Doungudomdacha S, Rawlinson A, Douglas C W
Department of Oral Pathology, School of Clinical Dentistry, University of Sheffield, UK.
J Med Microbiol. 2000 Oct;49(10):861-74. doi: 10.1099/0022-1317-49-10-861.
Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans are believed to play an important role in adult periodontitis, but the significance of their relative numbers and progress of the disease is still unclear. Traditional quantitative methods are generally time-consuming and inaccurate. The aim of this study was to develop a sensitive, quantitative PCR technique that would be useful for enumerating P. gingivalis, Pr. intermedia and A. actinomycetemcomitans in subgingival plaque samples from subjects with adult periodontitis. Primers to the following genes were employed: the fimbrial gene (fimA) of P. gingivalis, the 16S rRNA gene of Pr. intermedia and the leukotoxin-A (lktA) gene of A. actinomycetemcomitans. Competitive templates were constructed either by sequence deletion between primer binding sites or by annealing of the primer binding sites to an appropriate DNA core so as to yield products of a different size from that obtained with the target template. Coamplification of target and competitive templates yielded products of expected size and non-specific recognition by the primers was not found. The sensitivity of the designed primers was 100 cells of P. gingivalis, 100 cells of Pr. intermedia and 10 cells of A. actinomycetemcomitans. The three species were found in subgingival plaque samples collected from both healthy and diseased sites by the quantitative-competitive (QC)-PCR method and the technique was more sensitive than cultural methods. For determining the proportions of each of the three periodontopathogens, the total number of bacteria in the samples was enumerated by quantitative-PCR with 16S rRNA universal primers (27f and 342r). The findings indicate that QC-PCR is a useful method for enumerating bacteria in clinical oral specimens and the technique could play a role in the investigation of disease progression.
牙龈卟啉单胞菌、中间普氏菌和伴放线放线杆菌被认为在成人牙周炎中起重要作用，但其相对数量与疾病进展的意义仍不清楚。传统的定量方法通常耗时且不准确。本研究的目的是开发一种灵敏的定量PCR技术，用于对成人牙周炎患者龈下菌斑样本中的牙龈卟啉单胞菌、中间普氏菌和伴放线放线杆菌进行计数。使用了针对以下基因的引物：牙龈卟啉单胞菌的菌毛基因（fimA）、中间普氏菌的16S rRNA基因和伴放线放线杆菌的白细胞毒素A（lktA）基因。通过引物结合位点之间的序列缺失或使引物结合位点与合适的DNA核心退火来构建竞争模板，以产生与目标模板不同大小的产物。目标模板和竞争模板的共扩增产生了预期大小的产物，未发现引物的非特异性识别。所设计引物的灵敏度为牙龈卟啉单胞菌100个细胞、中间普氏菌100个细胞和伴放线放线杆菌10个细胞。通过定量竞争（QC）-PCR方法在从健康和患病部位采集的龈下菌斑样本中发现了这三种菌，且该技术比培养方法更灵敏。为了确定这三种牙周病原体各自的比例，使用16S rRNA通用引物（27f和342r）通过定量PCR对样本中的细菌总数进行计数。研究结果表明，QC-PCR是一种用于临床口腔标本中细菌计数的有用方法，该技术在疾病进展研究中可能发挥作用。
