Rapid detection of <I>Actinobacillus actinomycetemcomitans</I> using by Loolp-mediated Isothermal Amplification method

Actinobacillus actinomycetemcomitans has been implicated in the etiology of aggressive periodontitis. In this study, we developed a novel nucleic acid amplification method, termed loop -mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. To begin with we designed the primers for LAMP assays to detect A. actinomycetemcomitans and evaluated the specificities and sensitivities of A. actinomycetemcomitans. The specificities of the primers for A. actinomycetemcomitans were examined using chromosomal DNA of various oral bacteria. The sensitivities of A. actinomycetemcomitans were examined using agarose gel electrophoresis. Furthermore, LAMP assays were made use of for the rapid detection of A. actinomycetemcomitans in clinical specimens from 10 individuals. The LAMP primers used in this study successfully amplified the a to e serotypes of A. actinomycetemcomitans, while other oral bacteria were not amplified. The detection limits using real-time turbidimetry analysis were 10 to 106 copies for A. actinomycetemcomitans template DNA in one reaction tube. Also, the results for the LAMP method closely agree with those using conventional PCR. Our results suggest that LAMP-based assay is very useful for the rapid detection of A. actinomycetemcomitans and the diagnosis of aggressive periodontitis.