Table 1.

Amplification system of Pol gene.

	Amplification stage
	RT-PCR (25 μL)	Nest-PCR (50 μL)
Reaction conditions	50°C, 30 min	94°C, 2 min
	94°C, 3 min	54°C, 1 min
	35 cycles (94°C, 30 s; 54°C, 30 s; 72°C, 1 min 30 s)	72°C, 1 min 30 s
	72°C, 10 min	30 cycles ( 94°C, 30 s; 54°C, 45 s; 72°C, 45 s)
	4°C, Constant preservation	72°C, 10 min
		4°C, Constant preservation
Reagent (volume: μL)	2 × One step mix (12.5)	2 × Taq Plus Master Mix II (Dye Plus) (25)
	RT-21 (1)	RT-20 (2)
	MAW26 (1)	Pro-1 (2)
	RNase-free ddH2O (4.25)	RNase-free ddH2O (16)
	One Step Enzyme Mix (1.25)	Template DNA (5)
	Template RNA (5)
Primers	MAW26 (TTGGAAATGTGGAAAGGAAGGAC, HXB2: 2028-2050)	Pro-1 (CAGAGCCAACAGCCCCACCA, HXB2: 2147-2166)
	RT21 (CTGTATTTCTGCTATTAAGTCTTTTGATGGG, HXB2: 3509-3539)	RT20 (CTGCCAGTTCTATGCTTC, HXB2: 3441-3462)