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Combination of allele-specific detection techniques to quantify minority resistance variants in hepatitis B infection: a novel approach.
|Title||Combination of allele-specific detection techniques to quantify minority resistance variants in hepatitis B infection: a novel approach.|
|Publication Type||Journal Article|
|Year of Publication||2013|
|Authors||Bhattacharya D, Lewis MJ, Lassmann B, Phan T, Knecht G, Bickel M, Yang OO|
|Journal||Journal of virological methods|
|Date Published||2013 Jun|
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.
|Alternate Journal||J. Virol. Methods|