(3.80.6.131) 您好!臺灣時間:2021/05/14 01:34
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:::

我願授權國圖
: 
twitterline
研究生:宋子承
論文名稱:鑑定及偵測楊桃細菌性斑點病菌之聚合酶連鎖反應技術
論文名稱(外文):Application of polymerase chain reaction technique on identification and detection of pseudomonas syringae, the causal agent of bacterial spot of carambola
指導教授:徐世典
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2002
畢業學年度:89
語文別:中文
中文關鍵詞:聚合酶連鎖反應楊桃細菌性斑點病專一性引子對
相關次數:
  • 被引用被引用:2
  • 點閱點閱:157
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
由Pseudomonas syringae所引起的楊桃細菌性斑點病是近年來在台灣地區所發現的新病害,本研究利用以PCR為技術基礎的偵測楊桃細菌性斑點病菌之方法,並希望能將其應用於流行病學研究、檢疫及植物病害管理上。利用隨機增幅核酸多型性技術 (random amplified polymorphic DNA, RAPD) 增幅並篩選出Pseudomonas syringae楊桃菌株特有之DNA片段C14-300後,將其選殖出來並分析其核酸序列,再依據其核酸序列設計出引子對S1/R-S1。利用此引子對進行聚合酶連鎖反應 (polymerase chain reaction, PCR),對不同來源之Pseudomonas syringae楊桃菌株皆能增幅出125 bp的片段,而對其他供試細菌則否。當以引子對S1/R-S1測試楊桃細菌性斑點病菌之染色體DNA時,其靈敏度為10 pg,而用於偵測其細菌數時靈敏度可以達到約4 cfu。當應用於楊桃罹病葉片之診斷時,PCR反應物中添加dimethyl sulfoxide (DMSO) ,可測得特定的125bp片段,但若未添加DMSO則否。由楊桃葉表分離之三種腐生菌,不影響引子對S1/R-S1對楊桃細菌性斑點病菌之偵測。應用PCR技術偵測人工混菌之楊桃葉片組織萃取液時,未能增幅出125 bp的片段,若添加DMSO後則有特定的125bp片段出現,但只有在較高菌量時才能測得,表示楊桃葉片組織內可能含有PCR抑制物。由上述各項結果顯示引子對S1/R-S1具有快速鑑定楊桃細菌性斑點病菌之效果及診斷楊桃細菌性斑點病之潛力。
壹、前言-----------------------------------------------------------------------------1
貳、材料與方法--------------------------------------------------------------------6
一、菌株來源-----------------------------------------------------------------6
二、染色體DNA的抽取與濃度測定-----------------------------------6
1. 染色體DNA的抽取--------------------------------------------6
2. 染色體DNA濃度及純度測定---------------------------------7
三、P. syringaae楊桃菌株專一性DNA片段之篩選---------------8
1.RAPD增幅反應--------------------------------------------------8
2.引子的篩選-------------------------------------------------------9
四、P. syringae楊桃菌株專一性DNA片段之回收及純化--------9
五、南方雜合法 ( Southern hybridization ) -------------------------10
1.轉漬 (transfer) 及聯結 (UV-crosslinking) ---------------10
2.核酸探針之製備 ------------------------------------------------11
3.核酸雜合反應 (hybridization) 及偵測反應 (detection) 11
六、P. syringae楊桃菌株專一性DNA片段之選殖 ----------------12
七、小量質體的製備及選殖株篩選-------------------------------------13
八、選殖株重組質體DNA嵌入片段之核甘酸定序與其特性分析及專一性引子對之設計---------------------------------------------
14
九、聚合酶連鎖反應及引子對專一性與靈敏度之測定-------------15
1.聚合酶連鎖反應之引子黏合溫度測試及專一性測定---15
2.靈敏度測試 ------------------------------------------------------16
十、非標的細菌對PCR偵測楊桃細菌性斑點病菌之影響---------17
十一、楊桃細菌性斑點病菌之快速鑑定--------------------------------18
十二、應用PCR技術快速診斷葉片組織內楊桃細菌性斑點病菌 18
1.楊桃植株之接種-------------------------------------------------18
2.PCR反應樣品的製備與P. syringae楊桃菌株之偵測---18
十三、楊桃葉片含PCR抑制物之測試---------------------------------19
參、結果-----------------------------------------------------------------------------20
一、RAPD反應之引子篩選 ----------------------------------------------20
二、利用南方雜合法測試篩選專一性DNA片段---------------------20
三、楊桃細菌性斑點病菌專一性DNA片段之選殖 ----------------20
四、選殖株重組質體DNA嵌入片段之序列分析 --------------------21
五、楊桃細菌性斑點病菌之專一性引子對之設計 -------------------21
六、聚合酶連鎖反應之引子黏合溫度及引子對SL1/SR1之專一性與靈敏度 -----------------------------------------------------------------
22
1.聚合酶連鎖反應之引子黏合溫度及專一性 ---------------22
