Sensitive Assay for Quantification of Hepatitis B Virus Mutants by Use of a Minor Groove Binder Probe and Peptide Nucleic Acids

概要

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Lamivudine is the first nucleoside analogue that was shown to have a potent effect on hepatitis B virus (HBV). However, the emergence of resistant or cross-resistant mutants to nucleos(t)ide analogues remains a serious problem. Several assays for the detection and quantification of antiviral resistant mutants have been reported, but it has been difficult to measure the amounts of mutants accurately, especially when the target strain is minor in the mixed population. It has been shown that accurate measurement of a minor strain is difficult as long as matching reaction with a single probe was included in the assay. We developed a new method for the quantification of lamivudine-resistant strains in a mixed virus population by real-time PCR using minor groove binder probes and peptide nucleic acids, and we achieved a wide and measurable range from 3 to 10 log10 copies/ml and a high sensitivity with a discriminative limit of 0.01% of the predominant strain. The clinical significance of measuring substitutions of not only M204 but also L180 residues of HBV polymerase was demonstrated by this method. This assay increases the versatility of a sensitive method for the quantification of a single nucleotide mutation in a heterogeneous population.