| Project/Area Number |
06671918
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Conservative dentistry
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| Research Institution | Kyushu University |
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Principal Investigator |
HAMACHI Takafumi Kyushu Univ., Dentistry Research Associate, 歯学部, 助手 (80198811)
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| Co-Investigator(Kenkyū-buntansha) |
YONEDA Masahiro Kyushu Univ., Dentistry, Research Associate, 歯学部, 助手 (10253460)
HIROFUJI Takao Kyushu Univ., Dentistry, Assistant Professor, 歯学部, 講師 (10189897)
MAEDA Katsumasa Kyushu Univ., Dentistry, Professor, 歯学部, 教授 (00117243)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1995: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | Periodontal pathogens / PCR method / colorimetric assay / A.actinomycetemcomitans / P.gingivalis / 細菌検査法 / PCR / 細菌検査 / A.actinomycetemcomitans / P.gingivalis / 成人性歯周炎 / 歯肉縁下プラーク |
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| Research Abstract |
Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis has been implicated as causative organisms of periodontal disease. The aim of this research project was to develop a polymerase chain reaction (PCR) based method for detection and quantification of these periodontal pathogens in subgingival plaque samples. For specific detection of A.actinomycetemcomitans and P.gingivalis, leukotoxin gene and collagenase gene, respectively, were selected as the target sequence. Two pairs of oligonucleotide primers were synthesized to amplify the leukotoxin gene fragment (396bp) and the collagenase gene fragment (414bp) . Following PCR amplification, PCR products were analyzed by agarose get electrophoresis. PCR method was able to detect as few as 50 bacterial cells. To quantify the amount of A.actinomycetemcomitans and P.gingivalis, one primer was labeled with biotin and the other one was labeled with digoxigenin at 5'ends. Following amplification, the biotinylated PCR products were applied to a microtiter plate well percoated with avidin. The bound PCR products were detected colorimetrically with alkaline phosphatase conjugated anti-digoxigenin antibody and substrate. The detection limit of the colorimetric assay was found to be as few as 50 bacterial cells. The absorbance value in the colorimetric assay were log-linear between 50 and 10^5 bacterial cells. Therefore, this colorimetric assay was able to estimate the amount of periodontal pathogens in subgingival plaque. The colorimetric assay of the PCR product is very usuful method not only to detect the presence of A.actinomycetemcomitans and P.gingivalis but also to quantify the amount of these periodontal pathogens in subgingival plaque samples.
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