Quantitative detection of the M204V hepatitis B virus minor variants by amplification refractory mutation system real-time PCR combined with molecular beacon technology

Abstract

Mutations in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif are frequently associated with resistance to antivirals and represent a major concern in the treatment of hepatitis B virus (HBV) infection. Conventional methods fail to detect minority populations of drug-resistant viral quasispecies if they represent less than 25% of the total sample virus population. The amplification refractory mutation system real-time PCR (ARMS RT-PCR) was combined with molecular beacon technology using the LightCycler system. The samples from HBV patients selected for assay evaluation included (i) 57 samples from treatment-naïve patients for biological discriminatory ability (cutoff) estimation, (ii) 12 samples from patients with treatment failure that were M204V positive by sequencing, and (iii) 13 samples from patients with treatment failure that were negative for mutation at codon 204 by sequencing. The discriminatory ability of the assay was 0.25% when tested with laboratory-synthesized DNA target sequences. The median mutant-to-wild-type ratio for samples from naive patients tested positive for the wild type and for mutant variants was 0.01% (5th and 95th percentiles = 0.0001 and 0.04%, respectively). A value of 0.04% was selected as the biological cutoff of the assay of clinical samples. In all samples M204V positive by sequencing (12/12), the mutant variant was detected as the predominant population (range, 82.76 to 99.43%). Interestingly, in 5 (38%) of 13 samples negative by sequencing, the M204V variant was detected at a ratio above the biological cutoff (0.05 to 28%). The assay represents an efficient technique for the early detection and quantification of M204V variants before mutant strains emerge to dominate the population.

FIG. 1.

Fitted regression line of the concentrations obtained with the ARMS RT-PCR assay with molecular beacon technology for the Mut target sequence (A) and the WT target sequence (B) versus the expected concentrations (black line), the equality line (yellow line), and the 95% confidence interval of the fitted regression line (blue line).

FIG. 2.

Discriminatory ability of the ARMS RT-PCR assay with molecular beacon technology. The concentrations shown are 10⁶ copies per reaction mixture of the WT target sequence determined with the set of primers (prim) specifically designed for detection of the WT (blue line), 10⁶ copies per reaction mixture of the WT target sequence determined with the set of primers specifically designed for detection of the Mut (pink line), 10⁵ copies per reaction mixture of the Mut target sequence determined with the set of primers specifically designed for detection of the Mut (yellow line), 10⁴ copies per reaction mixture of the Mut target sequence determined with the set of primers specifically designed for detection of the Mut (light blue line), 5 × 10³ copies per reaction mixture of the Mut target sequence determined with the set of primers specifically designed for detection of the Mut (purple line), and 2.5 × 10³ copies per reaction mixture of the Mut target sequence determined with the set of primers specifically designed for detection of the Mut (brown line). The two-headed arrow shows the limits of the discriminatory ability of the assay.