Color optitaq pcr master mix

The Genomic Mini AX Bacteria Kit (A&A Biotechnology, Gdansk, Poland) was used to extract genomic DNA from AB isolates following the manufacturer’s protocol. The concentration and purity of the isolated DNA was assessed using a Nano Drop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA extracted from pure cultures was stored at −20 °C for further study.
The most common carbapenemases genes in AB in Poland were detected: bla_OXA-23,bla_OXA-40, bla_OXA-58, and bla_NDM [7 (link)]. Detection of carbapenemasse genes was carried out according out following the protocol described by Cerezales et al. [29 (link)]. In the multiplex polymerase chain reaction (PCR), bla_OXA-23 (718 bp), bla_OXA-40 (413 bp), bla_NDM (517 bp), and bla_OXA-58 (303 bp) genes were identified (Table 1). PCR amplification was performed using the Color OptiTaq PCR Master Mix (EURx Ltd., Gdańsk, Poland) in a final volume of 25 μL, with a final primer concentration of 0.1 μM for each primer. Bacterial DNA functioned as the template. PCR was conducted with an initial denaturation step of 3 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 15 s at 58 °C, and 1 min at 72 °C for amplification and a final extension step of 5 min at 72 °C. PCR products were analyzed via gel electrophoresis.

Genomic DNA was extracted as described above.
Detection of biofilm-associated genes was carried out in PCR. The genes bap, csuE, ompA, bfmS, and espA were identified (Table 2). PCR amplification was performed using Color OptiTaq PCR Master Mix (EURx Ltd., Gdańsk, Poland) in a final volume of 25 μL, with a final primer concentration of 0.1 μM for each primer. Bacterial DNA functioned as a template. For genes ompA, bfmS, and espA, PCR was performed with an initial denaturation step of 5 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 55 °C, and 45 s at 72 °C for amplification and a final extension step of 5 min at 72 °C. PCR products were analyzed using gel electrophoresis. For bap and csuE, PCR was performed with an initial denaturation step of 5 min at 96 °C, followed by 35 cycles of 1 min at 96 °C, 1 min at 56.5 °C for bap, and 57 °C for csuE and 1 min at 72 °C for amplification and a final extension step of 10 min at 72 °C. PCR products were analyzed using gel electrophoresis.