2.引子對SL1/SR1之靈敏度測試 -----------------------------24
七、非標的菌株對PCR偵測楊桃細菌性斑點病菌之影響---------24
八、楊桃細菌性斑點病菌之鑑定 ---------------------------------------25
九、葉片組織內之楊桃細菌性斑點病菌快速偵測-------------------25
十、楊桃葉片含PCR抑制物之測試-----------------------------------25
肆、討論 --------------------------------------------------------------------------- 27
伍、參考文獻 --------------------------------------------------------------------- 33
陸、中文摘要 --------------------------------------------------------------------- 38
柒、英文摘要 --------------------------------------------------------------------- 40
捌、圖表 --------------------------------------------------------------------------- 42
玖、附錄
1.文衍堂、黃智輝。1995。楊桃細菌性褐斑病研究初報。熱帶作物學報16:65-69。
2.陳谷婷。2000。楊桃細菌性斑點病菌之特性。國立中興大學植物病理學研究所第三十屆畢業碩士論文。
3.宋秉峰。1999。鑑定及偵測瓜類細菌性果斑病菌之聚合酵素連鎖反應技術。國立中興大學植物病理學研究所第二十九屆畢業碩士論文。
4.顏久焯,1998。應用聚合酵素連鎖反應技術偵測 Burkholderia caryophylli 。國立中興大學植物病理學研究所第二十八屆畢業碩士論文。
5.朱木貴。1995。Erwinia chrysanthemi 之遺傳差異性、藍色素基因選殖及PCR偵測。國立中興大學植物病理研究所畢業博士論文。
6.姚榮鼐。1994。台灣維管束植物植種名錄。國立台灣大學農學院實驗林管理處,南投縣。191pp。
7.蔡志濃、安寶貞、林俊義、吳雅芳。1998。楊桃細菌性斑點病之發生及防治。植物病理學會刊7:216(摘要)。
8.蔡志濃、安寶貞、許秀惠、林俊義。1999。楊桃細菌性斑點病之病因探討及防治成果,台灣省農業試驗所技術服務。37:7-8。
9.蔡雲鵬主編,1991。台灣植物病害名彙。修訂三版。中華植物保護學會及中華民國植物病理學會刊印。604pp。
10.蘇秋竹、徐世典。1998。在台灣由Pseudomonas syringae引起之楊桃細菌性葉斑病,植物病理學會刊7:216-217(摘要)。
11.Arnold, D. L., Athey-Pollard, A., Gibbon, M. J., Taylor, J. D., and Vivian, A. 1996. Specific oligonucleotide primers for the identification of Pseudomonas syringae pv. pisi yield one of two possible DNA fragments by PCR amplification : evidence for the phylogenitic divergence. 49:233-245.
12.Audy, P., Braat, C.E., Saindon, G., Huang, H. C., and Laroche, A. 1996. A rapid and sensitive PCR-based assay for concurrent detection of bacteria causing common and halo blights in bean seed. Phytopathology 86: 361-366.
13.Audy, P., Laroche, A., Sainadon, G., Huang, H. C., and Gilbertson, R. L. 1994. Detection of the bean common blight bacteria, Xanthomonas campestris pv. phaseoli and X. c. var. fuscans, using the polymerase chain reaction. Phytopathology 84:1185-1192.
14.Berswell, S., Pahl, A., Bellemann, P., Zeller, W., and Geider, K. 1992. Sensitive and species-specific detection of Erwinia amylovora by polymerase chain reaction analysis. Appl. Environ. Microbiol. 58:3522-3526.
15.Birch, P. R. J., Hyman, L. J., Taylor, R., Opio, A. F., Bragard, C., and Toth, I. K. 1997. RAPD PCR-based differentiation of Xanthomonas campestris pv. phaseoli and Xanthomonas campestris pv. phaseoli var. fuscans. Europ. J. Plant Pathol. 103:809-814.
16.Cazorla, F. M., Torés, J. A., Olalla, L., Pérez-García, A., Farré, J. M., and de Vicente, A. 1998. Bacterial apical necrosis of mango in southern Spain:A disease caused by Pseudomonas syringae pv. syringae. Phytopathology 88:614-620.
17.Darrasse, A., Prious, S., Kotoujansky, A., and Bertheau, Y. 1994. PCR and restriction fragment length polymorphism of a pel gene as a tool to identify Erwinia carotovora in relation to potato diseases. Appl. Environ. Microbiol. 60: 1437-1443.
18.De Bore, S. H., and Ward, L. J. 1995. PCR detection of Erwinia carotovora subsp. atroseptica associated with potato tissue. Phytopathology 85: 854-858.
