• 关键词：焦化废水  活性污泥  DNA提取  PCR扩增

• 基金项目：国家高技术研究发展计划(863)项目(No.2006AA062378);国家自然科学基金资助项目(No.50278036)

• 朱爽
• 广东药学院生命科学与生物制药学院, 广州 510006

• 周林
• 广东药学院生命科学与生物制药学院, 广州 510006

• 梁翠谊
• 广东药学院生命科学与生物制药学院, 广州 510006

• 吴海华
• 广东药学院生命科学与生物制药学院, 广州 510006

• 吴海珍
• 华南理工大学生物科学与工程学院, 广州 510006

• 韦朝海
• 华南理工大学环境科学与工程学院, 广州 510006

• 摘要：对焦化废水活性污泥中微生物建立高质量的总DNA提取方法是开展分子生态学研究的重要前提.通过反复冻融-蛋白酶K-SDS、溶菌酶-反复冻融-SDS及溶菌酶-反复冻融-蛋白酶K-SDS这3种综合方法对焦化废水活性污泥总DNA进行提取,以OD₂₆₀/OD₂₈₀、OD₂₆₀/OD₂₃₀、产率、片段完整性、片段大小5个指标来评价样品总DNA的提取效果.结果表明,溶菌酶-反复冻融-蛋白酶K-SDS法所提取的总DNA的OD₂₆₀/OD₂₈₀值约为1.8,产率为1.90～16.30μg·g^-1,片断完整性好,主带清晰,大小约为23kb,其PCR反应抑制物少,能够直接进行16SrRNA基因的PCR扩增.溶菌酶-反复冻融-蛋白酶K-SDS法能够为焦化废水活性污泥中微生物的分子生态学研究提供高质量的总DNA.

• Abstract：Development of an efficient method to extract high quality total DNA from activated sludge of coking wastewater is a prerequisite for understanding its molecular microbial ecology.In this study,three methods with different combinations of extraction steps(i.e.,Freezing and Thawing-Protease K-SDS,Lysozyme-Freezing and Thawing-SDS,and Lysozyme-Freezing and Thawing-Protease K-SDS)for extracting total DNA from activated sludge were compared.The extraction efficiency of total DNA was evaluated by the yield,purity,size,and integrity of the DNA fragments.The effect of PCR inhibitors on the extraction efficiency of total DNA was also investigated.The results showed that Method Ⅲ(Lysozyme-Freezing and Thawing-Protease K-SDS)was the most efficient extraction method.The DNA yields ranged from 1.90 μg·g^-1 to 16.30 μg·g^-1.The OD₂₆₀/OD₂₈₀ ratio was about 1.8 and the size of the extracted DNA was 23 kb.The total DNA extracted with Method Ⅲ can be directly applied to PCR amplification of 16S rRNA.These results indicate that Method Ⅲ efficiently provides high quality total DNA from activated sludge of coking wastewater,which allows study of the bacterial community structure and diversity.

• Key words：coking wastewater  activated sludge  DNA extraction  PCR amplification

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