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研究生:陳念群
研究生(外文):Nian-Qun Chen
論文名稱:台灣西南部沿海致病性腸炎弧菌之環境調查
論文名稱(外文):Environmental investigation of potential pathogenic Vibrio parahaemolyticus in the southwestern coast of Taiwan
指導教授:張福林張福林引用關係
指導教授(外文):Fu-Lin Chang
學位類別:碩士
校院名稱:中華醫事學院
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:93
語文別:中文
中文關鍵詞:腸炎弧菌 toxR基因 食物中毒
外文關鍵詞:V.parahaemolyticustoxR genefood -poisoning
相關次數:
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腸炎弧菌在台灣、日本及其他沿海國家是一種很重要的食物中毒致病菌,在台灣食物中毒案例中,腸炎弧菌歷年來都佔了70%以上,穩居細菌類食物中毒的第一名。本研究主要以傳統的培養方法及聚合酶連鎖反應(polymerase chain reaction)技術,以5組腸炎弧菌之專一性引子(toxR、tlh、tdh、trh、toxRS)對環境中的樣本進行分析,建立腸炎弧菌在台灣西南部海域的分佈與流行病學資料庫。實驗期間我們收集了台灣西南部沿海的水樣本及有機樣本來做偵測,腸炎弧菌的總數是藉由PCR擴大toxR基因及tlh基因來測定,而致病性腸炎弧菌的數量,則是藉由PCR擴大tdh、trh、及toxRS基因來偵測。研究結果顯示,利用傳統的培養方法只偵測到45.8%的樣本具有toxR基因,而PCR法則偵測到79.6%的樣本具有toxR基因,另外89.8%的樣本具有tlh基因。致於各式樣本中腸炎弧菌致病性基因的分佈情形分別是tdh (5.1%)、trh (9.3%)、toxRS (80.6%)。另外我們自環境中分離出來的腸炎弧菌菌株則只有一株具有trh基因,佔全部菌株的0.5%。若比較不同樣本型態中腸炎弧菌之數量則發現,有機樣本中腸炎弧菌的數量明顯高於水樣本。至於三個採樣地點平均是以安平港所分離之腸炎弧菌數量最多(51.85%),其次是興達港(38.18%)最少的是前鎮港(9.97%)。血清分型試驗則顯示台灣沿海腸炎弧菌以O2型所佔的比例最多。
Vibrio parahaemolyticus is one of the most important food-borne pathogens in Taiwan , Japan and other coastal countries. In Taiwan , this pathogen occupy more than 70% by the bacterial food -poisoning at the first occurring annually.
In this study, the traditional culture and polymerase chain reaction were employed based on five sets of primers (toxR、tlh、tdh、trh、toxRS) to analyze sample form environment , and establish distribution of Vibrio parahaemolyticus on southwest sea area in Taiwan and epidemiology database. We were collected seawater and organic material from coastal areas of southwestern Taiwan.to detect at experiment period. The total of V. parahaemolyticus was determined using PCR amplification of the toxR、 tlh genes ,however the pathogenic V.parahaemolyticus numbers was determined by using PCR amplification of the tdh、 trh、 toxRS genes. The study results showed traditional culture was only detected toxR(45.8%) , and the PCR can detected toxR(79.6%) and tlh (89.8%). As for distribution of the V. parahaemolyticus pathogenicity gene from various samples were detected tdh (5.1%)、 trh (9.3%) ,and toxRS (80.6%) ,respectively. In addition, we were isolated V. parahaemolyticus from environment, only 1 trh gene, occupy 0.5% in total bacteriun. If compare V. parahaemolyticus numbers from different sample types found organic samples greater than water samples. In the case of V.parahaemolyticus numbers isolated from three sampling site: Anping>Hsing-Da>Chien-Chen. The serum type experiment showed V.parahaemolyticus was the most serotype O2. in Taiwan coast.
目錄
致謝i
目錄ii
中文摘要iv
英文摘要v
表目錄vi
圖目錄vii
緒論1
1 食物中毒1
2. 腸炎弧菌1
3 研究目的8
材料與方法10
1. 實驗設備10
2. 實驗藥品12
3. PCR Primer14
4. 實驗流程15
5. 實驗方法16
結果38
1 不同檢測方法之比較38
2 毒性基因之檢測39
3 毒性基因分佈密度之差異40
4 季節與腸炎弧菌分佈密度之相關性40
5 腸炎弧菌在不同類型樣本分佈之比較40
6 安平港興達港與前鎮港之腸炎弧菌分離率比較41
7 血清型41
8 水質分析與腸炎弧菌數量之相關性41
9.毒性基因在不同採樣點之比較42
討論43
參考文獻46
表54
圖61
附錄1. MPN最確數表70
附錄2. 比重換算鹽度對照表71
附錄3. 生化鑑定對照表73

表目錄
表一. 台灣省歷年細菌性食物中毒案例統計表54
表二. 不同檢測方法偵測到腸炎弧菌之靈敏度比較55
表三. 三種腸炎弧菌毒性基因 (tdh、trh、toxRS)在樣本中之分佈情況56
表四. 三種毒性基因在分佈密度上的差異57
表五. 不同月份中腸炎弧菌密度之比較58
表六. 水質分析與腸炎弧菌數量之相關性分析59
表七. 毒性基因在不同採樣點之比較60

圖目錄
圖1. toxR-PCR之電泳圖61
圖2. tlh-PCR之電泳圖62
圖3. tdh-PCR之電泳圖63
圖4. trh-PCR之電泳圖64
圖5. toxRS-PCR之電泳圖65
圖6. 不同樣本類型間的比較66
圖7. 三個採樣點之菌落數的比較67
圖8. 腸炎弧菌之菌體抗原(O抗原)分型結果68
圖9. 比較不同採樣點所分離之腸炎弧菌菌體抗原(O抗原)之差異69
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