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研究生:陳明文
研究生(外文):Ming-Wen Chen
論文名稱:應用分子生物技術於活性污泥絲狀菌檢測之研究
論文名稱(外文):Detection of Filamentous Microorganisms in Activated Sludge by Molecular Biotechnology
指導教授:周裕然
指導教授(外文):Yu-Jan Chou
學位類別:碩士
校院名稱:輔英科技大學
系所名稱:生物技術系碩士班
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:2008
畢業學年度:96
語文別:中文
論文頁數:107
中文關鍵詞:陣列晶片限制片段長度多型性絲狀菌活性污泥
外文關鍵詞:activated sludgefilamentous microorganismsarray chiprestriction fragment length polymorphism
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傳統檢測活性污泥中絲狀菌的方法是利用污泥染色法以及顯微鏡鏡檢,觀察絲狀菌的染色特性及型態,以此加以辨別活性污泥所含絲狀菌之菌種,其缺點為常因檢測人員的標準不同而可能發生誤判菌種, 故可利用分子生物技術中限制片段長度多型性(restriction fragment length polymorphism, RFLP)與陣列晶片(array chip)進行絲狀菌菌種之檢測。本研究分析多種限制酵素後,建立絲狀菌限制酵素分析流程圖,可進行活性污泥絲狀菌之初步篩選,並以生物資訊軟體設計與收集國內外發表絲狀菌相關之核酸探針,製備絲狀菌檢測陣列晶片,終以實驗模型廠馴養之活性污泥基因選殖株16S rDNA 序列進行實測,同時本研究應用生物資訊軟體Vector NTI 模擬雜交結果與嚴苛度實驗,確認陣列晶片之專一性與感受性,並可正確地鑑定出活性污泥中Leptothrix discophora、Thiothrix nivea、Zoogloea ramigera 與Nostocoida limicola III 等四株絲狀菌。
Traditionally, filamentous microorganisms in activated sludge could be observed by stain and microscope examination. Although the characteristics and morphology of filamentous microorganisms could be identified, environmental engineers probably made mistakes in
examination procedures. Recent researches offered an another technique for filamentous microorganisms detection, including restriction fragment length polymorphism (RFLP) and array chip. This technique developed a serial of processes to screen filamentous microorganisms in activated sludge preliminary by restriction enzyme analysis. Furthermore, biological informational software was adapted to collect nucleic acid in bio-database for nucleic acid probe design in array chip construction. Purified 16S rDNA sequences of activated sludge cultivated in laboratory were considered as the test target in all experiments. Consequently,
sensitive and stringency tests were also accomplished in this research to confirm the accuracy of the developed array chip by Vector NTI functional analysis and simulated hybridization experiments. Finally, this study screened and identified accurately four filamentous microorganisms in activated sludge, including Leptothrix discophora, Thiothrix nivea, Zoogloea ramigera and Nostocoida limicola III.
誌謝-------------------------------------------------------------------------- i
中文摘要-------------------------------------------------------------------------- ii
英文摘要-------------------------------------------------------------------------- iii
目錄-------------------------------------------------------------------------- iv
表目錄-------------------------------------------------------------------------- vi
圖目錄-------------------------------------------------------------------------- vii
第一章緒論-------------------------------------------------------------------- 1
第一節研究緣起--------------------------------------------------------------- 1
第二節研究目的與內容------------------------------------------------------ 3
第三節研究架構--------------------------------------------------------------- 4
第二章文獻回顧--------------------------------------------------------------- 5
第一節活性污泥處理程序--------------------------------------------------- 5
一廢水生物處理原理及菌相------------------------------------------ 5
二絲狀菌膨化與菌種組成--------------------------------------------- 9
第二節環境分子生物檢測技術--------------------------------------------- 13
一傳統檢測技術原理--------------------------------------------------- 13
二分子生物檢測技術方法--------------------------------------------- 15
三分子生物檢測技術應用與限制------------------------------------ 17
第三節限制酵素檢測原理及應用------------------------------------------ 22
一限制酵素分析原理與方法------------------------------------------ 22
二限制酵素分析應用--------------------------------------------------- 24
第四節陣列技術檢測原理及應用------------------------------------------ 25
一陣列技術原理--------------------------------------------------------- 25
二陣列晶片應用--------------------------------------------------------- 26
第三章研究方法與設備------------------------------------------------------ 29
第一節基因選殖株來源------------------------------------------------------ 31
第二節分子生物實驗方法--------------------------------------------------- 33
一16S rDNA 萃取------------------------------------------------------- 33
二基因選殖--------------------------------------------------------------- 34
三限制酵素作用模擬與分析------------------------------------------ 35
四DNA 定序及親緣關係圖製作-------------------------------------- 36
五生物資訊軟體模擬雜交反應--------------------------------------- 36
第三節陣列晶片設計與檢測方法------------------------------------------ 38
一探針設計--------------------------------------------------------------- 38
二陣列晶片製備--------------------------------------------------------- 40
三活性污泥樣本標定與雜交反應------------------------------------ 42
四晶片清洗與呈色反應------------------------------------------------ 44
五訊號偵測--------------------------------------------------------------- 46
第四節研究儀器與設備------------------------------------------------------ 47
第四章研究結果與討論------------------------------------------------------ 49
第一節探針研發與晶片製備結果------------------------------------------ 49
第二節基因選殖株樣本之菌相分析--------------------------------------- 52
第三節限制酵素作用結果--------------------------------------------------- 57
一限制酵素作用之模擬結果------------------------------------------ 57
二限制酵素作用之實測結果------------------------------------------ 58
第四節嚴苛度實驗之模擬與晶片呈色結果分析------------------------ 61
一陣列晶片呈色模擬結果--------------------------------------------- 61
二陣列晶片呈色實測結果與分析------------------------------------ 64
第五節以污泥總16S rDNA 進行晶片實測結果------------------------- 72
一AO 程序(HRT=10 小時、SRT=15 天、R=0.0)污泥晶片實測72
二AO 程序(HRT=10 小時、SRT=15 天、R=1.5)污泥晶片實測78
三AO 程序(HRT=10 小時、SRT=30 天、R=0.0)污泥晶片實測78
第五章結果與討論------------------------------------------------------------ 80
參考文獻--------------------------------------------------------------------------- 83
附錄一傳統絲狀菌檢測方式------------------------------------------------ 93
附錄二探針專一性測試表--------------------------------------------------- 94
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董文蘭,2005,以聚合�○s鎖反應偵測水中之隱胞子蟲,逢甲大學環境工程與科學學系研究所,碩士論文。
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