マツカワのウイルス性神経壊死症原因ウイルス遺伝子の検出に及ぼすPCR条件の検討

Barfin flounder

Detection rate of viral nervous necrosis (VNN) virus gene using polymerase chain reaction was investigated. Tested specimens were: just hatched larvae, heads of larvae, eye or brain of juveniles, ovarian fluid and sperm obtained from brood fish. The specimens were mixed with a 10-fold serial dilution of virus solutions prepared from the eyes and brain of the Pacific Cod, Gadus macrocephalus, affected with VNN. For nucleic acid extraction, a comparison was made between 20-proteinase K, SDS- proteinase K, acid guanidium phenol chloroform, Isogen, TRIzol, RNA isolation kit, Catrimox-14, and High Pure Viral Nucleic Acid Kit. Isogen and/or RNA isolation kit showed the highest detection rate. PTC-200 and PJ 480 thermal cyclers were more effective than the PC-700 model. In comparison of reverse transcriptase, AMV, M-MLV, and Super Script II were tested; r Taq or Ex Taq was used as the DNA polymerase. Pairing of Super Script and Ex Taq was most effective. In PCR programs, 3-temperature PCR was more effective than 2-temperature PCR.