Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis

Both the viral titer and the genotype significantly determine clinical outcomes and responses to antiviral treatment in chronic hepatitis B virus (HBV) infection. A method was developed for large-scale A-to-G genotyping with simultaneous viral quantification. The assay was run on a LightCycler instrument using hybridization probes. The genotype was determined from the melting points of the probes in a two-step manner. Set 1 amplicons differentiated genotypes B, E, and F from A, C, D, and G and simultaneously quantified viremia by real-time PCR. Melting curve analysis using the set 2-1 amplicon or the set 2-2 amplicon reaction mixture was then used to differentiate these genotype groups into single genotypes. HBV DNA quantification was consistent with that of the Amplicor assay and linear in a range from 10² to 10¹³ copies/ml. By comparison with the restriction fragment length polymorphism method, 92.3% of 441 samples were accurately genotyped by the current assay. The method should be useful for genotyping and quantification of HBV DNA in areas where all genotypes exist.