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TitleNovel strategy to detect and locate periodontal pathogens: The PNA-FISH technique
Author(s)Mendes, Luzia
Rocha, Rui
Azevedo, Andreia S.
Ferreira, Catarina
Henriques, Mariana
Pinto, Miguel Gonçalves
Azevedo, Nuno Filipe
Tissue invasion
Aggregatibacter actinomycetemcomitans
Porphyromonas gingivalis
Periodontal disease
Issue dateNov-2016
JournalMicrobiological Research
CitationMendes, Luzia; Rocha, R.; Azevedo, Andreia S.; Ferreira, Catarina; Henriques, Mariana; Pinto, Miguel Gonçalves; Azevedo, Nuno Filipe, Novel strategy to detect and locate periodontal pathogens: The PNA-FISH technique. Microbiological Research, 192, 185-191, 2016
Abstract(s)Purpose: 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 150 min 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. Conclusion 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.
Publisher version
AccessOpen access
Appears in Collections:CEB - Publicações em Revistas/Séries Internacionais / Publications in International Journals/Series

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