羊粪沼液微生物总DNA提取方法优化比较研究  

The Optimization Extraction Methods of Total Microbial DNA of Sheep Dung Biogas Slurry

作　　者：陈竞[1,4] 苏艳[2] 冯蕾[3] 杨新平[3] 秦新政[3] 古丽·艾合买提[1,4] 

机构地区：[1]新疆农业大学动物医学学院 乌鲁木齐830052 [2]新疆农业大学动物医学学院 乌鲁木齐830052 [3]新疆农业科学院微生物应用研究所,乌鲁木齐,830091 [4]新疆农业科学院微生物应用研究所,乌鲁木齐830091

出　　处：《新疆农业科学》2013年第12期2280-2285,共6页Xinjiang Agricultural Sciences

基　　金：国家自然科学基金项目(31260016);新疆维吾尔自治区重大专项(201130104);新疆维吾尔自治区高技术研究发展计划项目(201010104);新疆乌鲁木齐市科技局项目(Y101210003)

摘　　要：【目的】目前沼液污泥中99%的微生物不可培养的,为开展羊粪沼液中微生物多样性的研究,须对沼液总DNA进行提取。【方法】研究以总DNA及其PCR产物的琼脂糖电泳结果为判定依据,以SDS结合酚氯仿抽提总DNA的方法为基础,采用逐步增加净化菌泥、溶菌酶裂解以及DNA纯化等优化方法,并测定优化效果。【结果】后续增加的步骤对提高总DNA的提取效率及质量十分有效,所得DNA在得率、大小以及纯净度方面都有所提高,可以满足后续分子实验的要求。【结论】以SDS法为基础,用TENP和PBS洗涤菌泥,添加适量溶菌酶,并适当延长反应时间至3 h,再经纯化柱吸附和洗脱的方法可以获得更高质量的羊粪沼液总DNA。【Objective】Because of more than 99% of the microorganisms in biogas slurry are uncultivable. This project aims to to research the microbial diversity of sheep manure biogas slurry. The total DNA of biogas slurry must be extracted.【Method】This study,based on the SDS combined phenol chloroform extraction method of total DNA,gradually increased the purifying bacterial sludge,lysozyme lysis,and DNA purification steps. The total DNA and PCR results were taken as the standard to determine the effects of the optimization.【Result】The results indicated that subsequent increase in steps was very effective to improve the quality of total DNA and extraction efficiency. The DNA size in the yield,purity was improved,and it could be used for subsequent molecular experiments.【Conclusion】Based on the method of SDS,using TENP and PBS washing bacterial sludge,and appropriate amount of lysozyme was added,and the reaction time was prolonged to 3 hours,and then the method of purification and elution column adsorption were used. As a result,more high quality total DNA from biogas slurry can be obtained.

关 键 词：沼液 总DNA提取 优化 PCR-DGGE检测 

分 类 号：S181[农业科学—农业基础科学]