100 bp size marker

DNA extraction was carried out according to a previous study with some modifications (Queipo-ortuno et al., 1997 (link)). Briefly, 0.5 ml of distilled water with 1 loop of bacteria were mixed and centrifuged at 13,000 g for 1 min and the supernatant was removed. Then, 100 µl of 50 mM NAOH was added and put in the water bath at 95 °C for 30 min. Finally 50 µl of 20 mM Tris buffer was added and centrifuged and the DNA obtained was used for PCR.
The omp2a gene was detected by PCR using Forward-5′-GGCTATTCAAAATTCTGGCG-3′ and Riverse-5′-ATCGATTCTCACGCTTTCGT-3′ specific primer pairs (Bahmani et al., 2016 ). The PCR amplification procedure was performed with 25 µl of master mix containing 0.2 µl of Taq polymerase 5 U/µl, 2.5 µl of 10X PCR buffer along with MgCl2, 1 µl of 10 pM from each primers, 2.5 µl of dNTPs MIX (2 Mm), 3 µl of DNA template, 14.8 µl of DNase and RNase-Free Distilled Water. The oligonucleotides and all reagents for PCRs were synthesized and purchased from Bioneer Co (South Korea). Agarose gel electrophoresis of the PCR product with 100 bp size marker (Fermentas, Korea) was carried out in a 2% agarose gel and stained with syber safe and visualized under UV transilluminator. (Al Dahouk et al., 2005 ). The B. melitensis (biovar 1, ATCC 23456) and distilled water were used as positive and negative controls, respectively.

The specific primers (Bioneer® Korea) including CTX-M, SHV and TEM (Table 1) were used for PCR amplification of the genes (Ellington et al., 2007; Kalai Blagui et al., 2007; Kolar et al., 2010; Turton et al., 2006 ). PCR amplification procedure was performed with 25 μl of master mix containing 0.2 μl of Taq polymerase 5 U/μl, 2.5 μl of 10× PCR buffer along with MgCl₂, 1 μl of 10 pM from each reverse and forward primers, 2.5 μl of dNTPs MIX (2 Mm), 3 μl of DNA template,14.8 μl of DNase-Free and RNase-Free Distilled Water. PCR amplification was done in the thermal cycler device. Agarose gel electrophoresis of the amplified DNA product with 100 bp size marker (Fermentas®, Korea) was carried out in a 2% agarose gel for 2 h at 80 V and stained with ethidium bromide.

The bacterial DNA was extracted by the alkaline lysis method (Kheyrodin and Ghazvinian, 2012 ). The pair of bla_OXA-51 primers (Bioneer® Korea); OXA-F 5′-TAATGCTTTGATCGGCCTTG-3′, and OXA-R 5′-TGGATTGCACTTCATCTTGG-3′ were used for PCR detection of the genes (Turton et al., 2006 (link)). PCR amplification procedure was performed using 25 μl of master mix containing 0.2 μl of Taq polymerase 5 U/μl, 2.5 μl of 10XPCR buffer along with MgCl2, 1 μl of 10 pM from each reverse and forward primers, 2.5 μl of dNTPs MIX (2 Mm), 3 μl of DNA template, 14.8 μl of DNase-Free and RNase-Free Distilled Water. PCR amplification was done in the thermal cycler device. Agarose gel electrophoresis of the amplified DNA product with 100 bp size marker (Fermentas®, Korea) was carried out in a 2% agarose gel for 2 h at 80 V and stained with ethidium bromide to manifest and detect the 353 bp band. Also, PCR reaction contained positive and negative control. The A. baumannii carrying bla_OXA-51-like gene (obtained from Dr. Bahador, Tehran University of Medical Sciences, Tehran, Iran) was used as the positive control.