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研究生:鄢靈湘
研究生(外文):Lin Shiang Yen
論文名稱:以多套式聚合酶連鎖反應快速檢測腸炎弧菌之研究
論文名稱(外文):Rapid detection of Vibrio parahaemolyticusby multiplex polymerase chain reaction
指導教授:陳幸臣陳幸臣引用關係林仲聖林仲聖引用關係
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:食品科學系
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:2003
畢業學年度:91
語文別:中文
論文頁數:103
中文關鍵詞:腸炎弧菌聚合酶連鎖反應熱穩定性溶血素熱不穩定性溶血素幾丁質酶牡蠣
外文關鍵詞:Vibrio parahaemolyticusmultiplex PCRthermostable direct hemolysinthermolabile hemolysinchitinaseoyster
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摘 要
腸炎弧菌 (Vibrio parahaemolyticus) 為人類主要病原菌之一,經常藉由攝入生鮮或未煮熟之海產品感染而造成腸胃炎。目前多以分子生物技術作為檢測本病原菌之方式。本研究是以多套式聚合酶連鎖反應 (multiplex polymerase chain reaction, m-PCR) 技術,以設計的三組引子組 (TDH-a/b, TL-1/2,及CHI-1/2) 對於20株非腸炎弧菌及40株腸炎弧菌作用,偵測此等菌之熱穩定性溶血素 (thermostable direct hemolysin, TDH) 基因、熱不穩定性溶血素 (thermolabile hemolysin, TL) 基因及幾丁質酶 (chitinase) 基因開發腸炎弧菌之快速檢驗方法。結果以設計出的一套以 1.5 mM Mg2+ 濃度及 45℃煉合溫度之m-PCR反應系統,具有極佳之特異性,對於腸炎弧菌菌懸液之靈敏度試驗可達100~101 CFU/assay。牡蠣中腸炎弧菌之檢測若配合以 alkaline peptone water 預培養8小時,其 m-PCR 靈敏度可達 100 CFU/assay。此外不但經濟,且可做為傳統方法檢測牡蠣中腸炎弧菌有用的參考數值。
Abstract
Vibrio parahaemolyticus is a notorious human pathogen which can cause gastroenteritis when consumed in raw or partially-cooked seafood.
The pathogen is presently detected using molecular biological technology. In this study, a multiplex PCR (m-PCR) was employed to develop a rapid method to detect 20 non-V. parahaemolyticus and 40 V. parahaemolyticus based on three sets of primers that specifically amplify segments of the tdh, tl and chi genes, which allowed simultaneous identification of thermostable direct hemolysin, thermolabile hemolysin and chitinase in a single reaction.
After the establishment of optimization condition, a m-PCR system was designed by using reaction buffer containing 1.5 mM MgCl2 and primer annealing temperature of 45℃. Primers amplified the respective genes without cross-reaction with other genes. The detection limit of the assay for pure bacterial targets was estimated at 100~101 CFU in this condition. In detection of V. parahaemolyticus in oyster samples, the detection sensitivity were 100 CFU if a pre-enrichment step using alkaline peptone water was performed prior to the PCR.
The experimental results demonstrated that the m-PCR assay developed in this study could provide a cost-effective and informative supplement to conventional microbiological methods for routine monitoring and risk assessment of oyster.
