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研究生:王昱澤
研究生(外文):Yu-TseWang
論文名稱:多通量定量聚合酶鏈鎖反應技術偵測水中有害藍綠菌之發展與現地應用
論文名稱(外文):Development and on-site Application of Multiplexed Quantitative PCR for Monitoring of Harmful Cyanobacteria in Reservoirs
指導教授:林財富林財富引用關係
指導教授(外文):Tsair-Fuh Lin
學位類別:碩士
校院名稱:國立成功大學
系所名稱:環境工程學系碩博士班
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:109
中文關鍵詞:即時定量聚合酶鏈鎖反應儀酵素免疫學分析方法微囊藻毒素柱孢藻毒素土味物質geosmin多重探針監測
外文關鍵詞:qPCRELISAmicrocystinscylindrospermopsingeosminMultiplexed detection
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  • 被引用被引用:1
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藍綠菌廣泛存在於台灣及世界許多淡水水體,藍綠菌代謝物微囊藻毒素(microcystins)、柱孢藻毒素(cylindrospermopsin)及土味物質geosmin對飲用水安全及可口,影響很大。台灣地區水庫水源眾多,且地區分散,發生藍綠菌藻華或代謝物問題時,通常將採集之樣品運送至分析單位,運輸及分析耗時久,對於水庫管理而言緩不濟急。因此,本研究擬建立現地產毒及產臭藍綠菌的快速監測方法,以有效掌握各分散水源中有害藍綠菌之發生潛勢,配合毒素現地監測方法,可以進一步保障民眾用水之安全。
本研究首先開發可同時偵測具產微囊藻毒素(Microcystins)以及具產geosmin之功能基因的分子生物監測方法,研究中應用快速DNA抽取技術、具專一性的引子與探針以及即時定量連鎖反應儀(qPCR),所開發之技術可在2小時內同時定量水體中該兩種基因的濃度。本研究並將開發之分子生物技術(qPCR)以偵測採集之水樣,同時搭配顯微鏡鏡檢技術、以及酵素免疫學分析方法,用於台灣地區5座水庫現地監測藍綠菌及毒素濃度,並配合實驗室氣相層析質譜儀(GC/MS)分析樣品中geosmin濃度。
研究結果顯示,本研究所建立之現地偵測方法,於現場採樣後2至3小時左右即可得到重要數據,並進一步將數據提供給水庫管理單位,使得各淨水廠有足夠的時間啟動緊急應變之程序,作為水庫藻華、毒素及臭味風險判定參考。研究期間監測中發現4座水庫水源中微囊藻毒素超過1 μg/L問題,後續也分別啟動持續監測採樣或是淨水廠清水採樣,以確保飲用水水質安全。

Prescence of cyanbacterial metabolites in drinking water, such as cyanotoxins and taste and odour (T&O) compounds, may pose health and aesthatic problems to the consumers. Microcystins and cylindrospermopsin are the two most commonly detected cyanotoxin groupds in Taiwan’s reservoirs, and geosmin is among the most detected T&O compounds. Conventionally, water samples potentially contaminated by these cyanobaterial metabolites are transported to and analyzed in the laboratories with sophiscated instruments. It usually requires more than 2 days to obtain analytical results, which might be too late as contaminated water may have entered consumers’ tap. Therefore, it is urgently needed to have a rapid and robust analytical scheme for the detection of cyanobacterial metabolites in drinking water systems.
In this study, a rapid on-site monitoring method for microcystins, cylindrospermopsin and geosmin-producers is developed. Quantitative real-time PCR (qPCR) is employed to quantify the targeted metabolite producers, by detecting the specific gene encoding target synthetase. Specific primers and probes were developed and tested for the targeted genes. The results showed the all the primers and probes developed are very specific to the targeted genes, with successful standard curved constructed between PCR standard and pure culture DNA. The method was then applied in 5 fresh water bodies in Taiwan, together with microscopic cell counting and cyanotoxin and geosmin concentration determination.
The results show that the method established is able tobe used on-site, with key information to be determined with 2-3 hours after sampling. The information may provide a basis for decision making for the managers of reservoirs and utilities. Four of the sampling runs conducted in the reservoirs were found to have microcystin concentration higher than 1 μg/L. To secure safe drinking water, samples for raw water and finished water were also collected for the water treatment plants. Fortunately, all the finished water samples show no contimation of cyanotoxins. This approach may provide a quick basis for the management of cyanobacteria and metabolites in drinkgin water systems.

摘要 I
ABSTRACT III
誌謝 V
目錄 VII
表目錄 XI
圖目錄 XIII
第一章 緒論 1
1.1 研究緣起 1
1.2 研究目的 3
第二章 文獻回顧 5
2.1 藍綠細菌之臭味物質 5
2.2 臭味物質分析方法 10
2.3 藍綠細菌之毒素 12
2.3.1來源、種類與化學構造 12
2.3.2 產毒藍綠細菌生長之環境條件 16
2.4 毒素分析方法 19
2.5 聚合酶鏈鎖反應 21
2.6定量聚合酶鏈鎖反應 23
2.7 多重探針監測之應用 28
第三章 實驗設備與方法 31
3.1 藻類培養 32
3.2 藻類計數 35
3.3 DNA RNA Protein 萃取 37
3.3.1 實驗設備與試劑 37
3.3.2 樣品前處理之方法 37
3.3.3 樣品萃取之方法 39
3.4 聚合酵素鏈鎖反應PCR 41
3.4.1 實驗設備與試劑 41
3.4.2 樣品配製之方法 41
3.5 DNA 純化 42
3.5.1 實驗設備與試劑 42
3.5.2 純化之方法步驟 42
3.6 即時定量聚合酵素鏈鎖反應(q-PCR) 44
3.6.1 實驗設備與試劑 44
3.6.2 實驗方法 44
3.7 酵素連結免疫吸附法(ELISA) 45
3.7.1 實驗設備與試劑 45
3.7.2 樣品前處理 45
3.7.3 實驗方法 45
3.8 臭味物質之分析 48
3.8.1 實驗設備與試劑 48
3.8.2 GC oven 升溫條件 49
3.8.3 實驗方法 49
第四章 結果與討論 51
4.1 基因萃取之方法建立 51
4.1.1 illustra triplePrep 商業套件之最佳化 51
4.2 q-PCR應用 55
4.2.1 多重探針測試目標基因之最佳化 55
4.2.2 DNA檢量線配製 63
4.2.3 不同藻數之微囊藻與魚腥藻之測試 68
4.3 現地應用 72
4.3.1 新竹寶山水庫 73
4.3.2 寶山第二水庫 76
4.3.3 蘭潭水庫 80
4.3.4 阿公店水庫 85
4.3.5 鯉魚潭水庫 90
4.3.6 成功大學成功湖 94
第五章 結論與建議 101
5.1 結論 101
5.2 建議 102
第六章 參考文獻 103

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曹翔崴,(2011),水庫中Geosmin產生源分子生物定量方法之開發與應用,國立成功大學環境工程學系碩士論文。
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張哲源,(2010),開發SYBR Green Real-time PCR 現地偵測Geosmin產臭基因之方法,國立成功大學環境工程學系碩士論文。

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