Detection of Putative Periodontal Pathogens in Subgingival Specimens by 16S Ribosomal DNA Amplification with the Polymerase Chain Reaction

Abstract

The utility of the 16S ribosomal RNA-based polymerase chain reaction (PCR) for detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola was examined and compared with that of anaerobic culture. Primer pairs consisting of 20-27 nucleotides amplified 404- to 688-bp regions of 16S ribosomal RNA genes of these organisms. This method had a lower detection limit of 50 target cells in a background of 10(7) cells. Its specificity for B. forsythus, P. gingivalis, and T. denticola seemed high. The primers for A. actinomycetemcomitans, C. rectus, and P. intermedia cross-reacted with some closely related species but did not reveal amplification products in tests with more distantly related organisms. The primers for E. corrodens did not seem to cross-react with oral organisms. This PCR technique was sensitive, reproducible, and easy to perform. PCR-based amplification may prove valuable for the detection of some periodontal pathogens in crude subgingival specimens.

PCR detection of four periodontopathogens from subgingival clinical samples

“…Organisms such as Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia or Eikenella corrodens have been considered to have a role in periodontal disease (9,11). Studies have demonstrated a strong correlation between the presence of putative periodontal organisms and the destruction of periodontal tissues (10).…”

“…Polymerase chain reaction (PCR) has been used for direct identification of periodontal pathogens in subgingival specimens (3,11) and also for elucidating the role of specific bacteria in the periodontal disease because of the ability to accurately detect species in mixed populations. 16S rRNA gene amplification has been used for detection of microorganisms that cannot be cultivated but these methods have given crossreactions with related organisms (11).…”

“…16S rRNA gene amplification has been used for detection of microorganisms that cannot be cultivated but these methods have given crossreactions with related organisms (11).…”

Prevalence of periodontopathogens and Candida spp. in smokers after nonsurgical periodontal therapy – a pilot study

“…The DNA was resuspended in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5, with 10 μg/mL RNAse). Molecular microbial identification was carried out by PCR with specific primers for Aggregatibacter actinomycetemcomitans, 25 Porphyromonas gingivalis, 26 Tannerella forsythia, 27 Candida albicans, 24 Candida glabrata, 24 Candida tropicalis, 24 and Candida dubliniensis. 28 PCR amplification was performed with a thermocycler AmpliTherm TX96 Gradient (Axygen, NY, USA) under thermal conditions specific for each pair of primers.…”

Transfer of Bacteroides splanchnicus to Odoribacter gen. nov. as Odoribacter splanchnicus comb. nov., and description of Odoribacter denticanis sp. nov., isolated from the crevicular spaces of canine periodontitis patients

“…The 16S rRNA gene from strain B106 T was PCR amplified in triplicate using the primers D134 (59-GAGTTTGAT-CCTGGCTCAGG-39) and D57 (Slots et al, 1995) (Invitrogen). The PCR products were pooled, purified and desalted, and subjected to direct DNA sequence analysis.…”