Single-step real-time PCR to quantify hepatitis B virus and distinguish genotype D from non-D genotypes

Hepatitis B virus (HBV) viral load and its genotype play important roles in clinical outcome, management of disease and response to antiviral therapy. In many parts of the world such as Europe or the Middle East, distinguishing HBV genotype D from non‐D is most relevant for treatment decisions, because genotype D‐infected patients respond poorly to interferon‐based therapeutic regimens. Here, we developed an in‐house real‐time PCR to concordantly assess HBV genotype (D vs non‐D) based on melt curve analysis and quantify the viral load. Genotype distinction was established with control plasmids of all HBV genotypes and validated with 57 clinical samples from patients infected with six different HBV genotypes. Our in‐house real‐time PCR assay could discriminate HBV genotype D from non‐D using single‐step melt curve analysis with a 2 °C difference in the melt curve temperature in all samples tested. Viral load quantification was calibrated with the WHO HBV international standard, demonstrating an excellent correlation with a commercial kit (r = 0.852; P