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Selective and easy detection of the Porphyromonas gingivalis fimA type II and IV genes by loop-mediated isothermal
Masae Kitagawa1, Kazuhisa Ouhara2, Hiroko Oka3
1Center of Oral Clinical Examination, Hiroshima University Hospital, Hiroshima, Japan.
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.
Insights
A new Loop-Mediated Isothermal Amplification (LAMP) method rapidly detects Porphyromonas gingivalis fimbrillin (fimA) types II and IV. This sensitive and specific technique aids in identifying periodontitis-associated bacteria in clinical samples.
Area of Science:
- Microbiology
- Molecular Biology
- Dental Research
Background:
- Porphyromonas gingivalis fimbrillin (fimA) types II and IV are key factors in periodontitis and linked to systemic diseases.
- Accurate and rapid detection of these specific fimA types is crucial for diagnosis and management.
Purpose of the Study:
- To develop and validate a rapid, selective, and sensitive Loop-Mediated Isothermal Amplification (LAMP) method for detecting Porphyromonas gingivalis fimA types II and IV.
- To assess the prevalence of fimA types II and IV in dental plaque samples from periodontitis patients.
Main Methods:
- Utilized Loop-Mediated Isothermal Amplification (LAMP) with specific primers for fimA types II and IV.
- Evaluated specificity against related oral pathogens and sensitivity using known DNA concentrations and bacterial cell counts.
- Applied the LAMP method to dental plaque samples from periodontitis patients.
Main Results:
- The LAMP method demonstrated high specificity, amplifying only target fimA types and not other bacterial DNA.
- Sensitivity was high, detecting as few as 1000 DNA molecules or 100 bacterial cells.
- In dental plaque samples, fimA type II was detected in 4.5% and type IV in 13.6% of patients.
- Simultaneous detection of both fimA types II and IV was achieved using multiplex primers.
Conclusions:
- A selective, rapid, and sensitive LAMP method for detecting Porphyromonas gingivalis fimA types II and IV has been established.
- This LAMP assay is suitable for clinical applications, including the analysis of dental plaque.
- The method allows for simultaneous detection of both fimA types, enhancing diagnostic efficiency.
