Accurate quantification of bacterial species in
dental plaque is needed for microbiological diagnosis of
periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher
dye, and
SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective
16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal
therapy, quantitative detection of tetQ gene, which confers resistance to
tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and
SYBR Green chemistry in their specificity, quantitativity and sensitivity. The
SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight
periodontal pockets) before and 1 week after the local
drug delivery of
minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the
antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in
periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the
antibiotic therapy should be included in the examination.