Novel Quantitative Detection Method for Aggregatibacter actinomycetemcomitans JP2

Objectives: Aggregatibacter actinomycetemcomitans strain JP2 is one of the etiological agents of aggressive periodontitis. A unique 530-bp deletion in the promoter region of the lkt operon results in the enhanced production of leukotoxin, which accounts for the virulence of this strain. Quantitative detection of strain JP2 is essential for the diagnosis of aggressive periodontitis, but thus far has been limited due to technical difficulties. This study, however, describes a specific and quantitative method for the detection of A. actinomycetemcomitans strain JP2. Method: Based on the results of a search of a defined genomic locus in A. actinomycetemcomitans strain JP2, real-time PCR primers specific for the bacterium were designed. The specificities of the primers were confirmed by conventional PCR using genomic DNA obtained from various strains of A. actinomycetemcomitans and by melting-curve analysis. In addition, a Ct-CFU graph was constructed from the relationship between the amount of genomic DNA and bacterial numbers. Results: This novel A. actinomycetemcomitans JP2 detection system was confirmed to be specific for strain JP2, identifying the clone from among various oral bacteria, including other strains of A. actinomycetemcomitans. The quantitative detection range was 6.06–6.06×10⁶ cells, corresponding to 15.8 pg–15.8 µg of genomic DNA, per sample. In addition, dental plaque negative for A. actinomycetemcomitans JP2 did not inhibit the PCR-based detection system. Conclusions: The specificity of this novel quantification system for A. actinomycetemcomitans strain JP2 is currently being confirmed in tests of subgingival plaque material obtained from patients with strain JP2-infected aggressive periodontitis.