跳到主要內容

臺灣博碩士論文加值系統

(3.236.84.188) 您好!臺灣時間:2021/08/06 10:45
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:吳琇婷
研究生(外文):Wu Hsiu-Ting
論文名稱:石斑魚病毒的分子檢測
論文名稱(外文):Molecular Detection of grouper viruses
指導教授:張繼堯張繼堯引用關係
指導教授(外文):Chang Chi-Yao
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:64
中文關鍵詞:石斑魚病毒檢測
外文關鍵詞:groupervirus detection
相關次數:
  • 被引用被引用:1
  • 點閱點閱:532
  • 評分評分:
  • 下載下載:104
  • 收藏至我的研究室書目清單書目收藏:1
石斑魚廣泛分布於世界各暖水域,種類繁多約有400種。因其肉質鮮美又富營養價值,迅速成為高經濟養殖魚類。近年來由於病毒感染,造成養殖上重大損失。主要感染的病毒有虹彩病毒與神經壞死病毒,其中有虹彩病毒科(Iridoviridae) Ranavirus屬的GIV (Grouper iridovirus)、SGIV (Singapore grouper iridovirus)和Megalocytivirus屬的OSGIV (Orange-spotted grouper iridovirus)、RSIV (Red sea bream iridovirus),為雙股去氧核醣核酸病毒(double strand DNA virus)。而另一造成重大疫情的神經壞死病毒(Nervous necrosis virus, NNV)屬於野田病毒科(Nodaviridae),Betanodavirus屬,為單股核糖核酸病毒(single strand RNA virus)。正確與快速的病原檢測為防治及檢疫上重要的工作,因此本研究的目的在發展出快速且靈敏的分子檢測方法。擬開發的檢測方法為恆溫圈環形核酸增幅法(Loop-mediated isothermal amplification, LAMP)與複合反轉錄酶-聚合酶鏈鎖反應法(Multiplex RT-PCR)。恆溫圈環形核酸增幅法是針對虹彩病毒GIV MCP (major capsid protein)進行設計,結果可以在65℃,1小時增幅出GIV DNA,10-7μg以上的GIV DNA就能夠進行檢測,比PCR方法靈敏十倍,而且57.5~70℃之間都有訊號出現。在複合反轉錄酶-聚合酶鎖鏈反應方面,針對Ranavirus的GIV與SGIV PNP (purine nucleoside phosphorylase)、MCP基因,Megalocytivirus的OSGIV與RSIV ATPase、MCP基因以及NNV的外套蛋白(coat protein)基因進行引子的設計,結果在一個反應中可以同時增幅出GIV PNP (purine nucleoside phosphorylase)、MCP以及NNV的外套蛋白(coat protein)基因轉錄片段。本研究建立的病毒分子檢測技術,將來可以應用在各種養殖魚種的檢疫以及海洋資源的感染調查。
Grouper distribute over the warm water. There are about more than 400 species of grouper around the world. Because of its delicious flavor and rich nutrition value, grouper become a popular commercial fish species. Recently, viruses infection causes tremendous economic losses in aquaculture, but the detection of these viruses is still remain inconvenient. The major infective viruses are iridovirus and nervous necrosis virus, especially Ranavirus, GIV (Grouper iridovirus), SGIV (Singapore grouper iridovirus), Megalocytivirus, OSGIV (Orange-spotted grouper iridovirus and RSIV (Red sea bream iridovirus). The accurate and rapid pathogen detection is very important in the prevention and quarantine of fish disease. Thus, the purpose of this study is to develop sensitive and fast methods of molecular detection for virus diagnosis-loop-mediated isothermal amplification(LAMP) and multiplex RT-PCR. LAMP primer set is designed for GIV major capsid protein (MCP) gene, and the signal can be observed after amplification at 65℃ for 1 hour. The signal can be also observed in the amplification temperature from 57.5 to 70℃. 10-7μg GIV DNA can be detected, the sensitivity is more than 10-fold of traditional PCR result. In multiplex RT-PCR, primers are designed for purine nucleoside phosphorylase (PNP) gene, MCP gene of Ranavirus, GIV and SGIV, ATPase, MCP gene of Megalocytivirus, OSGIV and RSIV, and coat protein (CP) gene of NNV, and it can detect GIV PNP, MCP, NNV CP simultaneously. In order to enhance the sensitivity and accuracy of the method, there should be more clinical samples to do the test. The molecular detection methods of viruses in this study can be used to quarantine all aquaculture fish species and to investigate the infection profile of ocean resources.
