Component name		Final conc	First round of PCR (μl) (n = 1)	Second round of PCR (μl) (n = 1)
PCR Buffer with 15 mM MgCl2	10x	1x	3	3
Q-Solution	5x	1x	6	6
MgCl2	25 mM	1 mM	1.2	1.2
HotStarTaq Plus DNA Polymerase	5 u/μl	2 units/reaction	0.4	0.4
dNTP	2 mM of each	0.2 mM of each	3	3
WNV f1 1330	18 μM	600 nM	1	–
WNV r1 2444	18 μM	600 nM	1	–
WNV f2 1400	18 μM	600 nM	–	1
WNV r2 2305	18 μM	600 nM	–	1
cDNA			5	–
PCR product of the first stage			–	3
RNase-free water			9.4	11.4