臺灣博碩士論文加值系統

　　柱孢藻毒素(Cylindrospermopsin,CYN)是一種常見的藍綠細菌有害二次代謝物，其結構穩定且具有高水溶性，在淨水處理程序中難以被去除，且會對人體造成許多負面影響。台灣本島水庫與金門地區水庫都曾發現過柱孢藻毒素的存在，顯示柱孢藻毒素對台灣飲用水體具有潛在的危害，相關應對措施的提出及預警策略的建立是必要的。因此本研究擬以即時定量連鎖聚合酶反應(qPCR) 之Taqman系統建立總柱孢藻細胞與產CYN細胞之現地偵測技術，以期可即刻掌握柱孢藻毒素對飲用水體的潛在危害，降低飲用水受到污染所造成的可能危害，進一步保障民眾用水安全。
　　本研究成功地以qPCR TaqMan系統建立偵測產CYN細胞功能性基因 (pks)與總柱孢藻細胞功能性基因(rpoC1)之單一定量與雙重定量分析技術，此外，本研究亦同時以酵素連結免疫分析法(ELISA)進行柱孢藻毒素濃度測量，和以顯微鏡細胞計數測量總柱孢藻細胞數，並進行三者間之相關性分析，整體分析時間僅需2~3小時。本研究將此現地偵測技術應用於台灣本島共11座水庫(共73個採樣點)和金門地區共11水庫與2座淨水場(共60個採樣點)，結果顯示，總柱孢藻細胞偵測基因(rpoC1)定量結果與顯微鏡細胞計數之數據間存在良好之正相關性；而產CYN細胞偵測基因(pks)定量結果與ELISA分析藻毒濃度雖無濃度趨勢上的相關性，但在60筆金門地區水庫分析結果中，有九成的數據可觀察到兩者間在水體中存在與否的一致性。另一方面，本研究中亦針對qPCR雙重定量系統進行分析條件的最適化，來改善因兩偵測基因間影響造成螢光檢測值偏低的情形，以期未來能將此技術廣泛應用於各種類型水體中總柱孢藻細胞與產CYN細胞的監測。

Cylindrospermopsin (CYN) is a toxic secondary metabolite produced by Cyanobacteria in water body. Because of its stable structure and high solubility in water, CYN is not easy to remove by conventional drinking water treatment processes, resulting in negative effect on human health. CYN has been discovered in a few of Taiwan’s reservoirs, including in main island and in Kinmen island. Therefore, a method to accurately and rapidly estimate the risk associated with the presence of CYN in drinking water sources in required for the protection of public health. Therefore, in this study we proposed to establish an on-site monitoring technology for Cylindrospermopsis cells and CYN-producing cells using qPCR combined with TaqMan system.
Both single and dual quantification systems targeted for the functional genes of Cylindrospermopsis cells, rpoC1, and CYN producing cells, pks, using qPCR combined with TaqMan system were successfully established. The qPCR-TaqMan method was further applied in the on-site monitoring of the targeted genes present in Taiwan’s reservoirs. A total of 73 and 60 samples were collected and analyzed for 11 reservoirs in main island and 11 reservoirs and 2 drinking-water treatment systems in Kingmen island, respectively. During the monitoring, an enzyme-linked immunosorbent assay (ELISA) and an optical microscope were also employed to analyze the concentrations of CYN and Cylindrospermopsis cells, respectively.
Monitoring results showed that good correlations between rpoC1 gene copy numbers detected with the qPCR method and cell numbers enumerated with optical microscope. For another targeted gene, pks, although no correlations were obtained, 90% of the Kinmen samples showed co-occurrence of pks gene copies and CYN concentrations measured with ELISA. A dual quantification system for both pks and rpoC1 genes was also developed. The experimental parameters were tuned to improve the detection of both genes, serving as a basis to better apply the dual detection system in the future.

