The following thermal programme was applied: a single cycle of DNA polymerase activation for 10 min at 95 °C followed by 40 amplification cycles of 15 s at 95 °C (denaturing step) and 1 min at 60 °C (annealing–extension step). Subsequently, melting temperature analysis of the amplification products was performed by gradually increasing the temperature
from 60 °C to 95 °C over 20 min (±0.6 °C/20 s).