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The study protocol conformed to the 1975 Declaration of Helsinki regulations. The procedures were approved by our ethics committee and informed, written consent was obtained from each participant. HBV serology and antibodies against hepatitis A, hepatitis C, hepatitis D and HIV were determined by ELISA using commercial test kits (Kehua Co., Shanghai, China). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured in www.selleckchem.com/products/isrib-trans-isomer.html a clinical laboratory (Modular P800; Roche, Basel, Switzerland) with commercial test kits (Olympus Diagnostica GmbH; Lismeehan, O��Callaghans��s Mills, County Clare, Ireland). Serum HBV DNA was quantified by real-time fluorimetry PCR (GmbH LightCycler1.5; Roche Diagnostics, Mannheim, Germany) using the Quantitative Hepatitis B Virus PCR Fluorogence Diagnostic Kit (PG Biotechnology, Shenzhen, China). Serum HBV DNA was extracted with a commercial kit (TIANamp, Beijing, China) according to the manufacturer��s instructions, suspended in 30?��L distilled water and 2?��L was used as template for HBV DNA amplification. First, nested PCR (Bioer XP Cycler; Bioer, Hangzhou, China) was performed using HBV A�CF genotype-specific primer pairs [19]. For the samples that were successfully genotyped, the HBV subgenotypes were further determined by multiplex CYTH4 PCR with HBV subgenotype-specific primers (for subgenotype C1, C2, B1 and B2) as described by Chen et?al. [20]. Taq DNA polymerase and the primers were purchased from MBI Fermentas (Glen Burnie, MD, USA) and Genecore Biological Technology Limited Company (Shanghai, China), respectively. The thermocycler was changed to an initial denaturation step of 5?min at 94��C, followed by 30 cycles of 94��C for 60?s, 48��C for 60?s, and 68��C for 60?s. The final elongation step was 72��C for 8?min. Amplified PCR products were electrophoresed on a 2% agarose gel, stained Metformin solubility dmso with ethidium bromide, and analysed by the Gel image system (GIS2010; Tanon, Shanghai, China). The ��2 and t-tests were used for statistical analysis, and statistical significance was assigned at p values?
