Polymerase chain reaction (PCR) amplification of DNA of red sea bream iridovirus (RSIV)

Abstract

The polymerase chain reaction (PCR) was used to amplify DNA of the red sea bream iridovirus (RSIV). Four oligonucleotide primer sets based on the ATPase gene, DNA polymerase gene, and a Pst I-restriction fragment of RSIV genomic DNA were synthesized. PCR products of the expected size were amplified with each primer set after 30 cycles using template DNA which was extracted from the supernatant of tissue-cultured GF cells infected with RSIV. These amplified products were shown to be specific for the genomic DNA of RSIV by Southern blot hybridization. In addition, PCR products were obtained from the DNAs of the spleen and blood of red sea bream, Pagrus major, artificially infected with RSIV. Furthermore, the PCR products were detected from the tissues of cultured marine fish naturally infected with RSIV and from the supernatant of tissue-cultured GF cells infected with RSIV isolated from cultured marine fish. However, PCR products were not obtained at 3 months post-challenge from the spleens of red sea bream infected by intraperitoneal inoculation of RSIV. None of the PCR products were obtained from the supernatant of tissue-cultured FHM cells infected with frog virus 3 or from the lymphocystis tissue of Japanese flounder, Paralichthys olivaceus, infected with fish lymphocystis disease virus.