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Detection of Hepatitis B Virus M204V Mutation Quantitatively via Real-time PCR

  • Jingjing Liang1 ,
  • Xinmiao Liang3,
  • Hong Ma1,
  • Leng Nie4,
  • Ying Tian5,
  • Guang Chen2 and
  • Yu Wang1,*
 Author information
Journal of Clinical and Translational Hepatology   2021;9(2):143-148

doi: 10.14218/JCTH.2020.00118


Background and Aims

Drug-resistant DNA mutations of the hepatitis B virus (HBV) affect treatment response in chronic hepatitis B patients. We have established a new, sensitive, specific, accurate and convenient real-time PCR method to detect HBV mutations quantitatively.


Blood samples were collected from patients showing viral breakthrough, primary nonresponse, or poor response during treatment, and mutations were detected via direct sequencing to assess our method. A plasmid containing the M204V mutation was synthesized and standard curves plotted.


The determination coefficient for linear correlation between Ct and log plasmid copy numbers was 0.996, where Ct value was −3.723log (DNA concentration) +48.647. Coefficients of variation indicated good reproducibility. Correctness was within tolerable bias. Limit of detection was 103 copies/mL. Specificity, accuracy, positive predictive value and negative predictive value were 92.86%, 100%, 96.88%, 100% and 94.74%, respectively.


These results show that our method can be used to detect HBV M204V mutations with the advantages of sensitivity, specificity and efficiency, providing a new choice for monitoring drug resistance.


HBV DNA, Drug-resistance mutation, Real-time PCR, DNA sequencing


Hepatitis B virus (HBV) infection remains a global disease, with significant morbidity and mortality.1,2 One of the main treatment options for patients infected with chronic hepatitis B (CHB) is the nucleotide analogs (NAs). Currently, NAs approved for clinical use in China include lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF).3 However, antiviral resistance is the most important factor in the failure of hepatitis B treatment.

The occurrence of drug resistance may lead to recurrence of hepatitis, and even cause progression of the disease, including virological breakthroughs, biochemical rebounds, hepatitis condition aggravation, and even hepatic failure.4,5 Long-term drug resistance monitoring of patients with CHB who are taking LAM showed the proportion of hepatitis recurrence increased significantly compared to patients without drug resistance; in addition, the incidence of compensation cirrhosis was increased in patients with long-term drug resistance.6,7 Not only for its impact on hepatitis, the occurrence of drug resistance will also lead to an increase in the incidence of hepatitis B-related liver cancer. A meta-analysis showed that the incidence of liver cancer in patients with LAM resistance is significantly higher than in treatment-naive patients (42/594 vs. 126/3,287, p=0.001).8 Thus, the occurrence of drug resistance mutations not only limits the choice of treatment options and increases the cost of treatment but is also closely related to the progression and prognosis of the disease.

Antiviral drug-resistant HBV variants occur spontaneously in CHB patients following exposure to NAs.9,10 Genotypic antiviral resistance refers to the presence of unique nucleotide mutations in drug target genes, which are the HBV polymerase genes that have been shown to be associated with antiviral resistance during HBV treatment with NAs. There are two types of mutations associated with drug resistance: primary resistance mutations and compensatory mutations. Primary resistance mutations directly lead to a decrease in the sensitivity to the drug, while the compensatory mutations can restore or enhance the replication of virus. The mutations rtM204V/I represent one of the most common primary resistance mutations in hepatitis B patients, which directly decrease the susceptibility to NAs, especially to LAM and LdT.11–15

LAM is the most widely used and longest-serving antiviral drug, but it also has the highest resistance rate. Recent studies have indicated that the percentage of LAM-associated resistance mutations that appear after 1 year may vary from 7% to 30%.15–17 The accumulation rate of LAM-associated drug resistance following 5 years of therapy is 70%.7 As a recommended first-line drug, ETV is also widely used in clinical practice. In addition to a background of the mutations rtM204V/I, the other mutations, such as rtI169T, rtS184G, rtS202I and rtM250V, are associated with the emergence of ETV resistance.18–21 A retrospective study conducted in China from 2009 to 2016 demonstrated that 73.7% of male CHB patients developed HBV genotypic resistance mutations to NAs, and that ETV-associated resistance increased to 17.1% in 2016.22 Moreover, rtM204V combined with some other mutations may lead to a resistance to TDF.23 Due to the widespread use of the above-mentioned drugs, drug-resistant specimens are the most readily available. Therefore, we chose mutations rtM204V/I to be detected.

