Fujimoto C, Maeda H, Kokeguchi S, Takashiba S, Nishimura F, Arai H, Fukui K, Murayama Y
Department of Patho-Physiology, Division of Periodontal Science, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
J Periodontal Res. 2003 Aug;38(4):440-5. doi: 10.1034/j.1600-0765.2003.02607.x.
Denaturing gradient gel electrophoresis (DGGE) was applied to the microbiologic examination of subgingival plaque.
The PCR primers were designed from conserved nucleotide sequences on 16S ribosomal RNA gene (16SrDNA) with GC rich clamp at the 5'-end. Polymerase chain reaction (PCR) was performed using the primers and genomic DNAs of typical periodontal bacteria. The generated 16SrDNA fragments were separated by denaturing gel.
Although the sizes of the amplified DNA fragments were almost the same among the species, 16SrDNAs of the periodontal bacteria were distinguished according to their specific sequences. The microflora of clinical plaque samples were profiled by the PCR-DGGE method, and the dominant 16SrDNA bands were cloned and sequenced. Simultaneously, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia were detected by an ordinary PCR method. In the deep periodontal pockets, the bacterial community structures were complicated and P. gingivalis was the most dominant species, whereas the DGGE profiles were simple and Streptococcus or Neisseria species were dominant in the shallow pockets. The species-specific PCR method revealed the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia in the clinical samples. However, corresponding bands were not always observed in the DGGE profiles, indicating a lower sensitivity of the DGGE method.
Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, it could visualize the bacterial qualitative compositions and reveal the major species of the plaque. The DGGE analysis and following sequencing may have the potential to be a promising bacterial examination procedure in periodontal diseases.
应用变性梯度凝胶电泳（DGGE）对龈下菌斑进行微生物学检测。
根据16S核糖体RNA基因（16SrDNA）上的保守核苷酸序列设计PCR引物，5′端带有富含GC的夹钳。使用这些引物和典型牙周细菌的基因组DNA进行聚合酶链反应（PCR）。扩增得到的16SrDNA片段通过变性凝胶进行分离。
尽管不同菌种扩增出的DNA片段大小几乎相同，但牙周细菌的16SrDNA可根据其特定序列进行区分。采用PCR-DGGE方法对临床菌斑样本的微生物群落进行分析，并对优势16SrDNA条带进行克隆和测序。同时，采用常规PCR方法检测伴放线放线杆菌、牙龈卟啉单胞菌和中间普氏菌。在深牙周袋中，细菌群落结构复杂，牙龈卟啉单胞菌是最优势菌种；而在浅牙周袋中，DGGE图谱简单，链球菌或奈瑟菌属为优势菌。种特异性PCR方法显示临床样本中存在伴放线放线杆菌、牙龈卟啉单胞菌和中间普氏菌。然而，在DGGE图谱中并非总能观察到相应条带，表明DGGE方法的灵敏度较低。
尽管DGGE方法的灵敏度可能低于常规PCR方法，但它能够可视化细菌的定性组成并揭示菌斑中的主要菌种。DGGE分析及后续测序有可能成为一种有前景的牙周疾病细菌检测方法。
