Extraction of Viral DNA and Genome-Length PCR

HBV DNA was extracted from 200 μl of serum samples from patients with RA CHB using QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. We performed semi-nested PCR to amplify four partially overlapping DNA fragments that encompass the complete HBV genome sequence using HotStarTaq plus DNA polymerase (QIAGEN, Hilden, Germany), primers, and conditions as previously described. The PCR products were then sequenced bidirectionally with second-round primers using an Applied Biosystems 3730XL (Applied Biosystems, CA, USA). The full-length HBV genomic DNA sequence was amplified by an Applied Biosystems 2720 thermal cycler (Applied Biosystems, CA, USA) in a 25 μl volume containing 1 μl of HBV DNA template, 0.5 μl of Taq DNA polymerase (5 U/μl), 2.5 μl of 10x Taq buffer, 2 μl of 25 mM MgCl₂, 0.5 μl of dNTPs (10 mM each), and 1 μl of each primer. The primers were designed according to the corresponding reference (9). The total genomes of 28 viral strains from the 28 patients were sequenced.