Open AccessQuantitative real‐time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans , Porphyromonas gingivalis , Prevotella intermedia , tetQ gene and total bacteria
Author(s) -
Maeda Hiroshi,
Fujimoto Chiyo,
Haruki Yasuhiro,
Maeda Takemasa,
Kokeguchi Susumu,
Petelin Millan,
Arai Hideo,
Tanimoto Ichiro,
Nishimura Fusanori,
Takashiba Shogo
Publication year - 2003
Publication title -
fems immunology & medical microbiology
Language(s) - English
Resource type - Journals
eISSN - 1574-695X
pISSN - 0928-8244
DOI - 10.1016/s0928-8244(03)00224-4
Subject(s) - taqman , prevotella intermedia , porphyromonas gingivalis , actinobacillus , biology , microbiology and biotechnology , sybr green i , real time polymerase chain reaction , dental plaque , prevotella , bacteria , gene , genetics
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 R 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.