The polymerase chain reaction was accomplished using a gradient thermocycler (Hercuvan, Milton, UK) under the following conditions: 3 min initial denaturation at 94 ◦C, 29 cycles denaturation for 1 min at 94 ◦C, 1 min of annealing at 58 ◦C, 2 min of extension for 10 min, and final extension at 72 ◦C for 10 min with incubation at 4 ◦C. Successfully amplified DNA fragments were analyzed on 1% (w/v) agarose gel.