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標題: 以 Real-Time PCR 建立紫色不含硫光合作用細菌之最佳定量方法
Optimization of a Real-Time PCR Method for the Quantifying of Purple Non-Sulfur Phototrophic Bacteria
作者: 吳樂竹
Wu, Yao-Chu
關鍵字: Phototrophic purple non-sulfur bacteria;紫色不含硫光合作用細菌;Molecular biotechnology;Real-Time PCR;Rhodopseudomonas palustris;分子生物技術;即時定量聚合酶鏈鎖反應;Rhodopseudomonas palustris
出版社: 環境工程學系所
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紫色不含硫光合作用細菌已經被成功地應用於高濃度有機廢水處理上,亦曾被利用於處理廢水或土壤中難分解之苯環物質;除了應用在污染物去除之外,紫色不含硫菌因具有產生氫氣的能力,近年來被許多學者將其應用於生物產氫之研究上;本實驗室於生物產氫的研究中,意外發現紫色不含硫菌具有在體內累積聚磷酸鹽的能力,其體內所累積之聚磷酸鹽濃度可達一般除磷菌的2 – 3倍以上。由上述內容可知,紫色不含硫菌於環工上具有相當多元之應用價值,因此至今仍為諸多環工學者進行研究之熱門對象,也將是本研究所針對的研究目標。
環境微生物學中,除了菌相之鑑定與生理特性研究外,菌種於環境中所佔有之數量也是相當重要之研究課題。早期以傳統方法進行菌量計算往往會發生低估的可能,即時定量聚合酶鏈鎖反應(Real-Time PCR)是分子生物技術應用於細胞定量上之一大突破,許多研究皆證實Real-Time PCR具有更高的靈敏度與準確性,近來已陸續應用於環境微生物的定量上,然而國內環工界仍鮮少看到相關之應用。因此,本研究之目的便以Real-Time PCR作為紫色不含硫光合作用細菌的定量工具,同時搭配不同的傳統定量方法進行比較,希望能強調出Real-Time PCR之定量優勢,建立出一套屬於紫色不含硫光合作用細菌的最佳定量方法。
研究中,首先以cloning的方式將已知濃度之研究目標序列(pufM gene)嵌入質體中,進行系列稀釋作為定量所需的標準品,由Real-Time PCR之螢光偵測,先建立出Real-Time PCR定量紫色不含硫菌之標準曲線。而樣本之準備上,許多學者曾提及,樣本DNA之萃取效率將影響到樣本以Real-Time PCR進行定量的準確性,為了決定DNA的最佳萃取方式,本實驗共挑選了六種不同之商業套組(編號:A – F)進行DNA萃取,以核酸偵測(A260 /A280)結果作為判斷依據,結果顯示A套組於相同菌液中萃取出之DNA濃度與純度皆最高,表示以此套組所萃取的DNA最能代表來自所有菌體,因此最適於用在樣本DNA之準備上。樣本DNA順利萃取後,便可直接以Real-Time PCR進行螢光偵測並換算出相對菌量,完成所有之定量程序。
本研究後續利用所建立之Real-Time PCR定量方法搭配其它定量方式,比較純種培養與環境樣本中紫色不含硫光合作用細菌的菌量。在純菌的定量上,實驗結果顯示平板計數法、DAPI染色與Real-Time PCR的定量結果相差不大,其中Real-Time PCR方法數值略高一些,判斷在純種培養的單純條件下,三種方法皆適於應用在菌株定量上,而Real-Time PCR則能克服平板計數法與DAPI染色於抹碟及濾膜轉貼時所造成部分菌體流失的缺點,使得定量結果更趨近實際菌量;此外,本研究針對多種環境水體與活性污泥進行定量比較,實驗結果顯示,Real-Time PCR的定量結果比平板計數高出101 – 104倍,本實驗證實傳統培養存在外來菌種競爭的問題,且以醋酸納為電子供給者之條件下,環境中部分紫色不含硫菌種亦可能無法利用其生長,因此平板計數法確實會造成嚴重低估的現象,而Real-Time PCR的結果可能與實際菌量較為接近。
除了準確性較高之外,以往傳統培養定量紫色不含硫菌的時間需求至少為5 – 7天,如今以Real-Time PCR操作則不需1小時,如此省時又準確之優勢條件,Real-Time PCR確實為紫色不含硫光合作用細菌之最佳定量方法。

Phototrophic purple non-sulfur bacteria had been applied to treat waste water with high concentration of organic matters and degrade aromatic hydrocarbons, such as benzoate or 3-chlorobenzoate, in waste water or soil. Besides contaminants removal, purple non-sulfur bacteria was also used in the study of biological H2 production. Other researches had observed that purple non-sulfur bacteria would accumulate polyphosphate (poly-P) inside the cells, and the capability of poly-P accumulating was 2 to 3 times higher than PAOs. Therefore, purple non-sulfur bacteria was a valuable microbial community for the environmental engineering field.
Not only species and physiological characteristics but also microbial population is important for studying environmental microbiology. In the past researches, quantification of microorganisms by traditional methods always led to underestimate. A new molecular biotechnique, Real-Time PCR, was expected to overcome this disadvantage, which with faster, reliable, sensitive and convenient advantage on cell quantification, and has recently been applied to quantify environmental microorganisms. Nevertheless, quantification of purple non-sulfur bacteria by molecular biotechnologies has not been reported to date. For this reason, the primarily goal of this study is to develop a Real-Time PCR assay to quantify the purple non-sulfur bacteria which is based on enumerating the copy number of pufM gene.
At first, known quantities of pufM gene inserted plasmid DNA was used as standard DNA, a series of 10-fold diluted standard DNA analyzed by Real-Time PCR for obtaining cycle threshold (CT), and then a standard curve was generated from these data. Due to the effect of sample DNA preparation on quantitative accuracy, six different commercial DNA extraction kits were used to compare the extracted DNA quality based on measuring nucleic acid assay (A260/A280). One of the commercial kit was finally chosen for further experimental using.
After Real-Time PCR assay was established, purple non-sulfur bacteria within both pure culture and environmental samples were quantified by using Real-Time PCR and other quantitative methods. From the quantification results of pure culture Rhodopseudomonas palustris GN11, there is no obvious difference between Real-Time PCR, DAPI staining and plate count methods. Real-Time PCR assay overcame the disadvantages which would lead cells losing on DAPI staining and plate count, and obtained more accurate amount. Quantification results of environmental samples indicated that date from Real-Time PCR were 101 to 104-folds higher than that from plate count method. This experiment confirmed that microorganisms in environmental samples would compete to each other while using traditional culture; moreover, a portion of purple non-sulfur bacteria probably couldn't grow on sodium acetate based medium. Using plate count method actually resulted in data underestimate; on the other hand, results from Real-Time PCR should more reliable.
Quantification of purple non-sulfur bacteria by using plate count method, cost at least 5 to 7 days, but less than 1 hour using Real-Time PCR method. Besides more reliable, quantification using Real-Time PCR method was faster. For this reason, Real-Time PCR method was really a proper method for the quantification of phototrophic purple non-sulfur bacteria.
其他識別: U0005-1308200713065700
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