Establishment of rapid method for detection of periodontitis-associated microorganisms

该项目的目标是建立可用于牙科治疗的微生物测试，特别是普通牙医的牙周治疗。本项目包括以下三个部分：第一项研究表明与牙周炎有关的假定病原体的复杂性。在后两项研究中，我们建立了多种病原菌快速、灵敏检测的实用方法，并对到普通医疗机构就诊的患者进行了检测。1)分析牙周炎及其相关疾病中特定微生物的关系。为了分析牙周炎、牙龈充血和牙髓-牙周病变的病因，我们在牙龈下袋或根管中检测了特定的微生物。我们选择了由于宿主防御功能、病原体获取和传播的基因同质性而表现出早发性牙周炎的家庭病例。更重要的是，他的结果表明，在每种情况下，特定病原体的可能作用。然而，它们的分布与临床诊断无关，甚至同一家庭成员也有不同的病原体。总之，评估牙周炎的治疗需要多种病原菌的检测。2)建立病原体快速检测方法。建立了简便、快速的多种病原菌检测方法。微生物的检测原理是基于细菌16S核糖体RNA （rRNA）序列的差异。通过杂交直接检测16S rRNA灵敏度低，且在保存样品过程中灵敏度降低。因此，我们开发了两种方法，一种是结合培养法的集落杂交法，另一种是扩增16S rRNA基因的基于16S rRNA的聚合酶链反应（PCR）法。两种方法对菌斑粗样中特定病原菌的检测均具有满意的灵敏度和准确性。特别是基于16S rrna的PCR快速、简便。3)微生物检测在普通医疗机构的应用。我们对就诊于医学院门诊或疗养院的胰岛素依赖型糖尿病和麻风病患者的龈下斑块菌群进行了分析。少

The goal of this project is to establish the microbiological test that can be used for dental treatments, especially for the periodontal treatments by general dentists. This project consists of the following three parts : First study indicated the complexity of putative pathogens relating to periodontitis. In the latter two studies, we established the practical methods for rapid and sensitive detection of multiple pathogens and carried out the test for patients visiting general medical facilities.1) Analyzing the relationships of specific microorganisms in periodontitis and its related diseases.To analyze the microbiological aspects for etiology of periodontitis, gingival hyperemia and endodontic-periodontic lesions, we detected specific microorganisms in subgingival pockets or in root canals. We selected family cases that each member manifested an early-onset periodontitis because of their genetic homogeneity for host defense functions, and acquisition and transmission of pathogens. T … More he results indicated that the possible roles of specific pathogens in each case. Their distributions, however, did not correlate with clinical diagnosis, and even the same family member had differnt pathogens. In conclusion detection of multiple pathogens is needed for assessing the treatment of periodontitis.2) Establishment of rapid method for detection of pathogens.We established the practical methods for detection of the multiple pathogens by easy and rapid procedures. The principle for the detection of microorganisms was based on differences of the sequence in bacterial 16S ribosomal RNA (rRNA). Direct detection of 16S rRNA by hybridization had low sensitivity and also decreased sensitivity during keeping the samples. Therefore we developed two kind of methods, the colony-hybridization method that combine with culture method and the 16S rRNA-based polymerase chain reaction (PCR) method that amplify the 16S rRNA gene. Both methods showed satisfying sensitivity and accuracy for detection of specific pathogens from crude plaque samples. Especially the 16S rRNA-based PCR was rapid and was easy to perform.3) Application of the microbiological test in general medical facilities.We analyze the microflora of subgingival plaques from the patients of insulin-dependent diabetes mellitus and leprosy that visited the clinics in medical school or sanatorium. Less