Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes.
Quantitative analysis, with identification of periodontopathic bacteria, is important for the diagnosis, therapeutic evaluation and risk assessment of periodontal disease. We developed a highly sensitive and specific method using real-time polymerase chain reaction (PCR) to detect and quantify six periodontal bacteria: Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans, Treponema denticola, Prevotella intermedia, and Prevotella nigrescens. Species-specific TaqMan probe/primer sets were designed according to 16S ribosomal RNA gene sequences. Plaque and tongue debris specimens were collected from 10 patients with advanced periodontitis and 10 periodontal healthy individuals and analyzed. All species, except for P. nigrescens, were detected in samples from diseased sites in significantly greater numbers than in those from healthy sites, whereas greater numbers of P. nigrescens were found in the controls. These results suggest that the present real-time PCR method with the designed probe/primer sets enabled sensitive detection of the six periodontal bacteria, and may also assist future microbial studies of periodontal diseases.
Kuboniwa
M
M
Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan. kuboniwa@dent.osaka-u.ac.jp
Amano
A
A
Kimura
K R
KR
Sekine
S
S
Kato
S
S
Yamamoto
Y
Y
Okahashi
N
N
Iida
T
T
Shizukuishi
S
S
D016976:Aggregatibacter actinomycetemcomitans / Q000302:isolation & purification; D001439:Bacteroides / Q000302:isolation & purification; D017931:DNA Primers; D015342:DNA Probes; D003773:Dental Plaque / Q000382:microbiology; D005456:Fluorescent Dyes; D006090:Gram-Negative Bacteria / Q000145:classification*; D006801:Humans; D008875:Middle Aged; D010510:Periodontal Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016966:Porphyromonas gingivalis / Q000302:isolation & purification; D018713:Prevotella intermedia / Q000302:isolation & purification; D045242:Prevotella nigrescens / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D013045:Species Specificity; D018709:Statistics, Nonparametric; D019914:Taq Polymerase; D014059:Tongue / Q000382:microbiology; D014208:Treponema / Q000302:isolation & purification
D017931:DNA Primers; D015342:DNA Probes; D005456:Fluorescent Dyes; D012336:RNA, Ribosomal, 16S; D019914:Taq Polymerase