Background:   Hepatitis B virus (HBV) drug resistance caused by the rtL180M+M204V+V173L triple mutation can result in an HBV vaccine escape phenotype. Conventional sequencing only detects mutations that are present in >25% of the population. Current techniques to detect minority resistance, such as pyrosequencing or clonal analysis are either expensive or labor intensive. Methods to detect allele-specific mutations include the locked-nucleic acid (LNA) primer design and minor groove binding (MGB) probe design, but these 2 techniques have not been combined. To detect the frequency of the triple mutation we designed a sensitive and specific allele-specific quantitative real-time PCR with the primer-probe by combining LNA allele-specific primers and quantitative PCR (qPCR) MGB probes.

Methods:  We created plasmids containing the HBV polymerase (wild type) and the reverse transcriptase mutations V173L, L180M, and M204V (triple mutant plasmid) by amplifying the polymerase gene from extracted HBV DNA from the Acrometrix (genotype A) control and inserting the gene into a plasmid vector (pCR 2.1-TOPO TA vector).  Site-directed mutagenesis was then performed for mutation insertions (Quikchange IIXL, Stratagene). Two LNA primers were designed for the rtV173L and M204V mutations and an MGB probe was designed for the rtL180M mutation.

Results:   We designed a novel method to detect minority variant populations of the triple mutation (V173L+L180M+M204V) in the HBV polymerase. Resistant mutations were detected to 0.01%, 0.001%, and 0.0001%, in 10⁴, 10⁶, and 10⁸ copies/mL, respectively. Our sensitivity was 100 copies/mL in 10⁴ while retaining 100% specificity with 10x9 copies/mL and was validated in human serum with drug-resistant HBV.

Conclusions:  We report the first known combination of LNA primer and MGB probe technologies in qPCR and for the detection of HBV minority drug-resistance populations. This assay will have applications in all genotypic evaluations where multiple mutation detection and quantification is desired.