Real-time PCR, a relatively new quantitative testing technique, includes a new platform that allows the initial concentration of the sample template to be estimated. In contrast to conventional PCR, it uses fluorescent dyes or specific fluorescent labeled probes to monitor the reaction process and illustrate the amount of DNA present, in real time, at each cycle of amplification. Two methods are used to calculate the initial DNA concentration: absolute quantitation and relative quantitation. Relative quantitation is used to determine the relative changes in expression in a similar target nucleic acid sequence and the correction sample. Absolute quantitative analysis determines the absolute value of a nucleic acid sequence in a sample.24–28 Real-time PCR is widely used in molecular diagnostics to detect and identify bacteria and viruses. In addition, because real-time PCR is quantifiable, it is used to evaluate disease progression and efficacy of antiviral/antibiotic therapies.

The current study developed a real-time PCR assay aimed at quantitatively detecting HBV mutations. The schematic diagram of this work is shown in Fig. 1.

Process of real-time PCR based on magnetic nanoparticles for detecting HBV DNA.
Fig. 1  Process of real-time PCR based on magnetic nanoparticles for detecting HBV DNA.


Sample preparation

We collected blood samples from patients treated with NAs, especially LAM and ETV, showing viral breakthrough (defined as a confirmed increase in HBV DNA levels of more than 1 log10 copies/mL compared to the lowest HBV DNA level on-therapy), primary nonresponse (defined as less than 1 log10 copies/mL decrease in the HBV DNA level from baseline following 12 weeks of therapy), or poor response (defined as more than 1 log10 copies/mL decrease in the HBV DNA level from baseline but detectable following at least 12 months of therapy) in response to NAs at the Beijing Friendship Hospital between 2015 and 2019. The exclusion criteria were as follows: co-infection with other hepatitis viruses or/and human immunodeficiency virus; poor treatment compliance; and patients who refused to participate in the experiment.

The study was approved by the Medical Ethics Committee of Beijing Friendship Hospital, affiliated with Capital Medical University.

Process for the detection system

DNA extraction

HBV DNA was extracted using nano-magnetic beads according to the following steps: nano-beads and heated lysis buffer were mixed in a 70 °C water bath to dissolve insoluble substances, and then mixed evenly before use. A 100 µL serum sample was added to a new centrifuge tube and mixed with 400 µL of cracking buffer solution, and was heated for 10 m at 70°C and mixed every 2–3 min. Subsequently, 300 µL of binding buffer and 20 µL of magnetic beads were added to the above centrifuge tube in turn, and mixed. The mixture was left to stand at room temperature for 5 m, with mixing once every 2 m. Next, 200 µL each of cleaning buffers I, II and III were added to the centrifuge tube, respectively, a magnet was used to adsorb the nano-beads for 30 s, and the supernatant was discarded. Centrifugation at high speed was used to separate the nano-beads from the mixture, followed by extraction. Next, 50 µL eluting buffer was added to the centrifuge tube, heated to 70°C and left for 5 m at 70°C. Nano-beads adsorbed on the magnets and supernatant fluid containing DNA were transferred to an RNA-free centrifuge tube and stored at –20°C for further use.

Primers and probes of M204V

Primers and probes were designed by the Wawasye Nanotech Company (Wuhan, China) and synthesized by Sangon Biotech (Shanghai, China). The probes were quenched using BHQ1 or MGB at the 3′-end (Table 1). Better probes were selected by subjecting the positive serum, containing the mutant gene of HBV polymerase M204V previously confirmed by Sanger sequencing, to a series of tests.

Table 1

Primer and probe sequences with their respective dye and quencher

Target geneNameSequences
Primerprimer forward5′-GCACTTGTATTCCCATCCCATCAT-3′

Preparation of the reaction system

We blended 12.5 µL of SYBR Premix (2x) with 0.5 µL of forward primer, 0.5 µL of reverse primer, 0.1 µL of specific probe and 9.4 µL of deionized water as a reaction system. Subsequently, we added 2 µL of template DNA to the experimental group and 2 µL of deionized water to the control group in order to obtain a final reaction volume of 25 µL each.

PCR amplification detection of mutations in 204 HBV nucleotides

Amplifications were processed using the FAMTM/SYBR® Green channel of the SLAN®-96P Real-Time PCR System (Shanghai Hongshi Medical Technology, Shanghai, China). Cycling conditions were as follows: 5 m at 95°C, followed by a two-step cycling stage of 40 cycles of 15 s at 95°C and 60 s at 59°C. Samples were considered positive when presenting a typical amplification curve with a Ct value of ≤35. Analyses of samples with Ct values of 35 and 38 were repeated.

