Table 3.

PCR Thermocycling Conditions

PCR type	Step	Conditions
Multiplex-PCR	Initial denaturation	98°C for 30 s
	Thermocycling: 35 cycles	98°C for 10 s
		55°C for 20 s
		72°C for 20 s
	Extension	72°C for 15 s
Nested conventional PCR	Initial denaturation	98°C for 30 s
	Thermocycling: 35 cycles	98°C for 10 s
		Ta⁎ for 30 s
		72°C for 10 s
Nested fast-COLD-PCR	Initial denaturation	98°C for 30 s
	Stage 1 cycling: 5 cycles	98°C for 10 s
		Ta⁎ for 20 s, fluorescent reading
		72°C for 10 s
	Stage 2 cycling: 20 cycles	Tc⁎ for 10 s
		Ta⁎ for 20 s, fluorescent reading
		72°C for 10 s
	Stage 3 cycling: 10 cycles	98°C for 10 s
		Ta⁎ for 20 s, fluorescent reading
		72°C for 10 s
	Melting curve	Ramping 0.2°C/s, 60°C to 90°C

^⁎Annealing temperatures and critical denaturation temperatures are defined in Table 2.