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

采用亚硫酸氢盐修饰的全基因组DNA探针，以碱性磷酸酶标记的抗SO3抗体免疫酶标法，建立了一种快速鉴定牙周病原菌的斑点杂交方法。亚硫酸氢盐修饰的牙龈假单胞菌和中间假单胞菌DNA探针对参比菌株的检测具有较高的特异性和敏感性。将牙龈假单胞菌探针应用于临床龈下菌斑标本，评价牙周炎患者牙周组织中牙龈假单胞菌的分布情况，并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个部位采集菌斑样本。同时记录探诊深度和探诊出血量(BOP)。对牙周炎患者组中牙周炎的检出百分率和检出量与牙周探诊深度和牙周压进行统计学比较。结果，所有被检查的患者和6名健康人中的3名均检出牙龈假单胞菌。患者组的平均检出率为31%。当探头深度大于4 mm或BOP为阳性时，牙龈假单胞菌的检出百分率显著增加。随着牙龈假单胞菌的发现越来越多，探诊深度较深或BOP阳性的部位所占比例显著增加。然而，在临床健康部位也发现了牙龈假单胞菌，同一患者中还存在探测深度较深的牙龈假单胞菌阴性部位或BOP阳性。这些结果表明牙龈假单胞菌在成人牙周炎中起重要作用，但不是引起成人牙周炎的唯一微生物。此外，我们还利用RFLPS分析了包括临床分离株在内的牙龈假单胞菌的fimbrins基因座。结果表明，fimbrins基因座内似乎存在遗传异质性。

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.