Novel strategy to detect and locate periodontal pathogens: The PNA-FISH technique

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• 作者：Luzia Mendes ; PhD DMD^a ; ^{lgoncalves@fmd.up.pt" class="auth_mail" title="E-mail the corresponding author} ; Rui Rocha ; MsD^b ; ^c ; ^d ; Andreia Sofia Azevedo ; PhD^b ; Catarina Ferreira ; DMD^a ; Mariana Henriques ; PhD^c ; Miguel Gonç ; alves Pinto ; PhD DMD^a ; Nuno Filipe Azevedo ; PhD^b
• 关键词：PNA-FISH ; Tissue invasion ; Aggregatibacter actinomycetemcomitans ; Porphyromonas gingivalis ; Periodontal disease
• 刊名：Microbiological Research
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：192
• 期：Complete
• 页码：185-191
• 全文大小：1427 K

文摘

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.