謝誌 --------------------------------------------------------------------------------- I
目錄 ------------------------------------------------------------------------------- III
中文摘要 ------------------------------------------------------------------------- VI
英文摘要 ----------------------------------------------------------------------- VIII
第一章 前言 --------------------------------------------------------------------- 1
1. 石斑魚之簡介 ----------------------------------------------------------- 1
2. 石斑魚虹彩病毒之簡介 ----------------------------------------------- 3
3. 神經壞死病毒之簡介 -------------------------------------------------- 5
4. 恆溫圈環形核酸增幅法(LAMP) ------------------------------------- 8
5. 複合反轉錄酶-聚合酶連鎖反應(Multiplex RT-PCR) --------- 11
6. 實驗目的 --------------------------------------------------------------- 13
第二章 材料與方法 ----------------------------------------------------------- 14
1. 生物性材料 ------------------------------------------------------------ 14
2. 病毒純化 --------------------------------------------------------------- 14
3. DNA萃取 -------------------------------------------------------------- 17
4. 恆溫圈環形核酸增幅法 --------------------------------------------- 19
5. Multiplex RT-PCR反應模板(template)製備 --------------------- 21
6. NNV RT-PCR與Multiplex RT-PCR ------------------------------- 22
7. GIV PCR ---------------------------------------------------------------- 26
第三章 結果 -------------------------------------------------------------------- 28
1. 恆溫圈環形核酸增幅法 --------------------------------------------- 28
3.1.1 病毒濃度測試與PCR比對 -------------------------------- 28
3.1.2 溫度範圍測試 ------------------------------------------------ 28
3.1.3 LAMP Real-time效應---------------------------------------- 29
3.1.4 反應呈色觀察 ------------------------------------------------ 29
2. 複合反轉錄酶-聚合酶鏈鎖反應法 ------------------------------ 29
3.2.1 基因比對結果 ------------------------------------------------ 30
3.2.2 單一基因RT-PCR測試 ------------------------------------ 30
3.2.3 引子綜合測試 ------------------------------------------------ 31
3.2.4 感染病毒樣品測試 ------------------------------------------ 32
第四章 討論 -------------------------------------------------------------------- 34
第五章 參考文獻 -------------------------------------------------------------- 38
第六章 圖表--------------------------------------------------------------------- 51
1. 圖一:LAMP與PCR連續10倍稀釋比較 --------------------- 51
2. 圖二:溫度範圍測試 ------------------------------------------------ 52
3. 圖三:LAMP反應Real-time效應 ------------------------------- 53
4. 圖四:LAMP反應白色沈澱的觀察 ------------------------------ 54
5. 圖五:EtBr呈色觀察 ------------------------------------------------ 55
6. 圖六:GIV、SGIV與Megalocytivirus屬的病毒MCP基因核苷酸序列比對 ------------------------------------------------------------ 56
7. 圖七:GIV、SGIV與Megalocytivirus屬的病毒ATPase基因核苷酸序列比對 --------------------------------------------------------- 58
8. 圖八:5種Betanodavirus外殼蛋白(coat protein)基因核苷酸序列比對 --------------------------------------------------------------------- 59
9. 圖九:單一基因RT-PCR測試 -------------------------------------- 61
10. 圖十:引子綜合測試 -------------------------------------------------- 62
11. 圖十一:感染病毒樣品測試 ----------------------------------------- 63
12. 表一:Megalocytivirus屬與Ranavirs屬病毒MCP基因核苷酸序列比對相似度表 --------------------------------------------------- 64
13. 附圖一:台灣地區石斑魚近年產值----------------------------------- 2
14. 附圖二:LAMP原理示意圖 ------------------------------------------ 9
15. 附圖三:Multiplex PCR原理示意圖 ------------------------------- 12
第五章參考文獻
1.吳明峰,2001,「石斑魚虹彩病毒之分子特性及其疫苗之開發」,國立台灣大學獸醫學研究所碩士論文。
2.吳佳瑞、賴春福,2004,「石斑」,菜市場魚圖鑑,大樹文化事業股份有限公司。
3.李錦城,2006,「點帶石斑抗黏液病毒與免疫球蛋白重鍊基因在胚胎時期之表現」,天主教輔仁大學生命科學研究所碩士論文。
4.涂堅,「魚類野田病毒之疫情及控制」,農政與農情 2004年1月 第139期
5.曾建璋、于乃衡,「海上箱網養殖技術之種苗生產與管理」,水產種苗協會月刊 2002年12月 第 54期
6.趙家本,「石斑魚虹彩病毒感染症」,水產種苗協會月刊 2003年9月 第 65期
7.蔡志東,2006,「石斑魚虹彩病毒完整基因體序列與其他虹彩病毒演化關係之分析」,國防醫學院生命科學研究所博士論文。
8.Arimoto, M., Mushiake, K., Mizuta, Y., Nakai, T., Muroga, K., and Furusawa, I. (1992) Detection of striped jack nervous necrosis virus (SJNNV) by enzyme- linked immunosorbent assay(ELISA). Fish Pathol. 27, 191-195.
