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研究生:張雅婷
研究生(外文):ZHANG, YA-TING
論文名稱:美人魚發光桿菌殺魚亞種特異性 PCR 檢測引子組之開發及應用
論文名稱(外文):Development and Application of a Specific Polymerase Chain Reaction Primers for Detection of Photobacterium damselae subsp. piscicida
指導教授:陳名倫陳名倫引用關係
指導教授(外文):CHEN, MING-LUN
口試委員:龐仁傑邱采新
口試委員(外文):PANG, JEN-CHIEHCHIU, TSAI-HSIN
口試日期:2017-12-04
學位類別:碩士
校院名稱:國立澎湖科技大學
系所名稱:食品科學系碩士班
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2017
畢業學年度:106
語文別:中文
論文頁數:69
中文關鍵詞:美人魚發光桿菌殺魚亞種隨機擴增多形性 DNA多套式 PCR
外文關鍵詞:Photobacterium damselae subsp. piscicidaRAPDmultiplex-PCR
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中文摘要................................................................................................................................................................Ⅰ
英文摘要 ...............................................................................................................................................................Ⅱ
目錄 ....................................................................................................................................................................... Ⅲ
表目錄.....................................................................................................................................................................VI
圖目錄...................................................................................................................................................................VII
壹、文獻整理.........................................................................................................................................................1
一、 美人魚發光桿菌 (Photobacterium damselae) 之簡介 .......................................................... 1
(一) P.damselae 之一般特性........................................................................................................................1
(二) P.damselae 分類學之地位....................................................................................................................2
二、美人魚發光桿菌殺魚亞種 (P. damselae subsp. piscicida) 之型態與生理生化特性..................3
三、美人魚發光桿菌美人魚亞種 (P. damselae subsp. damselae) 之型態與生理生化特性...........4
四、美人魚發光桿菌 (Photobacterium damselae) 之檢測.....................................................................5
(一) P. damselae 之傳統生化檢測...................................................................................................................5
(二) P. damselae 之分子檢測............................................................................................................................6
(三) P. damselae 其他檢測................................................................................................................................6
五、聚合酶鏈鎖反應 (Polymerase chain reaction , PCR)........................................................................7
(一) PCR 之發展....................................................................................................................................................7
(二) PCR 之應用....................................................................................................................................................7
六、隨機擴增多型性 DNA (Randomly amplified polymorphic DNA, RAPD).................................9
(一) RAPD 之原理.................................................................................................................................................9
(二) RAPD 之應用.................................................................................................................................................9
七、多套式聚合酶鏈鎖反應 (Multiplex-polymerase chain reaction)..............................................10
(一) 多套式 PCR 之原理....................................................................................................................................10
(二) 多套式 PCR 之應用....................................................................................................................................10
八、澎湖地區之經濟魚種-海鱺......................................................................................................................11
貳、前言...............................................................................................................................................................13
參、實驗架構......................................................................................................................................................14
肆、材料與方法.................................................................................................................................................15
一、材料...............................................................................................................................................................15
(一) 菌株...............................................................................................................................................................15
(二) 儀器設備......................................................................................................................................................15
(三) 藥品...............................................................................................................................................................16
(四) 溶液配置......................................................................................................................................................17
(五) 培養基..........................................................................................................................................................17
(六) 商業化測試套組.........................................................................................................................................18
(七) PCR 引子組.................................................................................................................................................19
二、實驗方法.......................................................................................................................................................20
(一) Genomic DNA 之萃取.............................................................................................................................20
(二) DNA分子鑑定之聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR).....................................20
(三) 隨機擴增多型性 DNA (Randomly amplified polymorphic DNA, RAPD)
分析...............................................................................................................................................................21
(四) PCR 產物之回收.........................................................................................................................................21
(五) PCR 產物之選殖及定序............................................................................................................................21
(六) PCR 引子組之設計.....................................................................................................................................22
(七) 單套式聚合酶鏈鎖反應 (PCR).................................................................................................................23
(八) 多套式聚合酶鏈鎖反應 (Multiplex-PCR)............................................................................................25
伍、結果與討論..................................................................................................................................................27
一、P. damselae 菌株之 PCR 鑑定...............................................................................................................27
二、隨機擴增多型性 DNA (Randomly amplified polymorphic DNA, RAPD) 之分析................28
三、RAPD 片段定序及特異性引子設計.......................................................................................................30
四、單套式聚合酶鏈鎖反應 (PCR)................................................................................................................30
(一) PDPf/PDPr 引子組之特異性分析..........................................................................................................30
(二) PDPf/PDPr 引子組之檢測靈敏度..........................................................................................................31
(三) 魚體樣品之應用..........................................................................................................................................31
五、多套式聚合酶鏈鎖反應 (Multiplex-Polymerase chain reaction)..............................................33
(一) PDPf/PDPr 與 PDDf/PDDr 引子組特異性之多套式 PCR...............................................................34
(二) 魚體樣品之應用..........................................................................................................................................35
陸、結論...............................................................................................................................................................37
柒、參考文獻.......................................................................................................................................................39

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