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 analyze the difference of the 16s rDNA “universal primer ” amplification target sequences in the study of intestinal microflora analysis and provide the basic data for selecting suitable universal primers for amplifying intestinal microflora. Methods The total DNA was extracted from the stool of a healthy adult. The 16S rDNA sequence was amplified by two pairs of “universal primers” 27F / 519R and 27F / 533R. The amplified products were cloned into PGEM-T vector and cloned A total of 65 clones were cloned and sequenced. The sequence alignment was used to construct phylogenetic tree. The differences between the two pairs of primers in the analysis of intestinal flora diversity were compared. Results The L519 and L533 clones were successfully constructed and the positive clonality rates were 95.3% and 92.3% respectively. The proportion of non-cultured or uncultureable clones in the 533 and 519 libraries accounted for 69.1% and 76.6%, respectively (P <0.05). The two dominant bacterial communities were Clostridium, Bacteroides, Bacillus and Proteus, but the distribution of each group was slightly different from that of the low-abundance group. In the population diversity, 533 and There were 22 and 17 operable taxa in the 519 library, respectively, and the 533 library had a richer population diversity than the 519 library (P <0.05). Conclusion In analyzing the diversity of intestinal flora, primer 27F / 533R has better merging than 27F / 519R, which is more suitable for the analysis of intestinal flora structure and diversity.