HBV cccDNA (double-stranded DNA without nick and gap) was quantified by real-time PCR using a protocol as previously described Werle-Lapostolle et al., 2004) with modifications. In particular, specific primers for cccDNA detection reported by Glebe et al. (2003) (ccc-1582F: 5′-TGCACTTCGCTTCACCT-3′; ccc-2316R: 5′-AGGGGCATTTGGTGGTC-3′) were validated and used for quantifying copy numbers of cccDNA using real-time PCR. Viral DNAs other than cccDNA, including single-stranded and relaxed circular DNAs, were degraded prior to amplification by treatment of the DNA templates with plasmid-safe adenosine triphosphate (ATP)dependent deoxyribonuclease DNase (Epicentre Technologies). In brief, HBV-infected cells were lysed for 4 hr at 65°C in lysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM EDTA, 100 mM NaCl, 1% SDS) supplemented with proteinase K (200 μg/ml) and followed by phenol-chloroform extraction. A total of 250 ng of the extracted DNA was digested with 5-10 units plasmid-safe DNase in a 50 μl volume for 8 hr at 37°C followed by DNase inactivation at 70°C for 30 min. 2 μl of the 50 μl reaction was then added to 20 μl of a real-time PCR reaction. Amplification of 735 bp cccDNA product was conducted by denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 30 s, 62°C annealing for 25 s, and 72°C elongation for 45 s. HBV cccDNA copy numbers were calculated with a standard curve from plasmid with known nucleic acid quantities. The detection limit for cccDNA is ∼10 copies cccDNA per reaction (equivalent to 10 ng of total cell lysate DNA). Realtime PCR was performed with SYBR Premix Ex Taq kit on an ABI Fast 7500 real-time system instrument.