Plasmid design and standard curve protraction

In order to obtain standard curves for the purposes of quantification and estimation of efficacy of the new real-time PCR protocol, a specific plasmid containing the M204V mutation was designed and synthesized by Sinogene Biotech (Beijing, China). Standard curves based on a 10-fold dilution of the plasmid were plotted. The formula [DNA (copies/µL)=6.02×1023 (copies/mol)×DNA concentration (µg/µL)/[DNA length (bp)×660(daltons/bp)] was used to calculate the DNA load. Next, we analyzed linear relationships based on amplification results. Log DNA concentration (horizontal axis; X) was plotted against Ct values (vertical axis; Y).

Results and discussion

We enrolled 32 CHB patients with drug-resistant gene mutations. The patients’ information is shown in Table 2.

Table 2

Detailed information of the 32 patients

No.SexGenotypeHBV viral load, copies/mLMutations
1MaleC2.03×105M204V, L180M
2MaleB1.317×105M204V, L180M
3MaleC1.2×103M204V, L180M
4FemaleC1.4×104M204I, L180M
5MaleB2.5×104M204I, L180M
7FemaleB1.99×102M204V, L180M, T184A
8FemaleC4.33×103M204V, M204I
9MaleC4.33×103M204V, M204I, L180M
10MaleC2.3×107M204V, L180M
13MaleC3.03×103M204V, L180M, S202G, T184A
14FemaleB4.79×103M204V, L180M, S202G
15MaleC3.6×107M204V, L180M, T184L
16FemaleC2.2×107M204V, L180M, T184A
17MaleB6.4×103A181T, N236T
18FemaleB4. 3×105A181V
20MaleC1.68×104A181T, A181V, N236T
22MaleC2.2×105M204V, L180M
23MaleC1.07×103M204V, L180M
24MaleC1.8×103M204V, L180M
25MaleC4.25×107M204I, L180M
26FemaleB3.51×103M204I, L180M
28MaleC4.2×104A181T, N236T
31FemaleB2×103A181T, A181V, N236T

We designed two different probes for 204 nucleotide mutations, to ensure the presence of a mutation, and subsequently designed two specific probes each for M204V or M204I. The specificity and sensitivity of the probes were confirmed via the amplification of positive serum samples, which had been verified as containing the M204V/I mutation via Sanger sequencing. We repeated a portion of the experiments on the same specimen in order to verify its accuracy and repeatability. An acceptance criterion of Ct <38 was used. According to the results of 16 positive serum samples, the 204-2-P probe was more specific and sensitive. The true positive rates were 100% and 62.4%, respectively. Both 204-2-VP and 204-2-IP were abandoned due to the absence of a fluorescent signal. The specificity and sensitivity of probes 204-2-VP2 and 204-2-IP2 were confirmed. The positive rate of detection was 100% and the false positive rate was 0%.

Amplification curves of the 10-fold dilution series of the plasmid are shown in Fig. 2. The efficiency of quantitative real-time PCR was confirmed via plasmid-based curves. The plasmid DNA concentration was 2,000 ng/µL and DNA length was 2,980 bp. We introduced relevant parameters to the formula, stated above, to obtain the initial DNA load. The serial dilution curves indicated that the relationship between Ct value and log DNA concentration was linear (Fig. 3). The linear coefficient of determination (R2) was 0.996, indicating a significant linear relationship for the quantitative real-time PCR. The slope of the standard curve was −3.723.

Amplification curves of plasmids.
Fig. 2  Amplification curves of plasmids.
Linear relationship between Ct values and log DNA concentrations.
Fig. 3  Linear relationship between Ct values and log DNA concentrations.

We diluted plasmid concentrations to 6.12×106, 6.12×105, and 6.12×104 copies/mL to determine the reproducibility of real-time PCR. Six repetitions of the dilution series were analyzed. The coefficients of variation of log DNA concentration were 0.43%, 0.69%, and 0.58%, respectively, which indicate good reproducibility.

The accuracy of real-time PCR was determined via a 10-fold dilution series ranging from 6.12×106 to 6.12×102 copies/mL. The plasmids were diluted using a mixture of serum samples from patients with CHB. The average log DNA concentration of five replicates tested simultaneously under similar operating conditions was estimated. The biases were −0.174, −0.085, 0.005, and 0.06, respectively, which indicated good correctness.

