The PCR amplification was performed in a Mastercycler ep cycler (Eppendorf Germany) under the following conditions:
heat denaturation at 95 C for 5 min followed by 35 cycles of heat denaturation at 95 C for 50 s, primer annealing at 54 C for 40 s and DNA extension at 72 C for 50 s. This was followed by incubation at 72 C for 7 min [18] and cooling at 4 C.