TY - JOUR
T1 - Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria
AU - Maeda, Hiroshi
AU - Fujimoto, Chiyo
AU - Haruki, Yasuhiro
AU - Maeda, Takemasa
AU - Kokeguchi, Susumu
AU - Petelin, Millan
AU - Arai, Hideo
AU - Tanimoto, Ichiro
AU - Nishimura, Fusanori
AU - Takashiba, Shogo
N1 - Funding Information:
We thank Dr. Progulske-Fox A. and Dr. Burks J. (Department of Oral Biology and Periodontal Disease Research Center, University of Florida) for providing the tetQ gene. This study was supported by Grants-in-Aid for Scientific Research (B12557192, C13672186 and C13671901) from the Japan Society for the Promotion of Science.
PY - 2003/10/24
Y1 - 2003/10/24
N2 - 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 GeneAmpR 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 10-107 cells (10-107 copies for tetQ gene), while the quantitative range for total bacteria was 102-107 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.
AB - 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 GeneAmpR 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 10-107 cells (10-107 copies for tetQ gene), while the quantitative range for total bacteria was 102-107 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.
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U2 - 10.1016/S0928-8244(03)00224-4
DO - 10.1016/S0928-8244(03)00224-4
M3 - Article
C2 - 14557000
AN - SCOPUS:10744219802
SN - 0928-8244
VL - 39
SP - 81
EP - 86
JO - FEMS Immunology and Medical Microbiology
JF - FEMS Immunology and Medical Microbiology
IS - 1
ER -
