Reactions were performed using Applied Biosystems Veriti Thermal Cycler (Applied Biosystems, Foster City, California, USA) with cycling conditions as follows: initial cycle of denaturation at 94C for 10 min, followed by 30 cycles of denaturation at 94C for 10 s, and annealing at 58C for 45 s and extension at 72C for 45 s followed by additional 7 min at 72C and a final hold at 4C for 5 min.