目 錄
目 錄 i
表 目 錄 v
圖 目 錄 vi
中文摘要 viii
英文摘要 ix
一、前言 1
二、文獻整理 3
1. V. parahaemolyticus之簡介 3
1-1. V. parahaemolyticus之生理與生化特性 3
1-2. V. parahaemolyticus之基因體 4
1-3. V. parahaemolyticus之幾丁質酶 5
1-4. V. parahaemolyticus之致病因子 6
2. V. parahaemolyticus的檢測方法 8
2-1. 傳統鑑定方法 9
2-2. 細菌自動鑑定儀Vitek Jr. 9
2-3. 免疫分析法(Immunoassay) 10
2-4. API 20E 鑑定法 10
3. 分子生物技術在V. parahaemolyticus檢測上的應用 10
3-1. DNA探針及rRNA探針分析法之發展 10
3-2. 聚合酶連鎖反應 12
3-3. 聚合酶連鎖反應之應用 14
3-4. 多套式聚合酶連鎖反應及其應用 15
三、研究目的 18
四、材料與方法 19
一、實驗材料 19
1. 實驗用菌與牡蠣 19
2. 培養基與化學藥品 19
2-1. 培養基 19
2-2. 試液與化學藥品 23
2-2-1. 萃取DNA試驗用 23
2-2-2. 電泳分析試驗用 23
2-2-3. PCR反應液 24
2-2-4. 傳統方法鑑定腸炎弧菌試藥 24
2-2-5. 傳統方法鑑定腸炎弧菌試劑 24
3. 儀器設備 25
二、實驗方法 25
1. 腸炎弧菌的培養與計數 25
2. PCR引子組設計 25
2-1. 寡核苷酸引子之合成 26
3. 製備染色體DNA 26
3-1. 精製法 26
3-2. 粗製法 27
3-3. 熱破碎處理法 27
3-4. 氫氧化鈉破碎處理法 27
4. PCR反應的溶液組成及反應條件 27
5. 電泳膠片分析 28
6. 聚合酶鏈鎖反應 (PCR) 28
6-1. 各引子之PCR特異性試驗 28
6-1-1. 各引子組對於腸炎弧菌之特異性 28
6-1-2. 各引子組對於非腸炎弧菌特異性之測試 28
6-2. 各引子之PCR靈敏度試驗 29
6-2-1. 不同前處理取得菌體DNA 29
6-2-2. 不同前處理對PCR靈敏度的影響 29
7. 多套式聚合酶鏈鎖反應 (Multiplex-PCR) 29
7-1. 多套式聚合酶鏈鎖反應之最適條件 29
7-1-1. 最適DNA聚合酶之選擇 29
7-1-2. 引子組間最適之相對濃度 30
7-1-3. 最適Mg2+濃度之選擇 30
7-1-4. 最適黏合溫度/時間之選擇 30
7-2. 多套式PCR引子組之特異性試驗 30
7-2-1. 三組引子組對於腸炎弧菌之特異性 30
7-2-2. 三組引子組對於非腸炎弧菌特異性之測試 31
7-3. 多套式PCR引子組之靈敏度試驗 31
7-3-1. 以純菌添加之傳統方法檢出率試驗 31
7-3-2. 以純菌添加之多套式PCR靈敏度試驗 31
8. 利用多套式PCR引子組檢測牡蠣中腸炎弧菌 32
8-1. 直接檢測牡蠣中腸炎弧菌 32
8-1-1 牡蠣檢液之製備 32
8-1-2. 總DNA的萃取 32
8-1-3. 利用多套式PCR偵測牡蠣中腸炎弧菌之靈敏度 32
8-2. 以預培養提高牡蠣中腸炎弧菌菌數之檢測 33
8-2-1. 牡蠣中腸炎弧菌的預培養 33
8-2-2. 利用多套式PCR偵測牡蠣中腸炎弧菌 33
9. 傳統方法檢測牡蠣中腸炎弧菌 33
9-1. 最確數之測定 33
9-2. 分離培養 34
9-3. 生化試驗 34
9-3-1. 初步試驗 34
9-3-2. 確認試驗 34
10. 聚合酶連鎖反應法對於腸炎弧菌之死菌及活菌之檢測 35
10-1. 腸炎弧菌生長曲線之測定 35
10-2. 活菌之m-PCR靈敏度 35
10-3. 死菌之m-PCR靈敏度 35
10-3-1. 熱處理 35
10-4. 預培養後進行PCR以區分活菌與死菌 36
五、結果與討論 40
1. 腸炎弧菌特異性PCR引子組之設計情形 40
2. 各引子組對V. parahaemolyticus菌株之特異性 43
3. DNA之製備 47
4. 建立多套式聚合酶連鎖反應之最適條件 51
5. 三組引子組對腸炎弧菌菌株之檢測 53
6. 多套式PCR引子組應用於牡蠣中腸炎弧菌之檢測 60
7. 聚合酶連鎖反應法對於腸炎弧菌之死菌及活菌之檢測 65
六、結論 81
七、參考文獻 82
附錄一、民國70年至90灣地區食品中毒案件病因物質分類表 93
附錄二、腸炎弧菌之生化及血清反應 94
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