Molecular Detection of Antibiotic Resistance Genes

The presence of AmpC genes (bla_CMY-2, bla_DHA-1, and bla_ACC) [18 (link)] and MBLs genes (bla_IMP, bla_VIM, and bla_NDM) [19 (link)] was detected by polymerase chain reaction (PCR). Genomic DNA extraction was performed using the boiling method [20 ]. The PCR was performed in a final volume of 25 μl consisting of DNA template (50 ng), dNTPs (100 μM), Taq buffer (5×), Taq DNA polymerase (1 U; Cinnagen, Iran), and forward and reverse primers (25 pM each). The PCR mixtures were subjected to thermal cycling. PCR reactions included 30 amplification cycles in a Mastercycler (Eppendorf, Germany) under the following conditions: denaturation at 95 °C/5 min, annealing at 55 °C/30 s, and extension at 72 °C/45 s, with a final extension at 72 °C/6 min. Amplified products were visualized using electrophoresis on a 1% agarose gel stained with safe stain (Sinaclon, Iran), in a Tris-Borate-EDTA buffer (Promega, USA). Water was used as a negative control in the study, and the positive controls were K.pneumoniae ATCC 700603, P.aeruginosa ATCC 27853, P.aeruginosa ST 147, K.pneumoniae KP696465, E.coli KX 342010 and E.coli KX342011.