A simplified method for the simultaneous detection of nervous necrosis virus and iridovirus in grouper Epinephelus spp.

Grouper nervous necrosis virus (GNNV) and grouper iridovirus (GIV) are major grouper-infecting viruses in southern China that can cause serious economic losses. A duplex reverse transcription-PCR (duplex RT-PCR) method was developed for the simultaneous detection of GNNV and GIV. Eight groups of primers specifically targeting the capsid protein genes of GNNV and GIV were designed and analyzed. The primer set GN4 was selected and used to amplify fragments of 887 bp and 319 bp in length from GNNV and GIV, respectively. Furthermore, the duplex PCR assay was shown to be sensitive because it could detect at least 20 pg of plasmid-viral DNA from a mixture of viruses. Using this assay, 18 GNNV infected groupers and 7 GIV infected groupers were detected amongst 41 suspected samples in Hainan. The duplex RT-PCR assay proved to be a rapid, specific, and sensitive method for detecting the two grouper viruses. This method could be used to facilitate better control of fish viruses through early detection