Plasmid Replicon Typing by PBRT

Bacterial DNA was obtained by the boiling lysis method, incubating the isolated colonies in distilled water for 10 min at 100 °C. The samples were then centrifuged at 15,000× g for 5 min and the supernatants were used for the following reactions.
All strains were typed by PCR-based replicon typing (PBRT) using the PBRT kit 2.0 (Diatheva, Fano, Italy) in order to identify plasmid replicons. This PBRT assay consists of eight multiplex PCRs and allows the detection of 30 replicons of the main plasmids in Enterobacterales.
All PCR reactions were carried out in accordance with the manufacturer’s instructions, including positive controls. The amplicons were detected through capillary electrophoresis on the AATI Fragment Analyzer (Agilent, Santa Clara, CA, USA) using the dsDNA 906 Reagent kit (Advanced Analytical, Ankeny, IA, USA). This amplicon analysis allows the combination of two multiplex PCRs in the same lane, resolving up to eight peaks, as previously published [58 (link)]. The positive peaks were successively analysed using the tool “PBRT plugin” [58 (link)] developed in cooperation with the Advanced Analytical Company that allows automatic peak calling and the recording of positive replicons.