Introduction of RNAscope method for detection of selected bacteria associated with apical periodontitis
| Autoři | |
|---|---|
| Rok publikování | 2023 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | Apical periodontitis (AP) is an inflammation of periapical tissues developing in response to bacterial infection in the root canal system. The anaerobic bacteria associated with AP include, for example,?Fusobacterium nucleatum?and?Porphyromonas endodontalis,?which are predominantly located in the apical part of the root canal.?Streptococcus mutans, a facultative anaerobe, is another bacterium associated with AP. Here, we designed and tested the RNAscope method for simultaneous detection of these three bacterial strains are associated with AP.? In this methodological study, these three bacteria were cultivated and then used as positive controls for the assay specificity verification. In addition,?Enterococcus?sp. bacteria were cultivated and?used as a negative control to confirm that the probe is specific for the target rRNA. The design of?in situ?hybridization assays was based on the knowledge of 16S rRNA sequences of selected bacterial strains. We prepared formalin-fixed paraffin-embedded (FFPE) low-gelling agarose blocks with?F. nuclatum,?P. endodontalis,?S. mutans,?and?Enterococcus sp.. These blocks were sectioned and RNAscope analysis was performed according to a modified protocol with probes specific to each of the embedded bacterial species. Initially, different times for boiling slides (15, 30, and 45 minutes) as well as for incubation with protease (10 and 30 minutes) were tested. Boiling times of slides were optimized with the aim to ensure sufficient disruption of the bacterial cell wall to enable the RNAscope reagents to bind to the target rRNA, while also making sure that the analyzed slides was not severely damaged during these steps. Three fluorescent assays were designed for visualising individual mRNA targets, commercially synthetized, and tested. All three selected bacteria were specifically detected. Based on the intensity of the detected fluorescent signal, 45 minutes for slide boiling followed by 30 minutes of their incubation with protease were used in the final protocol. These modified treatment parameters are suitable for use with human dental samples. In this work, we introduced and optimized the RNAscope method for the detection of?F. nuclatum,?P. endodontalis, and?S. mutans,?and showed the specificity of each assay for the selected bacteria. |
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