Development of non-radioactive DNA probe and its clinical use

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Actinobacillus actinomycetemcomitans (A.a..) is one of the important pathogens in the periodontal diseases. Recently, Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) has been proposed for detecting genetic polymorphism without prior knowledge of the DNA sequence. In this study, we examined the utility of AP-PCR for genotyping of A.a.. Whole genomic DNA from 8 laboratory strains was extracted by a modification of the method of Van Steenberen. A single 10-mer, synthetic, oligonucleotide of random sequence was used as primer in AP-PCR.One (GAAACGGGTG) of the 20 primers (Operon 10-mer kit A) was found useful in differentiating these strains. The result indicates that AP-PCR is capable of distinguishing different strains of A.a... In addition, we evaluated the potential of application of non-radioactive DNA probe method (Affirm DP^<TM>) in clinical diagnosis and evaluation of treatment efficacy. Sensitivity and specificity of cut-off level (10^4) of the method with bacterial culture in detection of Bacteroide forsythus, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were compared. The result showed that detection percentage of DNA probe method with 10^4 cut-off was equal to or higher than culture method for B.forsythus and P.gingivalis. It is suggested to be a useful chair-side DNA probe kit as an aid in clinical diagnosis and evaluation of treatment efficacy.