9.Ball, L. A., Hendry, D. A., Johnson, J. E., Rueckert, R. R. & Scotti, P. D. (2000) Family Nodaviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses , pp. 747-755. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego: Academic Press
10.Berry, E. S., Shea, T. B., Gablike, J. (1983) Two iridovirus isolates from Carassius auratus(L). J. Fish Dis. 6, 501-510.
11.Bloch, B., Gravningen, K., and Larsen, J. L. (1991) Encephalomyelitis among turbot associated with a picornavirus-like agent. Dis. Aquat. Org. 10, 65-70.
12.Casa, I., Pozo, F., Trallero, G., Echevarria, J. M., Tenorio, A. (1999) Viral diagnosis of neurological infections by RT multiplex PCR: a search for enteroand herpes viruses in a prospective study. J. Med. Virol. 57, 145–151.
13.Chamberlain, J. S., Gibbs, R. A., Ranier, J. E., Nguyen, P. N., Caskey, C. T. (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 16, 11141–11156.
14.Chen, L. M., Wang, F., Song, W., Hew, C.L. (2006) Temporal and differential gene expression of Singapore grouper iridovirus. J. Gen. Virol. 87, 2907-2915.
15.Chi, S. C., Shieh, J. R., Lin, S. J. (2003) Genetic and antigenic analysis of betanodaviruses isolated from aquatic organisms in Taiwan. Dis. Aquat. Organ. 55, 221-8.
16.Chou, H. Y., Chang, S. J., Chen, L. M. (1994) Investigation on the viral diseases of cultured grouper (Epinephelus sp.) and red sea bream (Pagrus major). Reports on Fish Disease Research (XIV), COA Fisheries Series 46, 41-49.
17.Chou, H. Y., Hsu, C. C., Peng, T. Y. (1998) Isolation and characterization of a pathogenic iridovirus from cultured grouper (Epinephelus sp.) in Taiwan. Fish Pathology 33: 201-206.
18.Chua, F. H. C., Ng, M. L., Loo, J.J., Wee, J. Y. (1994) Investigation of outbreak of a novel disease,‘Sleepy Grouper Disease’ ,affecting the brown spotted grouper, Epinephelus tauvina Forskal. J. Fish Dis. 17, 417-427.
19.Comps, M., Trindade, M., and Delsert, C. (1996) Investigation of fish encephalitis viruses (FEV) expression in marine fishes using DIG-labelled probes. Aquaculture 143, 113-121.
20.Crisan, D. Molecular diagnostic testing for determination of myeloid lineage in acute leukemias. Ann Clin Lab Sci 1994;24:355–363.
21.Cunningham, C. O. (2002) Molecular diagnosis of fish and shellfish diseases: present status and potential use in disease control. Aquaculture 206, 19-55.
22.Elnifro, E. M., Ashshi, A. M., Cooper, R. J., Klapper, P. E. (2000) Multiplex PCR: optimization and application in diagnostic virology. Clin. Microbiol. Rev. 13, 559-570.
23.Fukuta, S., Kato, S., Yoshida, K., Mizukami, Y., Ishida, A., Ueda, J., Kanbe, M., Ishimoto, Y. (2003) Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction. J. Virol Methods. 112, 35-40.
24.Glazebrook, J. S., Heasman, M. P., and de Beer, S. W. (1990) Picornalike viral particles associated with mass mortalities in larval barramundi, Lates calcarifer (Bloch). J. Fish Dis. 13, 245-249.
25.Grotmol, S., and Totland, G. K. (2000) Surface disinfection of Atlantic halibut Hippoglossus hippoglossus eggs with ozonated sea-water inactivates nodavirus and increases survival of the larvae. Dis. Aquat. Org. 39, 89-96.
26.Hall, D. W. (1985) Pathobiology of invertebrate isocahedrak cytoplasmic deoxyriboviruses (Iridoviridae), pp. 163-196. In K. Maramorsch and K. E. Sherman (eds.) Viral insecticides for biological control. Academic Press, Inc., New York.
27.Harris, E., Kropp, G., Belli, A., Rodriguez, B., Agabian, N. (1998) Single step multiplex PCR assay for characterization of New World Leishmania complexes. J. Clin. Microbiol. 36, 1989–1995.