摘要 I
ABSTRACT II
誌謝 IV
目錄 VII
表目錄 XI
圖目錄 XIII
第 一 章 諸論 1
1.1 研究緣起 1
1.2 研究目的 2
第 二 章 文獻回顧 3
2.1 藍綠細菌對用水安全之影響 3
2.2 柱孢藻毒 7
2.2.1 基本特性 8
2.2.2 柱孢藻毒對人體、生態及環境之影響 10
2.2.3 產柱孢藻毒素之菌種 11
2.2.4 生物合成途徑 17
2.2.5 影響柱孢藻毒產毒的相關因子 21
2.3 柱孢藻毒與C. RACIBORSKII的偵測方法 23
2.3.1 柱孢藻毒的偵測方法 23
2.3.2 C. raciborskii的偵測方法 25
2.4 分子生物技術 27
2.4.1 聚合酶鏈鎖反應 27
2.4.2 即時定量聚合酶連鎖反應 30
2.4.3 多重探針偵測之應用 34
第 三 章 實驗設備與方法 36
3.1 實驗流程圖 36
3.2 柱孢藻培養 38
3.2.1 菌源 38
3.2.2 實驗方法 38
3.3 藻類計數 39
3.3.1 實驗設備 39
3.3.2 實驗試藥與試劑 39
3.3.3 樣本前處理 39
3.3.4 藻類計數之方法與分析 40
3.4 DNA、RNA、PROTEIN萃取 42
3.4.1 實驗設備 42
3.4.2 樣品前處理 42
3.4.3 萃取流程 44
3.5 寡醣核酸引子 47
3.6 聚合酶鏈鎖反應 48
3.6.1 實驗設備與試劑 48
3.6.2 實驗流程 48
3.7 DNA純化與定量 49
3.7.1 實驗設備與試劑 49
3.7.2 實驗流程 49
3.8 即時定量聚合酵素鏈鎖反應 51
3.8.1 實驗設備與試劑 51
3.8.2 實驗方法 51
3.9 酵素連結免疫吸附法 53
3.9.1 實驗設備與試劑 53
3.9.2 樣品前處理 53
3.9.3 實驗方法 53
第 四 章 結果與討論 55
4.1 寡核酸引子與探針之適用性評估 55
4.2 即時定量PCR 的應用 57
4.2.1 兩組寡核酸引子/探針的功能性基因片段放大測試 57
4.2.2 寡核酸引子/探針的最佳接合溫度測試 59
4.2.3 單一定量系統中檢量線之建立 61
4.2.4 產毒柱孢藻與柱孢藻菌株之細胞數與其內Copy數之關係 63
4.2.5 建立同時分析pks和rpoC1的雙重定量系統I 64
– 同時監測可行性之初步評估 64
4.2.6 建立同時分析pks和rpoC1 gene的雙重定量系統II 65
- 最佳引子與探針劑量之評估 65
4.2.7 建立同時分析pks和rpoC1 gene的雙重定量系統III 67
4.3 應用柱孢藻雙重定量分析系統進行現地應用 69
4.3.1 台灣本島與金門地區水庫 69
4.3.2 現地監測技術應用於台灣本島水庫的分析結果 71
4.3.3 現地監測技術應用於金門地區水庫的分析結果 76
4.3.3.1即時監測系統應用於金門水庫的成果 79
4.3.3.2總柱孢藻細胞、產CYN細胞與藻毒的相關性分析 84
4.3.3.3總柱孢藻細胞數於總藻數中所佔比例之變化 86
4.4 QPCR雙重定量分析系統之影響情形與改善方法 88
4.4.1 qPCR雙重定量分析之影響情形 88
4.4.1.1雙重定量分析系統所觀察到之影響情形 88
4.4.1.2參考文獻(Rasmussen et al. (2008)於雙重定量(pks 和rpoC1基因)系統的實驗結果是否出現影響情形 90
4.4.2 雙重定量分析系統中兩偵測基因相互影響之原因探討 92
4.4.2.1假設一 影響因素主要是由樣本所造成 92
4.4.2.2假設二 影響因素主要是由引子與探針間之交互作用所引起的 ……………………………………………………………………….94
4.4.2.3 假設三 影響因素由引子、探針和樣本間之交互作用引起 96
4.4.3 qPCR檢測螢光值偏低的改善方法 99
4.4.3.1 qPCR反應最佳化－接合反應的溫度與反應時間 99
4.4.3.2 qPCR反應最佳化－探針之最適劑量 103
4.4.3.3 使用qPCR最佳化條件再次進行干擾測試 104
第 五 章 結論與建議 107
5.1 結論 107
5.2 建議 108
第 六 章 參考文獻 109

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