Selective and easy detection of the Porphyromonas gingivalis fimA type II and IV genes by loop-mediated isothermal amplification

Abstract

Porphyromonas gingivalis fimbrillin (fimA) type II and IV, the definitive factors for periodontitis, are also found to be associated with systemic diseases. To detect the fimA type II and IV genes easily and rapidly, we used the loop-mediated isothermal amplification (LAMP) method. The LAMP method showed high specificity as DNA from the P. gingivalis HW24D1 strain could only be amplified by the type II-specific primers and that from the W83 strain could only be amplified by the type IV-specific primers. Pathogens, namely, Streptococcus sobrinus, S. mutans, and Candida species, lack the type II and IV genes, and hence, were not detected by the LAMP reaction. Both bacterial cells and purified DNA could be used in the LAMP reaction. The LAMP reaction was highly sensitive and both type II and type IV genes could be detected in 1000 DNA molecules. In the bacterial suspensions of HW24D1 and W83 strains, type II and type IV genes, respectively, could be detected in 100 bacterial cells. We examined the type II and type IV genes in the dental plaques from 22 P. gingivalis-positive patients using the LAMP method. Only one person was found to be positive for the type II gene (4.5%). For the type IV gene, 3 positive cases (13.6%) were identified. Moreover, type II and type IV genes could be detected simultaneously using a multiplex amplification primer of fimA type II and type IV, under visible light. Thus, we established a selective and easy method to detect P. gingivalis fimA type II and IV genes using LAMP.

Keywords: Loop-mediated isothermal amplification; Porphyromonas gingivalis; fimA type II and IV.