Development of Oral Diagnosis Using Rapid and Simple Quantitative Gene Amplification of Oral Pathogens

采用聚合酶链反应（PCR）比色法对混合真菌悬液中伴放线放线杆菌、牙龈卟啉单胞菌和齿垢密螺旋体进行定量鉴定，并对混合悬液中的总细胞数进行定量。根据lktA、fim和atc基因的核苷酸序列分别制备了伴放线放线杆菌、牙龈卟啉单胞菌和齿垢毛癣菌的特异性引物。根据真细菌16SrRNA高度保守区的核苷酸序列设计了一套真细菌通用引物。将已知量的人工合成的非细菌DNA片段和相应的引物组加入到PCR混合物中，扩增的DNA片段用作内标。与内标相比，各靶DNA的比色增强得到补偿。通过该比色PCR试验，能够定量估计10^2至10^6个细胞之间的细菌细胞数。牙周病患者的临床牙菌斑样本进行了评估与此测定。估计所有患病部位的龈下菌斑样本中存在超过2*10^6个细菌总数。而半数以上健康部位龈下菌斑中细菌总数低于2 × 10^6个，表明患病部位龈下菌斑中细菌数量相对较多。伴放线菌在健康和患病部位均检出，牙龈卟啉单胞菌和齿垢毛癣菌仅在患病部位检出。这些牙周致病菌估计占龈下菌斑细菌总数的一小部分（小于0.01%）。目前我们正在开发一种类似的龋病诊断方法，评价变形链球菌和温和链球菌。

By using a colorimetric assay utilizing polymerase chain reaction (PCR), Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Treponema denticola in heterogeneous mixed eubacterial suspension were quantitatively identified and also total cell number in the mixed suspension was quantified. The sets of specific primers for A.actinomycetemcomitans, P.gingivalis, and T.denticola were prepared on the basis of nucleotid sequences of the lktA,fim and atc genes, respectively. The set of universal primers for eubacteria was designed from nucleotide sequences of highly conserved region in eubacterial 16S rRNA sequences. A known amount of non-bacterial DNA fragment which was artificially synthesized and the set of the corresponding primers were added to the PCR mixtures the amplified DNA fragment was used as an internal standard. The colorimetric enhacement from each target DNA was compensated as compared with that from internal standard. The bacterial cell numbers between 10^2 and 10^6 cells were able to be quantitatively estimated by this colorimetric PCR assay. Clinical plaque samples from patients with periodontal diseases were evaluated with this assay. More than 2*10^6 cells of total bacteria were estimated to be present in subgingival plaque samples from all diseased sites. On the contrary, less than 2*10^6 cells of total bacteria were estimated in those from more than half of healthy sites, suggesting that subgingival plaque in diseased site consists of relatively large number of bacteria compared with that in healthy site. While A.actinomycetemcomitans was detected in both the healthy and diseased sites, P.gingivalis and T.denticola were observed only in diseased site. These periodontopathic bacteria were estimated to occupy the minor portin (less than 0.01%) of total subgingival plaque bacteria. Now we developing the same kind of diagnosis method for dental caries evaluating Streptococcus mutans and Streptococcus sobrims.