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Development and clinical application of detection method of periodontopathic bacteria by using a non-radioactive DNA probe

非放射性DNA探针牙周病菌检测方法的开发及临床应用

基本信息

批准号：
03557081
负责人：
ISHIKAWA Isao
金额：
$ 6.91万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

A dot blot DNA hybridization assay for a rapid identification of periodontopathic bacteria in subgingival plaque samples was developed by using bisulfite modified whole genomic DNA probe from which was immunoenzymatically visualized by anti SO_3^- antibody conjugated with alkalinephosphatase. Bisulfite modified DNA probe of P.gingivalis and P.intermedia showed high specificity and sensitivity to detect reference strains. The probe from P.gingivalis was applied on clinical subgingival plaque samples to evaluate the distribution of P.gingivalis in the dentition, and to compare the presence of P.gingivalis with clinical parameters. Fifteen adult periodontitis patients and 6 healthy volunteers were examined. Subgingival plaque samples were taken from 4 sites of all the remaining teeth. At the same time, probing depth and bleeding on probing (BOP) were also recorded. The detection percentage and amounts of P.gingivalis present were statistically compared with probing depth and BOP in the patient group. As a result, P.gingivalis was detected in all patients examined and 3 out of 6 healthy individuals. The detection average was 31% in patient group. When the probing depth was over 4 mm or BOP was positive, the detection percentage of P.gingivalis significantly increased. As more P.gingivalis was identified, the percentage of sites with deep probing depth or that were BOP positive increased significantly. However, P.gingivalis was also detected in clinically healthy sites, and P.gingivalis negative sites with deep probing depth or that were BOP positive were existed in the same patient. These results indicate that P.gingivalis play an important role, but is not the only microorganism responsible for adult periodontitis. Moreover, using RFLPs analysis we examined P.gingivalis strains including clinical isolates on their fimbrillin gene locus. The results indicate that genetic heterogeneity seems to be existing within the fimbrillin gene locus.

应用亚硫酸氢盐修饰的全基因组DNA探针，以抗SO_3 ~（2-）抗体标记碱性磷酸酶免疫酶显色，建立了一种快速鉴定龈下菌斑中牙周致病菌的斑点杂交方法。亚硫酸氢盐修饰的牙龈卟啉单胞菌和中间型卟啉单胞菌DNA探针检测参考菌株具有较高的特异性和敏感性。将来自牙龈卟啉单胞菌的探针应用于临床龈下菌斑样本，以评估牙龈卟啉单胞菌在牙列中的分布，并将牙龈卟啉单胞菌的存在与临床参数进行比较。对15例成人牙周炎患者和6例健康志愿者进行了检查。从所有剩余牙齿的4个部位采集龈下菌斑样本。同时记录探诊深度和探诊出血（BOP）。将患者组中存在的牙龈卟啉单胞菌的检出百分比和量与探诊深度和BOP进行统计学比较。结果，在所有受检患者和6名健康个体中的3名中检测到牙龈卟啉单胞菌。患者组平均检出率为31%。当探诊深度超过4 mm或BOP阳性时，牙龈卟啉单胞菌的检出率显著增加。随着更多的牙龈卟啉单胞菌被识别，具有深探测深度或BOP阳性的位点的百分比显著增加。但在临床健康部位也可检测到牙龈卟啉单胞菌，且同一患者存在牙龈卟啉单胞菌阴性且探诊深度深的部位或BOP阳性部位。这些结果表明，牙龈卟啉单胞菌发挥重要作用，但不是唯一的微生物负责成人牙周炎。此外，使用RFLPs分析，我们检查了牙龈卟啉单胞菌菌株，包括临床分离株的菌毛蛋白基因座。结果表明，在菌毛蛋白基因座内似乎存在遗传异质性。