A sensitive method for detecting Porphyromonas gingivalis by polymerase chain reaction and its possible clinical application.
BACKGROUND
It is useful for the clinical diagnosis of periodontitis to monitor the colonization of periodontopathic bacteria in periodontal pockets. In this study, we attempted to establish and possibly identify the clinical application of a sensitive method to detect Porphyromonas gingivalis (P.g.), one of the putative periodontopathic bacteria related to chronic periodontitis.


METHODS
Genomic DNA extracted from cultured P.g. 381 and clinically isolated subgingival plaque samples were used as a template of polymerase chain reaction (PCR). We designed primers to amplify the genomic DNA coding 40 kDa outer membrane protein (OMP), one of the unique proteins to all strains of P.g. The efficiency and specificity of amplification were evaluated by agarose gel electrophoresis and subsequent Southern hybridization with a digoxygenin-labeled oligonucleotide probe.


RESULTS
Fewer than 100 P.g. bacterial cells in the specimen were reproducibly detected by PCR-hybridization assay. This PCR-hybridization assay was at least 100 times more sensitive than the conventional indirect immunofluorescence assay (IIF). Furthermore, the imaging analysis showed that there is a linear correlation between the strength of the signal and the cell number of P.g. from which the template DNA was extracted semiquantitatively. It is noteworthy that the PCR assay could also be applied to detect P.g. from clinical plaque samples and that it was approximately 100 times more sensitive than a conventional IIF assay.


CONCLUSIONS
The PCR assay established in this study can be a powerful tool to detect P.g. in periodontal pockets and monitor the colonization and/or recolonization of P.g. at the very early phase.
Nozaki
T
T
Department of Periodontology, Osaka University Graduate School of Dentistry, Suita, Japan. tnozaki@dent.osaka-u.ac.jp
Kusumoto
Y
Y
Kitamura
M
M
Hirano
H
H
Kohyama
A
A
Hayakawa
M
M
Takiguchi
H
H
Abiko
Y
Y
Murakami
S
S
Okada
H
H
D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000235:genetics; D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000032:analysis; D003773:Dental Plaque / Q000382:microbiology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D010514:Periodontal Pocket / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D016966:Porphyromonas gingivalis / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D018709:Statistics, Nonparametric
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial