表紙/

Size: px

Start display at page:

Download "表紙/"

あきひろそめや
5 years ago
Views:

1 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 条件の検討誌名水産増殖 = The aquiculture ISSN 著者巻 / 号渡辺, 研一吉水, 守 49 巻 1 号掲載ページ p 発行年月 2001 年 3 月農林水産省農林水産技術会議事務局筑波産学連携支援センター Tsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research Council Secretariat

2 SUISANZOSHOKU PCR Revelation of Effective Methods for Detection of Viral Nervous Necrosis Virus Gene using Polymerase Chain Reaction in Barfin Flounder, Verasper moseri Ken-ichi WATANABE 1 and Mamoru YOSHIMIZU 2 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. 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. Barfin flounder; PCR; Viral nervous necrosis; Viral detection ) viral nervous necrosis: VNN 2) VNN 7 2) 22 3) Verasper moseri VNN 4) VNN 5) ELISA 6) PCR 7) 8) 9) In situ hybridization 10) 11) PCR 12,13) 4,14-18) PCR PCR VNN 4,19) 10 VNN 16) 4) 1 Akkeshi Station of Japan Sea-Farming Association, Chikushikoi, Akkeshi, Hokkaido , Japan 2 Graduate School of Fisheries Science, Hokkaido University, Minato, Hakodate, Hokkaido , Japan

3 PCR PCR VNN 13) PCR PCR cdna PCR nested PCR 2 PCR PCR PCR VNN Nishizawa et al. 7) PCR 50 l 50 mg 1 VNN, Gadus macrocephalus unit/ml RNase Inhibitor (Takara) DDW 0.1% W/V Nishizawa et al. 7) 10 4 PCR RT 50U Super Script II (Gibco BRL) PCR DNA 5U Ex Taq (TaKaRa) (MJ Research) VNN tween 20- proteinase K 20) SDS-proteinase K 7) VNN SDS-proteinase K 17) AGPC 21) Isogen RNA isolation kit TRIzol (Gibco BRL) Catrimox-14 (Takara) High Pure Viral Nucleic Acid Kit (Behlinger-Manheim) l VNN VNN PCR 22) PCR 3 PCR VNN PCR 1 3 PCR AMV reverse transcriptase {AMV: Takara, RNA PCR kit (AMV) Ver. 2.1} M-MLV reverse transcriptase (M- MLV: Toyobo, RT-PCR high) Super Script II DNA Ex Taq r Taq (Takara) 1 Super Script II Ex Taq AMV 2.5 U M- MLV 10U r Taq 5U

4 VNN PCR dps PJ 480 (Perkin-Elmer Cetus) PC-700 (Astec) PCR Aldrich Chemical Company 1 PCR Table 1 Isogen TRIzol AGPC RNA isolation kit Isogen RNA isolation kit AGPC t Isogen p 0.5 RNA isolation kit p 0.4 Isogen RNA isolation kit RNA isolation kit Isogen TRIzol AGPC RNA isolation kit 10 5 VNN 10 4 PCR Table 2 PCR 2 PCR PCR Super Script II r Taq 0.5 Super Script II Ex Taq 0 1.5Super Script II Ex Taq MJ PTC- 200 PC PCR VNN Isogen TRIzol RNA isolation kit Sample Brain Head Eye Hatched larvae Ovarian fluid Sperm TP Effect of nucleic acid extraction on detection rate of NNV gene SP ssp 3 11 Method of nucleic acid extraction ISO TRI 5 AGPC RIK 7 CT ND 13 1 Tween 20-proteinase K method. 2 Sodium dodecyl sulfate-proteinase K method. 3 Sodium dodecyl sulfate-proteinase K method using supernatant of homogenized sample solution for nucleic acid extraction. 4 Isogen (Nippon gene) method. 5 TRIzol (Gibco BRL) method. 6 Acid guanidium phenol chloroform method. 7 RNA isolation kit (Toyobo) method. 8 Catrimox-14 (Takara) method. 9 High Pure Viral Nucleic Acid Kit (Behlinger-Manheim) method. 10 Mean of -log 10 (dilution rate of virus solution which detected the viral specific gene) S.D. 11 Not tested. 12 Not detected in some cases. 13 Not detected. HP

5 Results of experiments on detection rate of NNV gene Experimental object PCR program 3 Thermal cycler (MJ Research) Combination of enzyme Super Script II & Ex Taq PCR program Shuttle 4 Mineral oil Method of nucleic acid extraction 1,2 ISO TRI RIK 1.8 Combination of enzyme 6 AMV & r Taq AMV & Ex Taq M-MLV & r Taq M-MLV & Ex Taq Super Script II & r Taq Thermal cycler 8 Super Script II & Ex Taq PC-700 (Astec) Super Script II & Ex Taq PJ480 (Perkin-Elmer Cetus) Super Script II & Ex Taq Aldrich Chemical Company Aldrich Chemical Company Aldrich Chemical Company 1 See Table 1. 2 Brain, eye, heads, hatched larvae, ovarian fluid and sperm were used for nucleic acid extraction sample. 3 Results were compared with usual program. 4 (95 10sec, 60 40sec) 25 cycles. 5 ( log 10 (examined value of most diluted virus solution that detected the viral specific gene) log 10 (compared value of most diluted virus solution that detected the viral specific gene))/trial number. 6 Results were compared with combination of enzyme that use Super Script II & Ex Taq min, 95 2min, (95 40sec, 55 40sec, 72 40sec) 25 cycles, 72 5min. 8 Results were compared with that performed without mineral oil tween 20-proteinase K SDS-proteinase K SDS-proteinase K Isogen RNA isolation kit RNA isolation kit 150 Isogen 90 Isogen VNN RNA isolation kit Isogen RNA isolation kit RNA tween 20-proteinase K SDSproteinase K DNA PCR 23) VNN Catrimox-14 High Pure Viral Nucleic Acid Kit RNA VNN SDS-proteinase K trna 24) Isogen VNN PCR VNN SDS-proteinase K PCR PCR PCR 3 PCR Tm PCR F2 R3 Tm ) PCR 3 PCR PCR VNN 3 PCR Super Script II Ex Taq PJ 480

