SIMULTANEOUS DETECTION OF ACTINOBACILLUS-ACTINOMYCETEMCOMITANS AND PORPHYROMONAS-GINGIVALIS BY A RAPID PCR METHOD

The identification of periodontal pathogens by conventional methods is time-consuming and difficult. Therefore, a multiplex PCR method for s imultaneous detection of Actinobacillus actinomycetemcomitans (A.a.) a nd Porphyromonas gingivalis (P.g.) was developed for rapid and easy de termination of these risk-indicator bacteria in human periodontal dise ase. The PCR primers were designed to hybridize to various regions of 16S rRNA genes, and a hot-start technique was used to obtain maximum s ensitivity and specificity. This method can detect both of these bacte ria in subgingival plaque samples at concentrations as low as 5 to 50 cells per sample. The sensitivity, however, was even 10 times better w hen the bacteria were analyzed in a water suspension. Since the only s tep between sample collection and the actual analysis is a brief centr ifugation of the patient sample, the detection can be readily carried out in four hours. The performance of the method was studied with 36 p atient samples. The results showed that the PCR method detected A.a. ( 44% vs. 25%, respectively) and P.g. (56% vs. 42%, respectively) more o ften than the conventional culture in plaque samples. Thus, our multip lex PCR method is rapid and more effective than conventional protocols in detecting these periodontal pathogens.