Nexus gradient thermocycler

Each polymerase chain reaction (PCR) was conducted in 10 μL reaction volume containing 1.0 μL 10x reaction buffer (10 mM) (MgCl₂ included), 0.2 μl dNTPs (200 μM), 0.2 μL Taq polymerase (1 U/L), 1 μL of each primer (5 pM), 1 1 μLof template DNA (10 ng), and 5.6 μL of ddH₂O. The following temperature profiles and cycles were maintained using Eppendorf Nexus Gradient Thermocycler: 1 cycle at 94°C for 4 min, followed by 35 cycles of at 94°C for 45 s, annealing at 55°C or 61°C (depending on primers) for 45 s, 1 cycle at 72°C for 1 min, a final extension at 72°C for 10 min, and storage at 10°C. A list of primers is given in Table 2.

All PCR reactions were carried out with a HotStarTaq Plus Master Mix Kit (Qiagen) according to the manufacturer's protocols in a nexus gradient thermocycler (Eppendorf). The following genes were selected for the antimicrobial resistance tests: sul1, sul2, sul3, tet(A), tet(B), tet(G), bla_TEM‐1, floR, cat1, cat2, aac6, strA, and strB. In case of virulence, the investigated genes were: lpf, sivH, invA, agaF, and avaR. The list of investigated genes, primer sequences, product size, and annealing temperature are summarized in Table S2. All primers were synthesized by Genomed S.A. (Poland). Ten microlitres of PCR products were electrophoresed on a 2% agarose gel in the presence of Midori Green Advance (Nippon Genetics, Germany), at 120 V for 60 min. The results were read using the Quantum ST5 Gel Documentation System (Vilber). To confirm the specificity of the amplicons obtained, some PCR products of interest were randomly selected and purified using a CleanUp kit (A&A Biotechnology) for sequencing (Genomed).