Microbiological examination for periodontal therapy

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Real time PCR using GeneAmp^R sequence detection system; was applied to the microbiological examination in periodontal disease. Both TaqMan probe with reporter and quencher dye and SYBR Green dye were used for the source of fluorescence. The primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gigngivalis, Prevotella intermedia and total bacteria based on the nucleotide sequence of 16S ribosomal RNA gene. Since spread of antibiotic-resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confer resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans were linear over a range of 10 to 10^7 cells (10 to 10^7 copies for tetQ gene), while the quantitative range for total bacteria was from 10^2 to 10^7. There was no significant difference between the TaqMan and SYBR-Green chemistry systems in their specificity, quantitativity and sensitivity. The SYBR Green chemistry was then used for the clinical plaque samples. Subgingival plaque samples were obtained before and one week after the local drug delivery of minocycline. Although the number of P. gngivalis, P. intermedia and A. actinomycetemcomitans decreased after the antibiotic therapy in most cases, the plaque samples contained higher level of tetQ gene. The quantitative real time PCR will be a powerful tool for microbiological examination of periodontal disease and the quantitative monitoring of antibiotic resistance gene is thought to be necessary for periodontal therapy.