title: Combination of allele-specific detection techniques to quantify minority resistance variants in hepatitis B infection: a novel approach.
abstract: 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.
authors: Division of Infectious Diseases, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA. debikab@mednet.ucla.edu
authors: Debika
authors: D
authors: Bhattacharya
authors: Martha J
authors: MJ
authors: Lewis
authors: Britta
authors: B
authors: Lassmann
authors: Tina
authors: T
authors: Phan
authors: Gaby
authors: G
authors: Knecht
authors: Marcus
authors: M
authors: Bickel
authors: Otto O
authors: OO
authors: Yang
mesh_terms: D000483:Alleles; D000998:Antiviral Agents / Q000494:pharmacology; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics*; D024882:Drug Resistance, Viral; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D057131:Immune Evasion; D019259:Lamivudine / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods; D009154:Mutation; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity
chemical_list: D000998:Antiviral Agents; D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D019259:Lamivudine