28.Heemstra, P. C. and John E. R. (1993) Groupers of the World (Family Serranidae, Subfamily Epinephelinae): An Annotated and Illustrated Catalogue of the Grouper, Rockcod, Hind, Coral Grouper and Lyretail Species Known to Date. FAO Fisheries Synopsis, no. 125, vol. 16
29.Hendolin, P. H., Markkanen, A., Ylikoski, J., Wahlfors, J. J. (1997) Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions. J. Clin. Microbiol. 35, 2854–2858.
30.Henegariu, O., Heerema, N. A., Dlouhy, S. R., Vance, G. H., Vogt, P. H. (1997) Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques. 23, 504-511.
31.Heredia, A., Soriano, V., Weiss, S. H., et al. (1996) Development of a multiplex PCR assay for the simultaneous detection and discrimination of HIV-1, HIV-2, HTLV-I and HTLV-II. Clin. Diagn. Virol. 7, 85–92.
32.Kiyoshi, I., Yamano, K., Maeno, Y., Nakajima, K., Matsuoka, M., Wada, Y., Sorimachi, M. (1992) Iridovirus infection of cultured red sea bream, Pagrus major 魚病研究 Gyobyo Kenkyu 27, 19-27.
33.Lai, Y. S., Murali, S., Chiu, H. C., Ju, H. Y., Lin, Y. S., Chen, S. C. (2001) Propagation of yellow grouper nervous necrosis virus (YGNNV) in a new nodavirus-susceptible cell line from yellow grouper, Epinephelus awoara (Temminck & Schlegel), brain tissue. J. Fish Dis. 24, 299-309.
34.Lai, Y. S., Murali, S., Ju, H. Y., Wu, M. F., Guo, I. C., Chen, S. C., Fang, K., Chang, C. Y. (2000) Two iridovirus-susceptible cell lines established from kidney and liver of grouper, Epinephelus awoara (Temminck & Schlegel), and partial characterization of grouper iridovirus. J. Fish Dis. 23, 379-386.
35.Langdon, J. S., Huphrey, L. M., Hyatt, A. D., Westbyry, H. A. (1986) First virus isolation from Australian fisn an iridovirus-like pathogen from redfin perch Perca fluviatilis L. J. Fish Dis. 9, 263-268.
36.Langdon, S., Humphrey, J. D., Williams, L. M. (1988) Outbreaks of an EHNV-like iridovirus in cultured raninbow trout, Salmo gairdneri Richardson, in Australia. J. Fish Dis. 11, 93-96.
37.Lu, L., Zhou, S. Y., Chen, C., Weng, S. P., Chan, S. M., He, J. G. (2005) Complete genome sequence analysis of an iridovirus isolated from the orange-spotted grouper, Epinephelus coioides. Virology. 339, 81-100.
38.Lua, D. T., Yasuike, M., Hirono, I., Aoki, T. (2005) Transcription Program of Red Sea Bream Iridovirus as Revealed by DNA Microarrays. J. Virol. 79, 15151-15164.
39.Markoulatos, P., Georgopoulou, A., Kotsovassilis, C., Karabogia-Karaphillides, P., Spyrou, N. (2000) Detection and typing of HSV-1, HSV-2 and VZV by a multiplex polymerase chain reaction. J. Clin. Lab. Anal. 14, 214–219.
40.Markoulatos, P., Mangana-Vougiouka, O., Koptopoulos, G., Nomikou, K., Papadopoulos, O. (2000a) Detection of sheep poxvirus in skin biopsy samples by a multiplex polymerase chain reaction. J. Virol. Methods 14, 214–219.
41.Markoulatos, P., Samara, V., Siafakas, N., Plakokefalos, E., Spyrou, N., Moncany, M. (1999) Development of a quadriplex polymerase chain reaction for human cytomegalovirus. J. Clin. Lab. Anal. 13, 99–105.
42.Markoulatos, P., Siafakas, N., Katsorchis, T., Moncany, M. (2003) Multiplex PCR: rapid DNA cycling in a conventional thermal cycler. J. Clin. Lab Anal. 17, 108-112.
43.Markoulatos, P., Siafakas, N., Moncany, M. (2002) Multiplex polymerase chain reaction: a practical approach. J. Clin. Lab Anal. 16:47-51.
44.Mori, K., Nakai, T., Muroga, K., Arimoto, M., Mushiake, K., and Furusawa, I. (1992) Properties of a new virus belonging to Nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology 187, 368-371.
45.Mori, Y., Hirano, T., Notomi, T. (2006) Sequence specific visual detection of LAMP reactions by addition of cationic polymers. BMC Biotechnol. 6, 3.