19.Dye, D. W., Bradbury, J. F., Dickey, R. S., Goto, M., Hale, C. N., Hayward, A. C., Kelman, A., Lelliott, R. A., Patel, P. N., Sands, D. C., Schroth, M. N., Watson, D. R. W., and Young, J. M. 1975. Proposals for a reappraisal of the status of the names of plant-pathogenic Pseudomonas species. Int. J. Syst. Bacteriol. 25:p252-257.
20.Gardan, L., Shafif. H., and Grimont, P. A. D., 1997. DNA relatedness among pathovars of P. syringae and related bacteria. p445-448, In:K. Rudolph, T. J. Burr, J. W. Mansfield. D. Stead. A. Vivian, and J. Von Kietzell(ed.), Pseudomonas syringae pathovars and related pathogens. Kluwer Academic Publishers, London, United Kingdom.
21.Hartung, J. S., Daniel, J.F., and Pruvost, O. P. 1993. Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method. Appl. Environ. Microbiol. 59:1143-1148.
22.Iacobellis, N. S., Caponero, A., and Evidente, A. 1998. Characterization of Pseudomonas syringae ssp. savastanoi strains isolated from ash. Plant Pathol. 47:73-83.
23.Koike, S. T., Barak, J. D., Henderson, D. M., and Gilbertson, R. L. 1999. Bacterial blight of leek:A new disease in California caused by Pseudomonas syringae. Plant Dis. 83:165-170.
24.Koike, S. T., Henderson, D. M., Azad, H. R., Cooksey, D. A., and Little, E. L. 1998. Bacterial blight of broccoli raab:A new disease caused by a pathovar of Pseudomonas syringae. Plant Dis. 82:727-731.
25.Kovacs, N., 1965. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature, Lond. 178: 703.
26.Leite, R. P., Minsavage, G. V., Bonas, U., and Stall, R.E. 1994. Detection and identification of phytopathgenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria. Appl. Environ. Microbiol.60: 1068-1077.
27.Liop, P., Caruso, P., Cubero, J., Morente, C., and López, M. M. 1999. A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction. J. Microbiol. Methods. 37:23-31.
28.Louws, F. J., Rademarker, J. L. W., and de Bruijn, F. J. 1999. The three Ds of PCR-based genomic analysis of phytobacteria:diversity, detection, and disease diagnosis. Annu. Rev. Phytopathol. 37: 81-125
29.Maes, M., Garbeva, P., and Kamoen, O. 1996. Recognition and detection in seed of the Xanthomonas pathogens that cause cereal leaf streak using rDNA spacer sequences and polymerase chain reaction. Phytopathology 86:63-69.
30.Mäki-Valkama, T., and Karjalainen, R. 1994. Differentiation of Erwinia carotovora subsp. atroseptica and carotovora by RAPD-PCR. Ann. Appl. Biol. 125:301-309.
31.Mullis, K. B., and Faloona, F. A. 1987. Specific synthesis of DNA in vitro via a polymerase-catalysed chain reaction. Methods in Enzymology 155:335-350.
32.Opina, N., Tavner, F., Hollway, G., Wang, J. -F., Li, T. -H., Maghirang, R., Fegan, M., Hayward, A. C., Krishnapillai, V., Hong, W. F., Holloway, B. W., and Timmis, J. N. 1997. A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia Solanacearum (Formerly Pseudomonas Solanacearum). Asia Pacific Journal of Molecular Biology and Biotechnology Volume 5, No. 1, pp. 19-30.
33.Sambrook, J., Maniatis, T. I., and Fritsch, E. F. 1989. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
34.Samson, R., Shafik, H., Benjama, A., and Gardan, L. 1998. Description of the bacterium causing blight of leek as Pseudomonas syringae pv. porri(pv. nov.). Phytopathology 88:844-850.
35.Schaad, N. W., Azad, H., Peet, R. C., and Panopoulos. 1989. Identification of Pseudomonas syringae pv. phaseolicola by a DNA hybridization probe. Phytopathology 79:903-907.
36.Schaad, N. W., Cheong, S. S., Tamaki, S., Hatziloukas, E., and Panopoulos, N. J. 1995. A combined biological and enzymatic amplification (BIO-PCR) technique to detect Pseudomonas syringae pv. phaseolicola in bean seed extracts. Phytopathology 85: 243-248.
37.Southern, E. M., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503.
38.van Hall, C. J. J. 1902. Bijdragen tot de kennis der Bakterieele Plantenziekten. Inaugural Dissertation. Coöperatieve Drukkerij-Vereeniging “Plantijn”, Amsterdam.
39Wang, H., Qi, M., and Cutler, A. J. 1993. A simple method of preparing plant samples for PCR. Nucleic Acids Res. 21:4153-4154.
40.Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A., and Tingey, S. V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18: 6531-6535.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