MULTIPLEX PCR USING CONSERVED AND SPECIES-SPECIFIC 16S RIBOSOMAL-RNA GENE PRIMERS FOR SIMULTANEOUS DETECTION OF ACTINOBACILLUS-ACTINOMYCETEMCOMITANS AND PORPHYROMONAS-GINGIVALIS

Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are strongly associated with periodontitis, However, little is known about their distribution in periodontally healthy individuals, because cult uring techniques are not sufficiently sensitive, A modified multiplex PCR nas developed to address that question, Our method uses two specie s-specific forward primers in combination with a single reverse primer , These primers target variable and conserved regions of the 16S rRNA gene, Sensitivity was determined by testing serial dilutions of A. act inomycetemcomitans and P. gingivalis cells, Primer specificity was tes ted against (i) six A. actinomycetemcomitans strains and four P. gingi valis strains, (ii) seven different species of oral bacteria, and (iii ) supra- and subgingival plaque from 20 subjects, The multiples PCR ha d a lower limit of detection of 2 A. actinomycetemcomitans and 30 P. g ingivalis cells, Species-specific amplicons were obtained for all A. a ctinomycetemcomitans and P. gingivalis strains tested and did not occu r with seven other bacterial species unless A. actinomycetemcomitans a nd P. gingivalis were added, Neither target species was detected in su pragingival plaque; A. actinomycetemcomitans was detected in one subgi ngival specimen, and P. gingivalis was detected in another, When plaqu e samples were spiked with 10 A. actinomycetemcomitans cells and 100 P . gingivalis cells, species-specific amplicons were detected, These fi ndings show our multiplex PCR to be highly sensitive and specific whil e allowing simultaneous detection of A. actinomycetemcomitans and P. g ingivalis. This assay has potential applications in epidemiological st udies, diagnosis, treatment planning, and monitoring of periodontal pa thogens.