Establishment of rapid method for detection of periodontitis-associated microorganisms

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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