The PCR amplification was carried out in the thermo cycler PCR (Santa Clara, California, United States) according to the following programme: initial denaturation at 95 ◦C for 5 min, amplification for 30 cycles [95 ◦C/40 s (denaturation), 55 ◦C/40 s (annealing), 72 ◦C/1 min (extension)], then final extension at 72 ◦C for 10 min.