title: Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria.
abstract: 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.
authors.affiliation: Department of Patho-Physiology, Division of Periodontal Science, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8525, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Maeda
authors.forename: Chiyo
authors.initials: C
authors.lastname: Fujimoto
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Haruki
authors.forename: Takemasa
authors.initials: T
authors.lastname: Maeda
authors.forename: Susumu
authors.initials: S
authors.lastname: Kokeguchi
authors.forename: Millan
authors.initials: M
authors.lastname: Petelin
authors.forename: Hideo
authors.initials: H
authors.lastname: Arai
authors.forename: Ichiro
authors.initials: I
authors.lastname: Tanimoto
authors.forename: Fusanori
authors.initials: F
authors.lastname: Nishimura
authors.forename: Shogo
authors.initials: S
authors.lastname: Takashiba
mesh_terms: D016976:Aggregatibacter actinomycetemcomitans / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D052160:Benzothiazoles; D004269:DNA, Bacterial / Q000235:genetics; D003773:Dental Plaque / Q000382:microbiology; D003959:Diamines; D005456:Fluorescent Dyes; D005798:Genes, Bacterial; D006801:Humans; D009930:Organic Chemicals; D010510:Periodontal Diseases / Q000188:drug therapy / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D016966:Porphyromonas gingivalis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D018713:Prevotella intermedia / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D011804:Quinolines; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D013753:Tetracycline Resistance / Q000235:genetics
chemical_list: D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D019914:Taq Polymerase