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.

Aligning to the sample-specific reference sequence to optimize the accuracy of next-generation sequencing analysis for hepatitis B virus

“…We suggest using a sample-specific reference sequence as a mapping reference for NGS analysis in studies of P-gene variants for drug-resistance [ 24 , 25 ] and S-gene variants for the pathogenesis of HCC [ 26 , 27 ]. Emerging evidence supports the notion that certain drug-resistant HBV minor strains are crucial for the progression of liver diseases and are predictors of subsequent treatment failure [ 23 , 28 ]. They may accumulate and eventually dominate under a long-term selection effect during antiviral treatment [ 23 ].…”

Establishment of Real Time Allele Specific Locked Nucleic Acid Quantitative PCR for Detection of HBV YIDD (ATT) Mutation and Evaluation of Its Application

“…Bhattacharya D et al successfully developed an allele-specific quantitative PCR with combination of locked nucleic acid primers and a minor groove binder probe for the quantitative determination of minor viral quasispecies of the triple combination mutation rtV173L+rtL180M+rtM204V within one HBV genome. The assay could accurately detected 3×10 2 copies of the triple mutant in the 3×10 8 copies background of wild-type DNA [16] .…”

“…And there was not a specific guideline on choosing the last position versus the penultimate position for incorporation of the LNA base so far. To a great extent, the position of LNA was determined empirically [16] . Most studies had shown a 3′ LNA residue in the primer at the SNP site (i.e., the mismatch site) could improve mismatch discrimination excellently.…”

“…In a study of effect of locked nucleic acid (LNA) modification position upon representative DNA polymerase and exonuclease activities, Di Giusto et al reported single LNA at the penultimate (L-2) nucleotide position generated nuclease resistance activity and provided improved discrimination [19] . And a similar successful primer design had also been adopted by Bhattacharya D et al to quantify minority resistance variants in hepatitis B infection [16] . Inspired by their researches, we introduced an adenine (A) base into Primer L2 to allow LNA to locate at the penultimate to improve the specificity of Primer L2.…”