Novel strategy to detect and locate periodontal pathogens: The PNA-FISH technique.
OBJECTIVE
We aim to develop peptic nucleic acid (PNA) probes for the identification and localization of Aggregatibacter actinomycetemcomintans and Porphyromonas gingivalis in sub-gingival plaque and gingival biopsies by Fluorescence in situ Hybridization (FISH).


METHODS
A PNA probe was designed for each microorganism. The PNA-FISH method was optimized to allow simultaneous hybridization of both microorganisms with their probe (PNA-FISH multiplex). After being tested on representative strains of P. gingivalis and A. actinomycetemcomitans, the PNA-FISH method was then adapted to detect microorganisms in the subgingival plaque and gingival samples, collected from patients with severe periodontitis.


RESULTS
The best hybridization conditions were found to be 59°C for 150min for both probes (PgPNA1007 and AaPNA235). The in silico sensitivity and specificity was both 100% for PgPNA1007 probe and 100% and 99.9% for AaPNA235 probe, respectively. Results on clinical samples showed that the PNA-FISH method was able to detect and discriminate target bacteria in the mixed microbial population of the subgingival plaque and within periodontal tissues.


CONCLUSIONS
This investigation presents a new highly accurate method for P. gingivalis and A. actinomycetemcomitans detection and co-location in clinical samples, in just few hours. With this technique we were able to observe spatial distribution of these species within polymicrobial communities in the periodontal pockets and, for the first time with the FISH method, in the organized gingival tissue.
Department of Periodontology, Faculty of Dental Medicine, University of Porto, Porto, Portugal. Electronic address: lgoncalves@fmd.up.pt.
Luzia
L
Mendes
LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal; LIBRO, Laboratório de Investigação em Biofilmes Rosário Oliveira, University of Minho, Braga, Portugal; BIOMODE, Zona Industrial da Gandra, Apartado 4152, 4806-909 Guimarães, Portugal.
Rui
R
Rocha
LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal.
Andreia Sofia
AS
Azevedo
Department of Periodontology, Faculty of Dental Medicine, University of Porto, Porto, Portugal.
Catarina
C
Ferreira
LIBRO, Laboratório de Investigação em Biofilmes Rosário Oliveira, University of Minho, Braga, Portugal.
Mariana
M
Henriques
Department of Periodontology, Faculty of Dental Medicine, University of Porto, Porto, Portugal.
Miguel Gonçalves
MG
Pinto
LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal.
Nuno Filipe
NF
Azevedo
D001706:Biopsy; D003773:Dental Plaque / Q000382:microbiology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods / Q000592:standards; D020135:Peptide Nucleic Acids; D010510:Periodontal Diseases / Q000175:diagnosis* / Q000382:microbiology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D020135:Peptide Nucleic Acids
Aggregatibacter actinomycetemcomitans; PNA-FISH; Periodontal disease; Porphyromonas gingivalis; Tissue invasion