Identification of periodontal pathogen by DNA probe-biolobiclal and chemical luminescence system

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One approach to the development of a rapid, sensitive and specific assay for the detection of the pathogens is molecular hybridization by using DNA or RNA probe. We focused to detection and identification of periodontal pathogens, P.gingivalis and A.actinomycetemcomitans as disease-active site and A.viscosus as inactive site during the progression of periodontal disease. We cloned genes ; outer membrane protein and glycylprolyl amonopeptidase from P.gingivalis, leukotoxin from A.actinomycetemcomitans and sialidase from A.viscosus. Then non-radiolabeled riboprobes were developed using in vitro transcription RNA synthesizing system. All probes were highly specific for identification of each pathogen.We also examined the usefullness of alkaline phosphatase/lumigen PPD detection system using anti-DNA-RNA complex antibody. Result showed a good corelation between optical density and number of molecules until 10^4 molecules but poor corelation was found over 10^5 molecules. These unfortunate results may occur by contamination of RNA or DNA-RNA hybrid in the sample. Then we amplified the specific region from chromosomal DNA and measured by the same procedure, this experiment produced a good corelation for detection of pathogens.