一种基于PCR分析多样性的小鼠肠道微生物宏基因组提取方法  

A metagenome DNA extracting method of intestinal flora in mice for molecular diversity analysis based on PCR technology

作　　者：吴海[1] 周赛男[2,1] 郭纯[2,1] 谭周进[2,1] 蔡光先[2,1] 曾奥[1] 张华玲[1] 

机构地区：[1]湖南中医药大学,2009级研究生湖南长沙410208 [2]湖南省中药粉体与创新药物省部共建国家重点实验室培育基地,湖南长沙410208

出　　处：《中国微生态学杂志》2012年第7期648-651,共4页Chinese Journal of Microecology

基　　金：湖南省研究生科研创新项目(CX2010B348);湖南省科技厅项目(2010SK2002;2011RS4018);国家自然科学基金(81173214)

摘　　要：目的建立一种基于PCR分析分子多样性的小鼠肠道菌群宏基因组提取方法。方法比较、综合国内外小鼠肠道菌群宏基因组的提取方法后建立一种新方法,小鼠肠道内容物经丙酮洗涤,差速离心,溶菌酶、SDS裂解,CTAB处理,酚/氯仿抽提后可得到高质量的DNA,通过紫外分光光度计、琼脂糖凝胶电泳、细菌通用引物PCR和扩增核糖体限制性酶切片段分析(ARDRA)等检测该方法的实用性。结果该方法获得的小鼠肠道菌群宏基因组DNA大小在23 kb左右,A260/A280在1.8～2.0,经细菌通用引物PCR后能得到适用于ARDRA的目的产物。结论该方法经济适用性较强,具备一定的应用价值。Objective To establish a method of metagenome DNA extraction of intestinal flora in mice for molecular di- versity analysis based on PCR. Method The faecal samples were collected from intestinal tracts of mice. These samples were washed by acetone, and then centrifuged in different speeds. The cells were lysed by lysozyme and SDS, treated by CTAB, and then extracted with phenol/chloroform. The metagenome DNA of bacterial species was determined by a UV spectrophotometer, agarose gel electrophoresis. The 16S rDNA was amplified from these DNA samples through a set of bacterial universal primers. The PCR products were analyzed by Amplifed Ribosomal DNA Restriction Analysis （ARDRA）. Result The metagenome DNA of intestinal flora in mice extracted by this method was about 23 kb ; the A260/A280 of DNA was between 1.8 and 2.0 ; the PCR products were about 1.4 kb and can be applied to ARDRA for further analysis. Conclusion This method is cost-effective and easy, which is suitable for metagenome DNA extraction of intestinal flora.

关 键 词：小鼠肠道菌群 宏基因组DNA PCR 扩增核糖体限制性酶切片段分析 

分 类 号：Q78[生物学—分子生物学]