Structural analysis of oral microbiota by the molecular biology-technique, and its systematization

分子生物学技术对口腔微生物群的结构分析及其系统化

• 批准号: 13672202
• 负责人: SAKAMOTO Mitsuo
• 金额: $ 1.98万
• 依托单位: RIKEN
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672202/
• 关键词: Periodontal disease; Oral bacteria; 16S rRNA gene; T-RFLP; Quantitative PCR; 侵襲性歯周炎; 慢性歯周炎; スピロヘータ

Terminal restriction fragment length polymorphism (T-RFLP) analysis was applied to characterize oral bacterial flora in saliva from 18 healthy subjects and 18 patients with periodontitis. The 16S rRNA genes (rDNAs) of oral bacteria and spirochetes in saliva were amplified by PCR with a 6-FAM-labeled universal forward primer (27F) and a universal reverse primer (1492R) or the Spirochaeta-selective reverse primer. The 16S rDNAs were digested with restriction enzymes with 4-bp recognition sites (Hhal or MspI) and analyzed by using an automated DNA sequencer. T-RFLP patterns were numerically analyzed using a computer program. In the analysis of oral bacterial community, patterns derived from periodontally healthy subjects and patients with periodontitis were grouped into different clusters, though with some uncertainty. Samples from patients with periodontitis tended to cluster into their respective types (aggressive and chronic periodontitis), although this was not very clear. Analysis of spirochetal community using T-RFLP showed that the patterns derived from patients with periodontitis were grouped more as compared with the analysis of oral bacterial community. These results suggest that samples from patients with periodontitis contain an unexpected diversity. T-RFLP patterns of 16S rDNAs from saliva samples of two periodontally healthy subjects over a 5-week period showed host-specific relatively stable oral bacterial flora. Our study indicates that T-RFLP analysis is useful for the assessment of diversity of oral bacterial flora and rapid comparison of the community structure between subjects with and without periodontitis.

应用末端限制性片段长度多态性（T-RFLP）分析法对18名健康人和18名牙周炎患者唾液中的细菌植物群进行了分析。用6-FAM标记的通用正向引物（27 F）和通用反向引物（1492 R）或螺旋体特异性反向引物，对口腔细菌和唾液中螺旋体的16 S rRNA基因进行PCR扩增。用具有4-bp识别位点（Hhal或MspI）的限制酶消化16 S rDNA，并通过使用自动DNA测序仪分析。利用计算机程序对T-RFLP图谱进行了数值分析。在口腔细菌群落的分析中，牙周健康受试者和牙周炎患者的模式被分为不同的集群，虽然有一些不确定性。来自牙周炎患者的样本倾向于聚类到各自的类型（侵袭性和慢性牙周炎），尽管这不是很清楚。T-RFLP分析结果表明，牙周炎患者的螺旋体菌群比口腔细菌菌群更容易聚集。这些结果表明，牙周炎患者的样本含有意想不到的多样性。两名牙周健康受试者5周内的唾液样本的16 S rDNA的T-RFLP模式显示宿主特异性相对稳定的口腔细菌植物群。我们的研究表明，T-RFLP分析是有用的口腔细菌植物群的多样性和快速比较的社区结构之间的受试者与牙周炎。

项目成果

Sakamoto et al.: "Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis"Journal of Medical Microbiology. 52(1). 79-89 (2003)

Sakamoto 等人：“应用末端 RFLP 分析来表征健康受试者和牙周炎患者唾液中的口腔细菌菌群”《医学微生物学杂志》。

Sakamoto et aI.: "Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis"Journal of Medical Microbiology. 52 (1). 79-89 (2003)

Sakamoto 等人：“应用末端 RFLP 分析来表征健康受试者和牙周炎患者唾液中的口腔细菌菌群”《医学微生物学杂志》。