Sakamoto Mitsuo, Takeuchi Yasuo, Umeda Makoto, Ishikawa Isao, Benno Yoshimi
Japan Collection of Microorganisms, RIKEN, Wako, Saitama 351-0198 Japan 2Division of Periodontology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8549, Japan.
J Med Microbiol. 2003 Jan;52(Pt 1):79-89. doi: 10.1099/jmm.0.04991-0.
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 spirochaetes in saliva were amplified by PCR with a 6'carboxy-fluorescein (6-FAM)-labelled 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 (HhaI or MspI) and analysed by using an automated DNA sequencer. T-RFLP patterns were numerically analysed using a computer program. From analysis of the 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 spirochaetal community using T-RFLP showed that the patterns derived from patients with periodontitis were grouped more as compared with the analysis of the 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。唾液中口腔细菌和螺旋体的16S rRNA基因（rDNA）通过PCR进行扩增，使用6'-羧基荧光素（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。16S rDNA用具有4bp识别位点的限制性内切酶（HhaI或MspI）消化，并使用自动DNA测序仪进行分析。T-RFLP图谱使用计算机程序进行数值分析。通过对口腔细菌群落的分析，来自牙周健康受试者和牙周炎患者的图谱被分组到不同的簇中，尽管存在一些不确定性。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），尽管这不是很明显。使用T-RFLP对螺旋体群落进行分析表明，与口腔细菌群落分析相比，来自牙周炎患者的图谱分组更多。这些结果表明，牙周炎患者的样本包含意想不到的多样性。两名牙周健康受试者唾液样本在5周内的16S rDNA的T-RFLP图谱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分析可用于评估口腔细菌菌群的多样性，并快速比较有和没有牙周炎的受试者之间的群落结构。
