DETECTION AND STRAIN IDENTIFICATION OF ACTINOBACILLUS-ACTINOMYCETEMCOMITANS BY NESTED PCR

By using PCR, Actinobacillus actinomycetemcomitans strains were identi fied directly from plaque samples without the need to isolate or cultu re bacteria. DNA fragments were generated by a nested, two-step PCR am plification of the ribosomal spacer region between the 16S and 23S rRN A genes. For the first amplification, primers homologous to sequences common to all bacterial species were used. This was followed by a seco nd amplification with primers specific to A. actinomycetemcomitans. Th e ribosomal DNA spacer region was amplified from as few as 10 bacteria l cells within a total population of 10(8) cells (0.00001%), and cross -reactivity between species was not observed. DNA fragments specific f or Porphyromonas gingivalis were generated from the same samples by us ing a P. gingivalis-specific primer, and equivalent sensitivity and sp ecificity were observed. A. actinomycetemcomitans was detected in 60% and P.gingivalis was detected in 79% of 52 subjects tested. Sequence a nalysis of the spacer region DNA fragment for A. actinomycetemcomitans gave precise strain identification, producing unique sequences for se ven reference strains and identification of nine plaque derived isolat es. A phylogenetic tree based on quantitative sequence relationships w as constructed. Two-step PCR amplification directly from plaque sample s combined with sequence analysis of the ribosomal DNA spacer region p ro,ides a sensitive assay for detection and strain identification df m ultiple species directly from a single plaque sample. This simplified approach provides a practical method for large-scale studies on the tr ansmission and pathogenicity of periodontitis-associated bacteria.