Extracellular HBV DNA Quantitative Analysis by Combining Immunoprecipitation With qPCR

HepG2 cells (5 × 10⁴/well in 24-well plate) were transfected with 500 ng plasmid C, D or their mutants. Three days after transfection, virions were immunoprecipitation from 0.3 ml of culture media by 1 μl horse anti-HBs antibody (Abcam) conjugated to 10 μl bed volume of Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) at 4°C overnight. The immunoprecipitation was sequentially treated at 37°C with 2 μl Turbo DNase (Thermo Fisher Scientific) in manufacturer provided buffer for 1 h and 500 μl digestion buffer [25 mM Tris–HCl (pH 8.0), 0.5 mg/ml pronase (Merck KGaA), 0.5% SDS, 150 mM NaCl, and 10 mM EDTA] for 1 h, followed by DNA extraction with phenol. The extracted DNA was subjected to SYBR Green quantitative PCR using forward primer 5′-GACCACCAAATGCCCCTATC (2298–2317, numbering according to GTC) and reverse primer 5′-TGAGATCTTCTGCGACGCG (2412–2430, numbering according to GTC). The quantification range is 2.5 × 10³ to 2.5 × 10¹¹ copies/ml.