PCR

PCR amplification was performed using a Promega Go Taq Green Master Mix (USA). Briefly, the PCR mix included 12.5 μL of Go Taq Green Master Mix, 4.5 μL nuclease-free water, 2 μL MgCl₂, 2 μL of every forward and reverse primers, and 2 μL of genomic DNA. A total volume of 25 μL after that was initially incubated at 94°C for 4 min. PCR was then performed with 35 cycles with 1 min for DNA denaturation at 94°C, 1 min at 50°C for primer annealing for the OMP 2 primers set, and 56°C for the B. melitensis specific primer set, and for polymerase-mediated primer extension at 72°C for 1.5 min. The last (45) cycle included sample incubation for 10 min at 72°C and the retained at 4°C for an unlimited time. Seven microliters of the amplified product were analyzed by electrophoresis in 1.5% agarose gels in TBE buffer with ethidium bromide. DNA bands were visualized under UV light and photographed using an AlphaImager (Alpha Innotech) image documentation system [30].