Combination of allele-specific detection techniques to quantify minority resistance variants in hepatitis B infection: A novel approach

Detection of minor variant viral quasispecies of the rtV173L + rtL180M + rtM204V combination mutation in the hepatitis B virus (HBV) polymerase mediating both lamivudine resistance and vaccine escape is potentially important for tracking the development and evolution of resistance within both individuals and populations. A highly sensitive and specific assay to quantitate HBV genomes was developed with this mutation combination directly from viral DNA in serum using allele-specific quantitative PCR with locked nucleic acid primers and a minor groove binder probe. This combination of primers and probe yields a linear detection range down to 150 copies. This strategy has 100% specificity even in mixtures of predominately wild type genomes. The assay accurately detected 3 × 102 copies of the triple mutant spiked into 3 × 108 copies of the wild-type genomes (0.0001%), while maintaining 100% specificity. This approach was validated using serum from a subject infected with known lamivudine-resistant HBV. The triple mutant viral population was quantitated at 2.86 × 108 copies/ml within a total viral concentration of 1.03 × 1010 copies/ml of serum (2.8%). This quantitative allele-specific PCR strategy therefore is a useful method for highly sensitive and specific detection of point mutation combinations that are clinically important in the pathogenesis of drug resistance and/or immune escape.

► Measurement of point mutation combinations on the same viral genome is important.
► An allele-specific assay was developed for the HBV triple mutant rtV173L + rtL180M + rtM204 V.
► This combination of primers and probe yields a linear detection range down to 150 copies.
► This strategy has 100% specificity even in mixtures of predominately wild type genomes.