摘要
目的分析不同16s rDNA"通用引物"扩增靶序列在研究肠道微生物菌群分析时的差异,为选择合适的通用引物扩增肠道微生物菌群提供基础数据。方法从一健康成人的粪便中提取总DNA,以两对"通用引物"27F/519R和27F/533R扩增16S rDNA序列,扩增产物分别克隆到PGEM-T载体,建立克隆文库;两个克隆文库分别挑取65个克隆进行测序,通过序列同源性比对和构建系统发育树,比较两对引物在分析肠道菌群多样性上的差异。结果成功构建了L519和L533克隆文库,阳性克隆率分别达到95.3%和92.3%。533文库和519文库中非培养或不可培养的克隆比例分别占69.1%和76.6%(P<0.05)。两文库优势菌群相同,均为梭状芽孢杆菌、拟杆菌、芽孢杆菌和变形杆菌,但各菌群的比例及低丰度菌群的分布稍有差异;在种群多样性上,533文库和519文库中分别得到22个和17个可操作分类单元,533文库比519文库有更丰富的种群多样性(P<0.05)。结论在分析肠道菌群多样性时,引物27F/533R较27F/519R有更好的兼并性,更适宜于进行肠道菌群结构和多样性的分析。
Objective To compare the two sets of universal primers of 16S rDNA by investigating the difference of microbial communities of stool samples based on the amplification products.Methods The total of DNA from the stool of a healthy adult was extracted and used as template to amplify the conserved region of 16S rDNA with two sets of primers （27F/519R and 27F/533R）.After purification,the PCR products were cloned into PGEM-T vectors and sequenced.The obtained sequences were blasted in Genbank to analyze the community structure of bacteria.Results The L519 clone library based on primers 27F/519R and the L533 clone library based on primers 27F/533R were successfully constructed. The coverage of positive clones attached to 95.3% and 92.3% respectively.Among 65 clones of each library,62 and 60 positive clones were obtained respectively in L519 library and in L533 library.The sequence analysis indicated that the two libraries had similar proportion of predominant species including Clostridia,Bacteroidetes and Bacilli.As for minor species,there were differences between the two libraries.A clone belonging to Betaproteobacteria was detected in L533 library,but not in L519 library.Meanwhile a clone of unclassified bacteria was detected in L519 library,but not in L533 library.The number of operational taxonomic units （OUTs） in L519 library and in L533 library were 17 and 22 respectively （P0.05）.Furthermore,the frequencies of clones belonging to uncultured or unknown bacteria in L519 library and in L533 library were 69.1% and 76.6% respectively （P0.05）.Conclusions Primers 27F/533R was more effective than 27F/519R in analyzing the diversity of microbial communities of stool samples.
出处
《公共卫生与预防医学》
2010年第3期5-9,共5页
Journal of Public Health and Preventive Medicine
基金
科技部卫生行业科研专项(项目编号:200802016)
