Son-Jin Choe

Use of Insertion Sequence Element IS1126 in a Genotyping and Transmission Study of Porphyromonas gingivalis

ABSTRACT Porphyromonas gingivalis is strongly associated with periodontal diseases and is regarded as one of the risk factors for periodontitis. Insertion sequence element IS1126-based PCR was used to investigate the genetic heterogeneity of P. gingivalis from periodontitis patients and to examine the frequency of the parent-child and spouse-spouse transmission. Two sets of IS1126-specific primers were used for the PCR. The inward primer set (PI1 and PI2), which amplifies the IS1126 fragment of approximately 690 bp, was used to identify P. gingivalis. The outward primer set (PI1RC and PI2RC), which is reverse complementary to PI1 and PI2, respectively, and amplifies the gene fragments between the adjacent IS1126 elements was used to characterize the genotypes of the P. gingivalis strains. PCR of P. gingivalis with PI1RC and PI2RC resulted in the production of two to seven amplicons, which showed a unique electrophoretic pattern in each strain (4 laboratory strains and 37 clinical isolates cultured from 12 patients with aggressive periodontitis). The usefulness of the method for transmission study was confirmed by detecting identical genotypes between the isolates and the plaque samples from which the isolates were cultured and between the plaque samples from different tooth sites in the same patient. Thirty probands with periodontal diseases and their thirty immediate family members were included in the transmission study. In 11 of 14 parent-child pairs (78.6%), P. gingivalis revealed an identical or similar band pattern, whereas 5 of 16 spouse pairs (31.25%) had this similarity. These results show that IS1126-based PCR for genotyping P. gingivalis has a highly discriminating potential with reproducible data and is a simple and reliable method for a transmission study.

A new method for rapid screening of bacterial species- or subspecies-specific DNA probes

A simple assay for the rapid screening of bacterial species- or subspecies-specific DNA probes for the random cloning method is presented, involving the use of genomic DNAs as probes and recombinant plasmid DNAs containing genomic DNA digested with HindIII as targets. The optimal amount of target DNAs and the concentration of digoxigenin-labeled genomic DNA probes were 20 ng and 100 ng ml(-1) (or 10 ng and 200 ng ml(-1)), respectively. The method was applied to the development of Fusobacterium nucleatum subspecies-specific probes. Our results showed that four out of 96 probes were F. nucleatum subspecies-specific, which was confirmed by Southern blot analysis. Our results indicate that the new method can be used for the rapid screening of species- or subspecies-specific probes.