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，定量鉴定了异种混合真细菌悬浮液中的放线菌、牙龈卟啉单胞菌和齿密螺旋体，并定量了混合悬浮液中的总细胞数。分别根据菌株lktA、film和atc基因的核苷序列，制备了放线菌comitans、牙龈菌和牙齿菌的特异性引物。利用真菌体16S rRNA序列中高度保守区域的核苷酸序列设计真菌体通用引物。将人工合成的已知数量的非细菌DNA片段和相应的引物加入到PCR混合物中，扩增的DNA片段作为内标。与内标相比，每个目标DNA的比色增强得到补偿。该比色PCR法能够定量估计10^2 ~ 10^6个细胞之间的细菌细胞数。用这种方法对牙周病患者的临床菌斑样本进行评估。估计在所有患病部位的龈下菌斑样本中存在超过2*10^6个细菌细胞。而半数以上健康部位龈下菌斑的细菌总数小于2*10^6，提示病变部位龈下菌斑的细菌数量相对健康部位较多。在健康部位和病变部位均可检出放线菌，而在病变部位均可检出牙龈假单胞菌和牙齿假单胞菌。据估计，这些牙周病细菌占龈下菌斑细菌总数的一小部分（少于0.01%）。目前，我们正在开发一种评估变形链球菌和索姆链球菌的龋齿诊断方法。

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.