Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing

Abstract

Mutations in the reverse transcriptase (rt) region of the DNA polymerase gene are the primary cause of hepatitis B virus (HBV) drug resistance. In this study, we established a novel method that couples coamplification at lower denaturation temperature (COLD)-PCR and Sanger sequencing, and we applied it to the detection of known and unknown HBV mutations. Primers were designed based on the common mutations in the HBV rt sequence at positions 180 to 215. The critical denaturation temperature (T c ) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature (T m )-reducing mutation occurred (e.g., C-G¡T-A), the sensitivities of fast and full COLD-PCR for mutant detection were 1% and 2%, respectively; when the mutation caused no change in T m (e.g., C-G¡G-C) or raised T m (e.g., T-A¡ C-G), only full COLD-PCR improved the sensitivity for mutant detection (2%). For combination mutations, the sensitivities of both full and fast COLD-PCR were increased to 0.5%. The limits of detection for fast and full COLD-PCR were 50 IU/ml and 100 IU/ml, respectively. In 30 chronic hepatitis B (CHB) cases, no mutations were detected by conventional PCR, whereas 18 mutations were successfully detected by COLD-PCR, including low-prevalence mutations (<10%), as confirmed by ultradeep pyrosequencing. In conclusion, COLD-PCR provides a highly sensitive, simple, inexpensive, and practical tool for significantly improving amplification efficacy and detecting low-level mutations in clinical CHB cases.T he hepatitis B virus (HBV) gene is prone to undergoing mutations due to the lack of stringent correction mechanisms in the transcription process. High rates of nucleotide (nt) mismatches occur during treatment with antiviral drug nucleoside (or nucleotide) analogues (NAs), especially in the reverse transcriptase (rt) region of the HBV polymerase gene (1). These mismatches are genotypic resistance mutations that lead to antiviral drug resistance (2). In order to improve HBV treatment by monitoring and reducing the occurrence of drug resistance, it is essential to detect rt mutations in chronic hepatitis B (CHB) patients over the course of treatment (3).Current laboratory methods for the detection of HBV genetic mutations are nucleotide sequencing, reverse hybridization, PCRrestriction fragment length polymorphism (RFLP), allele-specific real-time PCR, restriction fragment mass polymorphism (RFMP), ultradeep pyrosequencing (UDPS), and gene chips (DNA microarrays). Direct PCR sequencing (Sanger sequencing) is considered to be the gold standard for detecting genotypic resistance mutations, because it can either detect multiple mutation sites during an assay or detect both known and unknown mutations simultaneously, with low false-positive rates (4). However, the sensitivity of populationbased sequencing often decreases when the method is used to detect mutants present at Ͻ20% prevalence in a given sample (1, 4). The reverse hybridization assay has higher sensitivity, to a...

Characterization and Clinical Significance of Natural Variability in Hepatitis B Virus Reverse Transcriptase in Treatment-Naive Chinese Patients by Sanger Sequencing and Next-Generation Sequencing

“…In this study, except rtA181T, the classical primary drug resistance mutations (i.e., I169T, A181T/V, T184A/C/F/G/I/L/M/S, A194T, S202C/G/I, M204I/V/S, N236T, and M250I/L/V) were not detected by Sanger sequencing in treatment-naive patients (Table 3), which was consistent with previous reports (6, 25, 26). Considering that Sanger sequencing is a population-based sequencing approach used to detect mutations with an intrahost rate of more than 20% (6, 27), the minor mutations with an intrahost rate of less than 20% were likely to be ignored by Sanger sequencing. In this study, using next-generation sequencing, mutations were found at rt202 (rtS202R/I/N/C/G), rt204 (rtM204L/R/I), rt236 (rtN236T/K/H), and rt250 (rtM250I/L) (Table 5).…”

COLD-PCR Method for Early Detection of Antiviral Drug-Resistance Mutations in Treatment-Naive Children with Chronic Hepatitis B

“…With the design of COLD-PCR for the amplification of P1 and P2 fragments, corresponding to the B, C, D, and E domains in HBV RT, the developed method can be applied for sensitive detection of major NA-drug resistance mutations, including three mt (rtV173L, rtL180M, and rtM204I/V) that are associated not only with LMV and ETV resistance but also with vaccine escape [ 12 , 14 ]. Additional mt including rtS256G for resistance to LMV and rtL229V, rtI233V, rtP237T, and rtN238A/K/T for resistance to ADF were detected based on the work of Liu and his colleagues [ 21 ]. To confirm the reliability of mt detection in clinical samples by the COLD-PCR method, we cross-checked the two representative samples having the lowest mt peaks constituting ≤20% of the wt peak heights based on real-time PCR assay using Taqman LNA probes.…”

“…The size of P1 and P2 fragments were intentionally designed to be <200 nt, as recommended previously [ 21 , 22 ], to ensure the difference in melting temperature between homoduplex (mt-mt; wt-wt) and heteroduplex (mt-wt).…”

“…The developed COLD-PCR does not need additional reagents or equipped light cyclers and, thus, can easily replace conventional PCR and, at the same time, improve the mutation detection sensitivity limit of downstream Sanger DNA sequencing, enriching low-level variants (<5%) within a mixture of wild-type (wt) and mt sequences. The reliability of COLD-PCR has been validated by ultradeep pyrosequencing, and COLD-PCR has been successfully applied for detection of several important NA-resistance mt in HBV [ 21 , 22 ]. The aim of this study was to develop a modified COLD-PCR method that identifies most of the known HBV NA-resistance mt and to apply it on clinical samples to describe the current frequencies of these mt in treatment-naive HBV-infected children.…”

The Polymerase Chain Reaction

“…Therefore, the denaturation temperature of the mutant DNA is often lower than that of wild type DNA. The assay is often used for viral gene mutation [59] detection, cancer associated gene mutations (p53) [60,61], EGFR, KRAS, etc. ), beta globulin (HBB) mutations that cause beta thalassemia [62], etc.…”