Semiquantitative analysis of periodontopathogens by gene amplification

Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, which are known as major causative organisms of periodontitis, were semiquantitatively identified by two-step polymerase chain reaction (PCR). The sets of specific primers for A. actinomycetemcomitans and P. gingivalis were prepared on the basis of the nucleotide sequences of the lktA and the fimA genes, respectively. The set of universal primers for eubacteria was designed from the nucleotide sequence of a highly conserved region in the eubacterial 16S rRNA sequence. The number of bacteria detectable by one-step PCR assay was no fewer than 10(3) cells. Less than 10(3) bacterial cells were detectable by two-step PCR assay. Subgingival plaque samples from 37 sites of 16 patients were obtained with paperpoints and analyzed by two-step PCR assay. More than 2 x 10(6) bacterial cells were found in the subgingival plaque samples from all diseased sites. In contrast, the number of total bacteria in those from more than half of healthy sites estimated by PCR assay was less than 2 x 10(6) cells, suggesting that subgingival plaque in diseased sites consists of a relatively larger number of bacteria compared with the population in healthy sites. While A. actinomycetemcomitans was detected in both healthy and diseased sites, P. gingivalis was observed only in diseased sites. Both periodontopathic bacteria occupied a minor part (less than 0.1%) of the total subgingival plaque bacteria.