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研究生:陳丘錞
研究生(外文):Chiu-Chun Chen
論文名稱:養殖水產生物及池水霍亂弧菌之調查研究
論文名稱(外文):The Survey of Vibrio cholerae in Aquaculture Practice and Water
指導教授:林正輝林正輝引用關係
指導教授(外文):Cheng-Hui Lin
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:39
中文關鍵詞:霍亂弧菌養殖環境聚合酶連鎖反應
外文關鍵詞:Vibrio choleraeaquatic environmentpolymerase chain reaction
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最近一起本土性霍亂是由生食甲魚卵所感染,這對於養殖業而言無疑是一種隱憂,由於霍亂弧菌不易培養,在檢測時常常低估其菌數亦無法分辨其是否具病源性,本篇主要目的是針對養殖環境建立有效且簡便又快速的霍亂弧菌檢驗方法,本研究一共採集173家養殖場水樣,樣品分布範圍包括十個縣市,樣品依其粒徑大小分為兩部分,第ㄧ組為大於11�慆粒徑的物質,第二組為介於0.22�慆~11�慆粒徑的物質,共計346個樣品,其中有效樣品315個,無效樣品31個。霍亂發生是由帶有霍亂毒性基因的霍亂弧菌致病株O1或O139型所引起,霍亂弧菌O1型分別在雲林及高雄檢出陽性反應,霍亂弧菌O139型在雲林及屏東檢出陽性反應;在霍亂毒素基因檢驗均無檢出霍亂毒素基因陽性反應,而檢出霍亂弧菌陽性反應的四間養殖場所使用的養殖的水體均使用海水養殖,傳統霍亂弧菌鑑定整組實驗流程所需時間至少需花費四到六天,而使用聚合酶連鎖反應之檢測方法僅需花費三至四小時,而且並不會受限於休眠時期之限制;由於霍亂弧菌為法定傳染病,無法取得活菌株做偵測極限試驗,故本篇則以V. carchariae及V. harveyi做偵測極限模擬,在Trypticase soy broth(TSB)中V. carchariae偵測極限為110CFU/ml,溶於海水的菌體偵測極限下降至106CFU/ml,V. harveyi偵測極限為2.7×103CFU/ml,而溶於海水的菌體偵測極限下降至107CFU/ml以上,偵測極限差了10000以上,顯示海水可能會干擾聚合酶連鎖反應之進行。
The standard microbiological method used for aquatic environment cholera detection can hardly be adapted in environment background test for its no economic benefit. However, it would result in omission in epidemic control. Due to its cultivation difficulty, the bacterial number is often underestimated and it will result in discrimination difficulty on pathogenicity. The main purpose of this study is to establish a simple, effective and fast detection method for cultivation environment. In this study, there are 173 farm samples from 10 counties. Samples are divided into two groups on particle size. There are 346 samples in total which 315 are effective and 31 are non effective. Two samples of Vibrio cholerae O1 are positive in Yunlin and Kaohsiung respectively. Two samples of Vibrio cholerae O139 are also positive in Yunlin and Pingtung. ctx gene of V. cholera had not been found. The four farms with V. cholera are all marine cultures. In standard cassical microbiological methods, the whole procedure takes at least 3 to 4 days. Using polymerase chain reaction method, it takes just only 3 to 4 hours, and will not limited by viable but non-culturable (VBNC). According to the difficulty to obtain living strains for detection limit test,the V. carchariae and V. harveyi are used for simulation of detection limit test. In Trypticase soy broth, the detection limit of V. carchariae is 110CFU/ml and 2.7×103CFU/ml for V. harveyi, but it will rise to 106CFU/ml in sea water. It indicates that sea water will interfere the process of polymerase chain reaction.
目錄

謝辭…………………………………………………………………... i
中文摘要……………………………………………………………... ii
英文摘要……………………………………………………………... iii
目錄…………………………………………………………………... iv
表目錄………………………………………………………………... vii
圖目錄………………………………………………………………... viii

附錄…………………………………………………………………... ix

壹、前言……………………………………………………………… 1
一、霍亂弧菌分類地位……………………………………………… 1
二、霍亂弧菌生化特性……………………………………………… 2
三、霍亂弧菌致病機制……………………………………………… 3
四、霍亂弧菌傳染途徑……………………………………………… 4
五、傳統霍亂弧菌鑑定方法………………………………………… 4
六、弧菌16S rRNA基因…………………………………………… 5
七、研究目的………………………………………………………… 6
貳、材料方法………………………………………………………… 7
ㄧ、池水樣品採集………………………………………………….. 7
二、檢體DNA萃取………………………………………………….. 7
三、16S rRNA基因之PCR檢測……………………………………. 8
四、霍亂弧菌O1型之PCR檢測…………………………………… 8
五、霍亂弧菌O139型之PCR檢測………………………………… 9
六、霍亂毒素基因(ctx gene)之PCR檢測………………………… 9
七、PCR產物電泳分析…………………………………………….. 10
八、聚合酶連鎖反應偵測極限……………………………………… 11
參、實驗結果………………………………………………………… 12
ㄧ、水樣有效樣品分析……………………………………………… 12
二、霍亂弧菌的分布………………………………………………… 12
三、霍亂弧菌存在環境分析………………………………………… 12
四、霍亂弧菌在生物體體表檢驗結果 …………………………… 12
五、霍亂毒素分析 …………………………………………………. 13
六、與傳統霍亂弧菌鑑定方法比較……………………………….. 13
七、水產弧菌對16S rRNA引子PCR反應偵測極限……………… 13
肆、討論……………………………………………………………… 15
ㄧ、水樣有效樣品分析……………………………………………… 15
二、霍亂弧菌的分布………………………………………………… 15
三、霍亂弧菌存在環境分析………………………………………… 16
四、霍亂弧菌在生物體體表檢驗結果 …………………………… 16
五、霍亂毒素分析 …………………………………………………. 16
六、與傳統霍亂弧菌鑑定方法比較……………………………….. 17
七、水產弧菌對16S rRNA引子PCR反應偵測極限……………… 18
伍、參考文獻…………………………………………………………. 20
陸、圖表……………………………………………………………… 24










表目錄

表一、PCR特異性引子序列及反應條件…………………………… 24
表二、PCR檢驗結果總表…………………………………………… 25
表三、霍亂弧菌分布圖……………………………………………… 32
表四、傳統霍亂弧菌鑑定與PCR鑑定方法所需時間比較………… 33
表五、V. carchariae和 V. harveyi在不同基質的PCR偵測極限比較 …………………………………………………………………… 34










圖目錄

圖一、霍亂毒素(Cholera-toxin)特異性引子設計………………… 35
圖二、V. carchariae在TSB及海水中偵測極限…………………….. 36
圖三、V. harveyi在TSB及海水中偵測極限………………………. 37













附錄

附錄一、霍亂毒素作用機制模式…………………………………… 38
附錄二、霍亂弧菌傳統生化鑑定結果……………………………… 39
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