PCR reactions contained 12.5 μL of GoTaq Green Master Mix 2× (Promega), 1 μL of the primer at 50 pmol, 2 μL of DNA and DNA-free water (Promega) until a final volume of 25 μL. PCR amplification conditions were: initial step at 95 °C for 30 s, 30 cycles of annealing at 40 °C for 30 s and 65 °C for 8 min, and a final extension at 65 °C for 16 min