Development of New Real-time PCR Assays for Detecting Megalocytivirus Across Multiple Genotypes

In the present study, two real-time PCR assays (Cummins and Kawato-1) were newly developed for the detection of broad range of Megalocytivirus (MCV) genotypes. The analytical sensitivity (ASe) and analytical specificity (ASp) of four real-time PCR assays, including two previously reported assays (Kawato-2 and Mohr), targeting the major capsid protein gene were compared across the four MCV genotypes including infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), and threespine stickleback iridovirus (TSIV). The four assays were tested using artificially synthesized plasmids containing seven different representative nucleotide sequences of the target region from the genotypes. The ASe and ASp for the detection of each MCV genotype differed between assays while the three assays other than Kawato-2 detected all ISKNV, RSIV, and TRBIV targets to a detection limit of approximately 2 plasmid copies per reaction. Diagnostic capability of each assay was validated using spontaneously RSIV-infected Japanese amberjack Seriola quinqueradiata. Diagnostic sensitivity in the dead fish of RSIV infection was equivalent among the four assays. However, in the asymptomatic fish, the detection rate of Cummins assay was higher than that of Kawato-1 assay and had equivalent sensitivity to previously reported assays. Thus, in addition to screening with synthesized plasmids, diagnostic capability of the assays should be tested using fish infected with the other MCV genotypes depending on target of host and the viral genotypes.

Automated summary

Two new real-time PCR assays were developed for detecting a broad range of Megalocytivirus genotypes, with different sensitivities and specificities compared to previously reported assays. The diagnostic capabilities of the assays were validated in RSIV-infected Japanese amberjack, showing equivalent sensitivity in dead fish and higher detection rate in asymptomatic fish for Cummins assay. Testing diagnostic capabilities of the assays using fish infected with other MCV genotypes is recommended.