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

应用末端限制性片段长度多态性(T-RFLP)技术对18例健康人和18例牙周炎患者唾液中的细菌菌群进行了分析。用6-FAM标记的通用正向引物(27F)和通用反向引物(1492R)或螺毛虫选择性反向引物，对口腔细菌和唾液中螺旋体的16S rRNA基因(RDNA)进行了聚合酶链式反应(PCR)。用带有4个碱基识别位点的限制性内切酶(HHA1或MspI)对16S rDNA进行酶切，并用自动DNA测序仪进行分析。使用计算机程序对T-RFLP图案进行了数值分析。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归为不同的类群，尽管存在一定的不确定性。牙周炎患者的样本倾向于分成他们各自的类型(侵袭性牙周炎和慢性牙周炎)，尽管这一点不是非常清楚。用T-RFLP对螺旋体群落的分析表明，与口腔细菌群落分析相比，牙周炎患者的螺旋体群落类型更具聚类性。这些结果表明，牙周炎患者的样本含有意想不到的多样性。从两名牙周健康受试者的唾液样本中提取的16S rDNA的T-RFLP图谱显示，宿主特异性的口腔细菌菌群相对稳定。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，并快速比较患有牙周炎和不患有牙周炎的受试者的群落结构。

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.