Multiplex PCR method for distinguishing two genotypes of Megalocytivirus isolates in Korea

Megalocytivirus is a genus of piscine viruses that belongs to the Iridoviridae family, and this family includes red seabream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), and infectious spleen and kidney necrosis virus. RSIV causes high mortality and economic losses in the rock bream (Oplegnathus fasciatus) Korean aquaculture industry. The World Organization for Animal Health’s Office International des Epizooties has provided a manual for RSIV detection. However, distinguishing it from TRBIV has not been confirmed. In this study, a multiplex PCR method was established to detect two genotypes of Megalocytivirus, RSIV and TRBIV. New primer pairs were optimized for the PCR reaction. A mixture of one universal and two specific primer pairs could amplify three distinct products targeting ATPase, DPO, and LRP genes. The sensitivities of primer pairs were evaluated, showing a detection limit of 2.0 × 105 copies, using a plasmid construct, against the three target genes of Megalocytivirus. Moreover, specificity evaluation indicated that primer pairs were not subjected to interference by other virus strains. This evaluation of multiplex PCR showed that it can distinguish two genotypes of Megalocytivirus from 21 Korean Megalocytivirus isolates, including 20 RSIV and one TRBIV. Finally, we describe a new multiplex PCR method to detect different genotypes of Megalocytivirus simultaneously, which makes the diagnosis of viral diseases occurring in the Korean aquaculture industry more convenient.