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

基本信息

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

项目摘要

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) 分析来表征 18 名健康受试者和 18 名牙周炎患者唾液中的口腔细菌菌群。使用 6-FAM 标记的通用正向引物 (27F) 和通用反向引物 (1492R) 或螺旋体选择性反向引物，通过 PCR 扩增口腔细菌和螺旋体的 16S rRNA 基因 (rDNA)。使用具有 4 bp 识别位点（Hhal 或 MspI）的限制性酶消化 16S rDNA，并使用自动 DNA 测序仪进行分析。使用计算机程序对 T-RFLP 模式进行数值分析。在口腔细菌群落分析中，来自牙周健康受试者和牙周炎患者的模式被分为不同的簇，尽管存在一些不确定性。牙周炎患者的样本往往分为各自的类型（侵袭性牙周炎和慢性牙周炎），尽管这一点还不是很清楚。使用 T-RFLP 对螺旋体群落的分析表明，与口腔细菌群落的分析相比，源自牙周炎患者的模式分组更多。这些结果表明牙周炎患者的样本包含意想不到的多样性。 5 周内两名牙周健康受试者唾液样本的 16S rDNA 的 T-RFLP 模式显示宿主特异性相对稳定的口腔细菌菌群。我们的研究表明，T-RFLP 分析有助于评估口腔细菌菌群的多样性，并快速比较患有和未患有牙周炎的受试者之间的群落结构。