this study, RPA (recombinase polymerase amplification) was used to solve this problem. Detection methods using RPA are expected to be more simplified, fast, specific and sensitive than ordinary PCR methods. For testing RPA primer amplifying target virus genes, plasmid pCTCON which has a RSIV coat protein gene is used. RPA was performed at a constant temperature at 37℃, and therefore can be carried out outside the lab. In addition RPA was completed in 30min, much faster than conventional PCR. Furthermore, we showed that only RSIV gene was amplified, whereas VHSV, MABV, NNV, PVY, PLRV, TYLCV and PRV DNA were not. Thus, RPA can be appropriate diagnostic methods for RSIV.