6 VNN PCR PC-700 PC-700 PCR PC-700 PCR Isogen RNA isolation kit Super Script II Ex Taq 3 PCR PCR 7) 26) VNN SSN-1 11) 27) (IHN) 1 PCR IHN 28,29) VNN PCR PCR Isogen RNA Isolation Kit cdna 3 Super Script II DNA Ex Taq PJ pp (2) (4) (2) Yoshikoshi, K. and K. Inoue (1990): Viral nervous necrosis in hatchery-reared larvae and juveniles of Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel). J. Fish Disease,, Arimoto, M., K. Mushiake, Y. Mizuta, T. Nakai, K. Muroga, and I. Furusawa (1992): Detection of striped jack nervous necrosis virus (SJNNV) by enzyme-linked immunosorbent assay (ELISA). Fish Pathol., (4), Nishizawa, T., K. Mori, T. Nakai, I. Furusawa, and K. Muroga (1994): Polymerase chain reaction (PCR) amplification of RNA of striped jack nervous necrosis virus (SJNNV). Dis. Aquat. Org., (2), Nguyen, D. N., T. Nakai, and K. Muroga (1996): Progression of striped jack nervous necrosis virus(sjnnv) infection in naturally and experimentally infected striped jack Pseuedocaranx dentex larvae. Dis. Aquat. Org.,, Office International des Epizooties (OIE) (1997): Diagnostic manual for aquatic animal diseases. 195p. 10 Comps, M., M. Trindade, and C. l. Delsert (1996): Investigation of fish encephalitis viruses (FEV) expression in marine fishes using DIG-labeled probes. Aquaculture,, (4) Mori, K., K. Mushiake, and M. Arimoto (1998): Control measures for viral nervous necrosis of striped jack. Fish Pathol., (4), Watanabe, K., S. Suzuki, T. Nishizawa, K. Suzuki, M. Yoshimizu, and Y. Ezura (1998): Control strategy for viral nervous necrosis of barfin flounder. Fish Pathol., (4), Mushiake, K., M. Arimoto, T. Furusawa, I. Furusawa, T. Nakai, and K. Muroga (1992): Detection of antibodies against striped jack nervous necrosis virus (SJNNV) from brood stocks of striped jack. Fisheries Sci., (2),

7 (3) (1) Mushiake, K., T. Nishizawa, T. Nakai, I. Furusawa, and K. Muroga (1994): Control of VNN in striped jack: Selection of spawners based on the detection of SJNNV gene by polymerase chain reaction (PCR). Fish Pathol., (3), VNNSJNNV (1), Nishizawa, T., K. Muroga, and M. Arimoto (1996): Failure of the polymerase chain reaction (PCR) method to detect striped jack nervous necrosis virus (SJNNV) in striped jack Pseudocaranx dentex selected as spawners. J. Aquat. Anim. Health, 8(4), PCR 8 B 21 Yoshinaka, T., M. Yoshimizu, T. Sawabe, and Y. Ezura (1997): Detection and identification of infectious hematopoietic necrosis virus (IHNV) by reverse transcription (RT)-polymerase chain reaction (PCR). Fisheries Sci., (4), PCR PCR PCR ADNA PCR 1. PCR DNA PCR DNA Innis, M. A., and D. H. Gelfand (1994) 1. PCR PCR HBJ Nishizawa, T., K. Mori, M. Furuhashi, T. Nakai, I. Furusawa, and K. Muroga (1995): Comparison of the coat protein gene of five nodaviruses, the causative agents of viral nervous necrosis in marine fish. J. Gen. Virol., (4), Yoshimizu, M., T. Kimura, and J. R. Winton (1985): An improved technique for collecting reproductive fluid samples from salmonid fish. Prog. Fish-Cult, 47, RT-PCR IHNV Yoshimizu, M., Y. Hori, Y. Yoshinaka, and Jo-Ann Leong (2000): Evaluation of methods used for the surveillance and monitoring of fish health for risk assesment. Rev. Sci. Tech. Off. Int. Epiz., : in press.

農林水産業における自然エネルギーの効率的利用技術に関する総合研究（グリーンエナジー計画）第1期成果の概要

( グリーンエナジー計画 ) 第一期成果の概要誌名農林水産業における自然エネルギーの効率的利用技術に関する総合研究著者農林水産技術会議事務局, 掲載ページ p. 1-207 発行年月 1982 年 3 月農林水産省農林水産技術会議事務局筑波事務所 Tsukuba Office, Agriculture, Forestry and Fisheries Research Council Secretariat