臺灣博碩士論文加值系統

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，本研究以PCR為技術基礎，發展楊桃細菌性斑點病菌鑑定與偵測之方法。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，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，而若利用管柱純化法則可提高靈敏度至約102 cfu/ml，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
十四、利用管柱純化楊桃細菌性斑點病菌DNA進行PCR測試 20
參、結果----------------------------------------------------------------------------- 21
一、RAPD反應之引子篩選 ---------------------------------------------- 21
二、利用南方雜合法測試篩選專一性DNA片段--------------------- 21
三、楊桃細菌性斑點病菌專一性DNA片段之選殖 ---------------- 21
四、選殖株重組質體DNA嵌入片段之序列分析 -------------------- 22
五、楊桃細菌性斑點病菌之專一性引子對之設計 ------------------- 22
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 -----------------------------------------------------------
23
1. 聚合酶連鎖反應之引子黏合溫度及專一性 --------------- 23
2. 引子對SL1/SR1之靈敏度測試 ----------------------------- 25
七、非標的菌株對PCR偵測楊桃細菌性斑點病菌之影響--------- 25
八、楊桃細菌性斑點病菌之快速鑑定 ---------------------------------- 26
九、葉片組織內之楊桃細菌性斑點病菌快速偵測------------------- 26
十、楊桃葉片含PCR抑制物之測試----------------------------------- 26
肆、討論 --------------------------------------------------------------------------- 28
伍、參考文獻 --------------------------------------------------------------------- 35
陸、中文摘要 --------------------------------------------------------------------- 40
柒、英文摘要 --------------------------------------------------------------------- 42
捌、圖表 --------------------------------------------------------------------------- 44
玖、附錄

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