46.Mori, Y., Kitao, M., Tomita, N., Notomi, T. (2004) Real-time turbidimetry of LAMP reaction for quantifying template DNA. J. Biochem. Biophys. Methods. 59, 145-157.
47.Mori, Y., Nagamine, K., Tomita, N., Notomi, T. (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 289, 150-154.
48.Munday, B. L., Kwang, J., Moody, N. (2001) Betanodavirus infections of teleost fish: a review. J. Fish Dis. 25, 127-142.
49.Nagamine, K., Hase, T., Notomi, T. (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell Probes. 16, 223-229.
50.Nagamine, K., Watanabe, K., Ohtsuka, K., Hase, T., Notomi, T. (2001) Loop-mediated isothermal amplification reaction using a nondenatured template. Clin. Chem. 47, 1742-1743.
51.Nishizawa, T., Mori, K., Furuhashi, M., Nakai, T., Furusawa, I., Muroga, K. (1995) Comparison of the coat protein genes of five fish nodaviruses, the causative agents of viral nervous necrosis in marine fish. J. Gen. Virol. 76, 1563-9.
52.Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, E63.
53.OIE (2000) Chapter 2.2.2. Viral encephalopathy and retinopathy. In: OIE Diagnostic Manual for Aquatic Animal Diseases, 3rd edn, pp. 69-73. OIE, Paris.
54.Parida, M. M., Santhosh, S. R., Dash, P. K., Tripathi, N. K., Saxena, P., Ambuj, S., Sahni, A. K., Lakshmana, Rao. P. V., Morita, K. (2006) Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of Japanese encephalitis virus. J. Clin. Microbiol. 44, 4172-4178.
55.Periasamy, M., Niazi, FR., Malathi, V. G. (2006) Multiplex RT-PCR, a novel technique for the simultaneous detection of the DNA and RNA viruses causing rice tungro disease. J. Virol. Methods. 134, 230-236.
56.Rithidech, K. N., Dunn, J. J., Gordon, C. R. (1997) Combining multiplex and touch down PCR to screen murine microsatellite polymorphisms. Bio- Techniques 23, 36–45.
57.Savan, R., Kono, T., Itami, T., Sakai, M. (2005) Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens. J. Fish Dis. 28, 573-581.
58.Shuber, A. P., Skoletsky, J., Stern, R., Handelin, B. L. (1993) Efficient 12-mutation testing in the CFTR gene: a general model for complex mutation analysis. Hum. Mol. Genet. 2, 153–158.
59.Soliman, H., El-Matbouli, M. (2005) An inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV) infection by loop-mediated isothermal amplification. Virol. J. 2, 83.
60.Song, W. J., Qin, Q. W., Qiu, J., Huang, C. H., Wang, F., Hew, C. L. (2004) Functional genomics analysis of Singapore grouper iridovirus: complete sequence determination and proteomic analysis. J. Virol. 78, 12576-12590.
61.Ting, J. W., Wu, M. F., Tsai, C. T., Lin, C. C., Guo, I. C., Chang, C. Y. (2004) Identification and characterization of a novel gene of grouper iridovirus encoding a purine nucleoside phosphorylase. J. Gen. Virol. 85, 2883-92.
62.Tsai, C. T., Ting, J. W., Wu, M. H., Wu, M. F., Guo, I. C., Chang, C. Y. (2004) Complete genome sequence of the grouper iridovirus and comparison of genomic organization with those of other iridoviruses. J. Virol. 79, 2010-2023.
63.Varga, A., James, D. (2006) Use of reverse transcription loop-mediated isothermal amplification for the detection of Plum pox virus. J. Virol. Methods. 138, 184-190.
64.Williams, T. (1996) The iridoviruses. Adv. Virus Res. 46, 345-412.
65.Xeros, N. (1954) A second virus disease of the leatherjacket, Tipula paludosa. Nature 174, 562-563.
66.Yoshikoshi, K., and Inoue, K. (1990) Viral nervous necrosis in hatchery-reared larvae and juveniles of Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel). J. Fish Dis. 13, 69-77.
67.Yoshimizu, M., Suzuki, K., Nishizawa, T., Winton, J. R., and Ezura, Y. (1997) Antibody screening for the identification of nervous necrosis carriers in flounder broodstock. In: Proceedings NRIA International Workshop on New Approaches to Viral Diseases of Aquatic Animals, Kyoto, pp. 124-130.
68.Zimmermann, K. D., Schogl, B., Plaimauer, B., Manhalter, J.W. (1996) Quantitative multiple competitive PCR of HIV-1 DNA in a single reaction tube. BioTechniques 21, 480–484.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top