A method for direct DNA amplification of uncharacterized DNA viruses and for development of a viral polymerase chain reaction assay: application to the red sea bream iridovirus.
A method is described for isolating a DNA segment of a virus for which no protein or DNA sequence information is available. This segment can then be used to develop a PCR-based assay for the virus. The method is based on the widespread presence and strong conservation of the ribonucleotide reductase gene among DNA viruses. The validity of the procedure is demonstrated by development of an assay for the fish iridovirus. We report the direct isolation from infected fish of a 738-bp segment of the iridovirus ribonucleotide reductase small subunit gene without prior virus purification. Using the sequence information obtained, a PCR-based diagnostic system was developed for detecting iridovirus infection.
Oshima
S
S
Central Research Laboratory, Nippon Suisan Kaisha Ltd., Tokyo, Japan.
Hata
J
J
Segawa
C
C
Hirasawa
N
N
Yamashita
S
S
D000595:Amino Acid Sequence; D000818:Animals; D017756:Aquaculture; D017931:DNA Primers / Q000138:chemical synthesis; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005393:Fish Diseases / Q000175:diagnosis*; D005814:Genes, Viral; D019156:Indian Ocean; D017994:Iridovirus / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D010473:Perciformes / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012264:Ribonucleotide Reductases / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014777:Virus Diseases / Q000662:veterinary*
D017931:DNA Primers; D004279:DNA, Viral; D012264:Ribonucleotide Reductases