Table III.

Reaction parameters and systems used for the amplification of HBV Pre-S/S genes by nested PCR.

Reaction step	Amplification parameters	Reaction system
First step	95°C denaturation for 3 min followed by 5 cycles at 95°C for 30 sec. The Ta was then reduced from 57°C to 53°C over 4 cycles with a reduction of 1°C per cycle. The annealing time was 30 sec and extension was conducted at 72°C for 30 sec. Subsequently, 30 cycles were conducted at 95°C for 30 sec, Ta 53°C for 30 sec, 72°C for 30 sec, and a final extension step was performed at 72°C for 2 min.	Each 25-μl reaction tube contained 5 μl 5X KAPA2G buffer A, 5 μl 5X KAPA enhancer, 0.1 μl KAPA2G Robust HotStart DNA polymerase (Kapa Biosystems, Inc., Wilmington, MA, USA), 0.5 μl 10 μM dNTP Mix (Takara Bio, Inc., Otsu, Japan), l μl each 10 M primer, and 3 μl DNA template. The total volume was brought to 25 μl with PCR-specific water.
Second step	The same as the first step.	The same as the first step. The amplified DNA product obtained from the first step was used as the DNA template for the second step.

PCR, polymerase chain reaction; Ta, annealing temperature.