and plasmid ICall (500 plasmids per reaction). The duplex real-time PCR was performed in a Stratagene MxPro 3005 real-time cycler (Agilent Technologies, Loveland, CO, USA) starting with an initial activation of the polymerase at 95 C for 10 min, followed by 45 cycles of denaturation (95 C, 15 s) and annealing/elongation (60 C, 1 min)