2024-03-28T15:59:19Zhttps://eprints.lib.hokudai.ac.jp/dspace-oai/requestoai:eprints.lib.hokudai.ac.jp:2115/530952022-11-17T02:08:08Zhdl_2115_20049hdl_2115_141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Revelation of effective methods for detection of viral nervous necrosis virus gene using polymerase chain reaction in barfin flounder, Verasper moseri渡辺, 研一Watanabe, Ken-ichi1000040122915吉水, 守Yoshimizu, Mamoruopen access© 2001 日本水産増殖学会Barfin flounderPCRViral nervous necrosisViral detection663Detection 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.マツカワを材料とした場合の,核酸抽出法,PCR反応温度,反応酵素,機器等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影響について比較検討した。核酸抽出法としてはIsogenおよびRNA Isolation Kitが,cDNAの増幅に際して変性,アニーリング,伸長反応を3種類の反応温度により行う方法が,逆転写酵素としてはSuper Script II,DNAポリメラーゼはEx Taqが,サーマルサイクラーはPTC-200およびPJ480が優れていた。日本水産増殖学会2001-03-20jpnjournal articleVoRhttp://hdl.handle.net/2115/53095https://doi.org/10.11233/aquaculturesci1953.49.850371-42172185-0194AN00124667水産増殖4918590https://eprints.lib.hokudai.ac.jp/dspace/bitstream/2115/53095/1/article102.pdfapplication/pdf983.56 KB2001-03-20