环氧丙烷皂化废水活性污泥DNA提取方法的比较与优化  

Comparison and optimization of DNA extraction methods from activated sludge in wastewater of epoxy propane saponification

作　　者：曲衍洪 田琳 李玉梅[3] 李强[3] 方嘉碧 宋冬雪[3] 王佳佳[1] 郝大可[3] 陈中合[1] 

机构地区：[1]济南大学化学化工学院,山东济南250022 [2]中海沥青股份有限公司,山东滨州256601 [3]济南大学生物科学与技术学院,山东济南250022

出　　处：《生物加工过程》2015年第1期42-46,共5页Chinese Journal of Bioprocess Engineering

基　　金：国家自然科学基金(31100088);山东省科技发展计划(2013GSF12006)

摘　　要：探索适宜的环氧丙烷废水中活性污泥微生物总DNA提取的方法并进行优化。采用11种方法提取环氧丙烷废水中活性污泥微生物总DNA,即Na Cl溶液-提取缓冲液-溶菌酶-十二烷基磺酸钠(SDS)法、蛋白酶K-SDS法、SDS-反复冻融法、Tris-EDTA-Na Cl-聚乙烯吡咯烷酮(PVP)(TENP)法和反复冻融-SDS-蛋白酶K法等。通过对这11种方法进行比较,以确定最佳试验方案并结合单因素法对最佳方案进行优化。琼脂糖凝胶电泳结果显示:Na Cl溶液-提取缓冲液-溶菌酶-SDS法提取DNA亮度高,有大片断DNA的存在且拖带较少,条带集中;酶法所提取的DNA效果仅次于提取缓冲液-溶菌酶-SDS法,其他方案没有提取出DNA。单因子试验结果表明:10 mg/m L溶菌酶,100g/LSDS,反应时间为30 min,反应温度为45℃时效果最佳。Na Cl溶液-提取缓冲液-溶菌酶-SDS提取法为环氧丙烷废水中微生物群落在分子水平上的研究提供了一种简便、可靠的DNA提取方法。The optimal extracting method for genomic DNA from the epoxypropane wastewater sample was investigated in this paper. Eleven methods were used to extract total DNA of the activated sludge samples, and they were sodium chloride solution-extraction buffer-lysozyme-sodium dodecyl sulfate （SDS）, proteinase K-SDS method, SDS-repeated freezing and thawing method, Tris-EDTA-NaCl-polyvinylpyrrolidone （ TENP ） method,repeated freezing and thawing-SDS-proteinase K method, etc. According to the result from the analysis and comparison of these methods, the optimal experimental design was obtained, and it was the extraction buffer-lysozyme-SDS method which resulted in high quality genomic DNA. The results of single- factor experiments indicated that the optimal level for each factor was 10 mg/mL of lysozyme, 100 g/L SDS, the reaction time for 30 min, the reaction temperature at 45 ℃. These data suggested sodium chloridesolution-extraction buffer-lysozyme-SDS method was a simple and reliable DNA extraction method for the microbial communities in epoxypropane wastewater at the molecular level.

关 键 词：活性污泥 环氧丙烷 DNA提取 废水 

分 类 号：X703.1[环境科学与工程—环境工程] Q93-33[生物学—微生物学]