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 treatmentnaïve 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.Nucleoside analogs inhibit hepadnavirus replication by terminating viral DNA synthesis and have been used in the treatment of patients with hepatitis B virus (HBV) infection. Antiviral treatment has been shown to rapidly reduce serum HBV DNA to levels below the detection limit of standard commercial assays and to be well tolerated without major adverse events (23). However, as with other antivirals, resistance may occur at a rate mainly dependent on the nucleoside/nucleotide analogue used. Taking lamivudine as an example, resistance increases with longer durations of treatment, from 24% at year 1 to 65 to 70% of treated patients at year 5 (17).Resistance to lamivudine has been frequently associated with mutations that reside in the highly conserved tyrosinemethionine-aspartate-aspartate (YMDD) motif of the C domain (M204I, M204V) and secondarily with mutations residing outside the C domain but mainly in the B domain (V173L, L180M) of the HBV polymerase region (Pol/RT). The M204V mutation has also been associated with resistance to other antivirals such as entecavir and emtricitabine; selection of M204I/V mutants affects future treatment options (4,16). In vitro studies have demonstrated that mutant (Mut) viral variants exhibit a diminished replication capacity compared w...

Characterization of antiviral resistance mutations among the Eastern Indian Hepatitis B virus infected population

“…The limited numbers of NAs available and development of drug resistance conferred by viral mutations in the HBV reverse transcriptase (rt) domain during long term treatment remains the major concern leading to treatment failure [1]. Recent reports showed presence of antiviral resistance even in HBV isolates from therapy naive patients [2-4]. …”

Ultrasensitive Amplification Refractory Mutation System Real-Time PCR (ARMS RT-PCR) Assay for Detection of Minority Hepatitis B Virus-Resistant Strains in the Era of Personalized Medicine

“…ARMS RT-PCR with molecular beacon assay has been evaluated using serial dilutions of WT and Mut target sequences from 10 6 copies per reaction to 10 copies per reaction with the appropriate set of primers for every one of the HBV resistance-associated mutations (14). Repeated measurements showed that the assay successfully detected WT and Mut target sequences in concentrations as low as 10 copies per reaction an analytical sensitivity of 90 to 100% for each one of the studied mutations.…”

“…Primers were designed according to the ARMS technique so as to be refractory for the PCR amplification of the nonmatching target sequences (12)(13)(14). A series of experiments where mutant primers with four different nϪ1 substitutions have been used under the same conditions of temperature and time allowed us to choose the best set of primers that gave sufficient discriminatory ability without decreasing the sensitivity and specificity of the method.…”

“…Based on our previous study, the biological cutoff the ARMS RT-PCR assay has been determined to be 0.04%, meaning that the assay was able to detect the viral subpopulation in a clinical sample if it was represented in at least 4 copies per 10 4 copies of the total viral population (14). For this reason, we decided that most of our clinical samples should have a viral load Ͼ10 4 copies per reaction after dilution in order to be included in our study.…”

Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique

“…Over the past two decades, most of the research on enhancing the detection of minority (mutant) alleles in clinical samples has focused on improving the selectivity of PCRbased technologies and/or enriching the mutant sequences. A number of approaches have been developed, including amplification refractory mutation system (ARMS) [10,11], coamplification at lower denaturation temperature PCR (COLD-PCR) [12][13][14], locked nucleic acid/peptide nucleic acid clamp PCR (LNA/PNA-PCR) [15,16], mutant-enriched PCR [17,18], one-step enzyme-enriched ARMS PCR [19] and restriction endonuclease-mediated real-time digestion-PCR (RTD-PCR) [19]. Most of these assays were reported to show moderate to high selectivity.…”