Optimization of a Real-Time PCR Method for the Quantifying of Purple Non-Sulfur Phototrophic Bacteria

• Main Authors: Yao-Chu Wu, 吳樂竹
• Other Authors: 洪俊雄
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/80138205362853713069

碩士 === 國立中興大學 === 環境工程學系所 === 95 === 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.