Mgcl2

Detection of enterotoxin D gene protocol was performed by using the specific primer pairs which amplified a 294 bp fragment based on the standard strain genome. For DNA amplification, the master mix was made in 200 μL microtubes by using a 25 μL reaction mixture that contained 1 μL DNA template (50 ng/μL), 0.3 U of Taq DNA polymerase (1 μL), 2.5 μL of 10X PCR buffer, which contained 0.16 mM of each dNTPs, 2 mM MgCl₂ (all reagents were from CinnaGen Co. Tehran, Iran), 1 μL (10 µmol) of the primer pairs (synthesized by CinnaGen) and double-distilled water, to a final volume of 25 μL. All amplifications were carried out in a thermal cycler (Bio-Rad, C1000, USA) with initial denaturation at 95°C for 3 minutes followed by 35 cycles of denaturation at 94°C for 30 seconds, primer annealing at 61°C for 40 seconds and extension at 72°C for 40 seconds, followed by a final extension at 72°C for 5 minutes. The amplified PCR products were electrophoresed in 1.5% agarose gel for 45 minutes, and then stained with ethidium bromide for 20 minutes (0.05 mg/mL; Sigma Aldrich). The gels were photographed under ultraviolet light using Gel Doc (Bio-Rad Universal Hood II, USA). Also, molecular size markers (50 and 100 bp) were included in each agarose gel. Finally, all samples of buffy coat genomes were assayed by the optimization PCR.

Genomic DNA extraction was performed using the standard phenol-chloroform extraction method ^{(13 )}. Specific primers targeting insertion sequence 481 (IS481) and pertussis toxin promoter (ptxP) were used to amplify 181 base pair (bp) and 573 bp products ^{(14 (link), 15 (link))}, respectively (Table 1). Amplification of IS481 region was carried out in a total reaction volume of 20 µl containing 2 µl 10× PCR buffer (CinnaGen co., Iran), 1.5 mM MgCl₂ (CinnaGen co., Iran), 0.2 mM deoxynucleotide (CinnaGen co., Iran), 0.25 mM of each primer (Bioneer, Seoul, South Korea), 0.5 U Taq polymerase (CinnaGen co., Iran), and 10 ng DNA. The Thermocycler (Peqlab, Germany) was set with the following conditions: Initial denaturation for 5 min at 95 °C and 30 cycles including denaturation for 30 s at 95°C, annealing for 30 s at 54°C, extension for 2 min at 72°C, and final extension for 10 min at 72°C. For amplification of ptxP, in a total volume of 25 µl, the same reaction mix was used as described above, with the exceptions that Taq polymerase was increased to 1 U, 10% of dimethyl sulfoxide (CinnaGen co., Iran) was added, and annealing temperature was increased to 58°C. Electrophoresis was performed in a 1% agarose (Invitrogen, USA) gel and was stained with 0.5 µg/ml ethidium bromide (Sigma, USA).

Identification of A. baumannii was confirmed using bla_OXA-51-like PCR assay by specific primers (Gene Fanavaran, Iran) (18 (link)). To amplify the bla_OXA-51-like gene, each reaction final volume of 25 μL was considered, containing 1x PCR buffer (CinnaGen, Iran), 1 U Taq polymerase (CinnaGen, Iran), 1.5 mM Mgcl₂ (CinnaGen, Iran), 200 μM of dNTP (CinnaGen, Iran), 10 pmol of each primer and 1 μL of extracted DNA. PCR conditions were programed in an Eppendorf Mastercycler as follows: Initial denaturation at 94°C for 3 min, 35 cycles of 94°C for 45 s, 57°C for 45 s, 72°C for 1 min and 5 minute final extension of 72°C. PCR products were electrophoresed (Bio Rad, USA) on 1.2% agarose gel (CinnaGen, Iran), stained with ethidium bromide solution (1 mg/mL) (CinnaGen, Iran) and placed under UV gel transilluminator. A. baumannii reference strain ATCC 19606 was used as positive control for bla_OXA-51-like. Negative control was also included in each PCR reaction, containing all components except the DNA template which was replaced by distilled water (6 (link), 14 (link)).