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目的对比Sanger和Pyrosequencing测序法分析健康人口腔菌群组成。方法收集6例健康成人唾液、舌背、黏膜、龈上及龈下菌斑并构建16SrRNA基因文库,分别用Sanger和Pyrosequencing测序法分析。结果 Sanger测序所得已知的序列有5,794条(占6,535总序列数88.7%)、75个属,396个序列划分操作分类单元(operational taxonomic units,OTUs,占总OTUs的61.4%)。Pyrosequencing测序所得已知的序列有10,771条(占11,103总序列数97.0%)、66个属,322个OTUs(占总OTUs的68.0%)。Sanger和Pyrosequencing测序法所得口腔菌群在门、属的水平分布趋势基本一致,但在种的水平分布差异显著。Sanger和Pyrosequencing测序法构建的口腔菌群文库均匀度值分别为0.016和0.007,说明Pyrosequencing分析口腔菌群物种数量分布比Sanger测序方法的文库均匀性稍差,但优势种更显著。结论 Pyrosequencing测序时所构建基因文库能代表口腔菌群的多样性且经济、省时,可以应用于口腔细菌物种的分析。
Objective To compare Sanger and Pyrosequencing methods to analyze the oral flora of healthy individuals. Methods Salivary, tongue back, mucosa, supragingra and subgingival plaque were collected from 6 healthy adults and the 16SrRNA gene library was constructed. The sequences were analyzed by Sanger and Pyrosequencing methods respectively. Results There were 5,794 sequences (88.7% of 6,535 total sequences), 75 genera and 396 sequences of operational taxonomic units (OTUs, 61.4% of total OTUs). There are 10,771 known sequences (97,10% of the total number of 11,103 and 97.0% of the total), 66 genera and 322 OTUs (68.0% of the total OTUs) obtained by Pyrosequencing. The distribution of oral flora in Sanmen and Pyrosequencing was basically the same, but there was significant difference in species distribution. The results of Sanger and Pyrosequencing showed that the homogeneity of oral flora was 0.016 and 0.007, respectively, indicating that Pyrosequencing analysis showed that the number distribution of oral flora was slightly worse than that of Sanger sequencing method, but dominant species were more significant. Conclusion The gene library constructed by Pyrosequencing can represent the diversity of oral flora and is economical and time-saving. It can be applied to the analysis of oral bacterial species.