Table 1. Nested PCR protocol 1, 2, 3, 4

	Protocol 1		Protocol 2		Protocol 3		Protocol 4
	Outer	Inner	Outer	Inner	Outer	Inner	Outer	Inner
Buffer qiagen (10X)	2.5	2.5	2.5	2.5			1.5	1.5
5X Q5 reaction buffer					5	5
Q5 hot start DNA polymerase					0.25	0.25
dNTPs [10 mM]	0.5	0.5	[25 mM] 1.25	[25 mM] 1.25	0.5	0.5	0.6	0.25
MgCl2 [25 mM]	0.8	0.8	0.25	0.25			0.3	0.25
Tag polymerase	0.2	0.2	0.2	0.2			0.15	0.15
HEV38/HEV39 [10 μM]	0.5		0.5		1		0.6
HEV37/HEV27 [10 μM]		0.5		0.5		1		0.9
Nuclease-free H₂O	15	15	14.8	14.8	12.5	12.5	9.25	9.05
cDNA	5	5	5	5	5	5	2	2
Total volume (µL)	25	25	25	25	20	20	15	15

Nested PCR, nested polymerase chain reaction; HEV, hepatitis E virus; cDNA, complementary DNA.