Dsdna 906 reagent kit

Plasmid characterization was performed by PBRT [10 (link)] using the PBRT kit 2.0 (Diatheva, Fano, Italy). This system, consisting of eight multiplex PCR assays, allows the identification of the following 30 replicons found in the Enterobacteriaceae family: HI1, HI2, I1, I2, X1, X2, X3, X4, L, M, N, FIA, FIB, FIC, FII, FIIS, FIIK, FIB KN, FIB KQ, W, Y, P1, A/C, T, K, U, R, B/O, HIB-M and FIB-M. All PCR reactions were performed according to 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. One µL of multiplex PCR 1 (M1) was combined with 1 µL of multiplex PCR 3 (M3), followed by M2 and M7, M6 and M8; the remaining M4 and M5 were loaded separately (2 µL each). The positive peaks were analyzed using the “PBRT plugin” developed in cooperation with the Advanced Analytical Company. This tool allows automatic peak calling and the recording of positive replicons.

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.