In order to determine the limit of detection, the plasmids were diluted to 106, 105, 104, 103, 102, and 10 copies/mL. A Ct value <38 and a standard deviation of 0.5 were considered as the reference standard. The coefficients of variation of log DNA concentration were 0.43%, 0.69%, 0.58%, 2.79%, 5.54%, and 4.89%, respectively. The standard deviations of log DNA concentration were 0.03, 0.04, 0.03, 0.11, 0.16, and 0.12, respectively. The positive rates of detection were 100%, 100%, 100%, 100%, 83.3%, and 50%, respectively. Thus, the limit of detection for real-time PCR was determined to be 103 copies/mL.

A comparative analysis of 32 serum samples from CHB patients suspected of showing signs of drug-resistance was conducted. Both real-time PCR and direct sequencing protocols indicated that 13 out of the 32 samples (40.625%) were M204V-positive. One sample (3.125%) was identified as M204V-positive by direct sequencing, whereas the real-time PCR results could not be evaluated. Eighteen samples (56.25%) were identified as M204V-negative by both real-time PCR and direct sequencing. None of the samples were found to be M204V false positives. Direct sequencing is considered as the gold standard. Overall, the sensitivity of real-time PCR for detecting the M204V mutation was 92.86% (13/14), with a specificity of 100%.


The current study established a practical quantitative real-time PCR that monitors HBV DNA polymerase gene mutations in patients undergoing treatment with NAs, with particular reference to the M204V mutation. Compared with direct sequencing, quantitative real-time PCR is effective, low-cost, and convenient. It does not involve complex and expensive instruments. Furthermore, our quantitative detection system, which gathers a fluorescence signal from each cycle, is rapid, as each DNA test requires only 6–7 m. Moreover, the limit of detection is similar to that of direct sequencing. This new method is able to monitor HBV DNA variations quantitatively, thus providing a new method that monitors drug resistance during the early stages of therapy and assesses the relationship between genetic mutations and phenotypic resistance.

Tests that determine the limit of detection are insufficient, requiring many further tests to confirm the limitation. Further assays are required to develop systems designed to monitor other mutations, and also to establish a system for multiplex detection purposes.



adefovir dipivoxil


chronic hepatitis B




hepatitis B virus








tenofovir alafenamide


tenofovir disoproxil fumarate



This work was supported by funds from the Beijing Municipal Science & Technology Commission (No. Z171100001017037) and the Chinese National Science and Technology Major Project (No. 2017ZX10105015002).

Conflict of interest

The authors have no conflict of interests related to this publication.

Authors’ contributions

Study design (JJL, HM, YW), performance of experiments (JJL, LN), analysis and interpretation of data (JJL, GC), manuscript writing (JJL, GC), statistical analysis (JJL, GC, XML), critical revision of the manuscript (HM, YW), critical funding (HM, YW), and technical or material support (HM, LN, YW).


  1. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017;67:370-398 View Article
  2. Hermans LE, Svicher V, Pas SD, Salpini R, Alvarez M, Ben Ari Z, et al. Combined analysis of the prevalence of drug-resistant hepatitis B virus in antiviral therapy-experienced patients in Europe (CAPRE). J Infect Dis 2016;213:39-48 View Article
  3. Terrault NA, Bzowej NH, Chang KM, Hwang JP, Jonas MM, Murad MH. AASLD guidelines for treatment of chronic hepatitis B. Hepatology. 2016;63:261-283 View Article
  4. Lin CL, Kao JH. Natural history of acute and chronic hepatitis B: The role of HBV genotypes and mutants. Best Pract Res Clin Gastroenterol 2017;31:249-255 View Article
  5. Wang W, Shu Y, Bao H, Zhao W, Wang W, Wang Q, et al. Genotypes and hot spot mutations of hepatitis B virus in northwest Chinese population and its correlation with diseases progression. Biomed Res Int 2019;2019:3890962 View Article
  6. Liaw YF, Sung JJ, Chow WC, Farrell G, Lee CZ, Yuen H, et al. Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med 2004;351:1521-1531 View Article
  7. Lok AS, Lai CL, Leung N, Yao GB, Cui ZY, Schiff ER, et al. Long-term safety of lamivudine treatment in patients with chronic hepatitis B. Gastroenterology. 2003;125:1714-1722 View Article
  8. Papatheodoridis GV, Lampertico P, Manolakopoulos S, Lok A. Incidence of hepatocellular carcinoma in chronic hepatitis B patients receiving nucleos(t)ide therapy: a systematic review. J Hepatol 2010;53:348-356 View Article
  9. Lok AS, Zoulim F, Locarnini S, Bartholomeusz A, Ghany MG, Pawlotsky JM, et al. Antiviral drug-resistant HBV: standardization of nomenclature and assays and recommendations for management. Hepatology 2007;46:254-265 View Article
  10. Gupta N, Goyal M, Wu CH, Wu GY. The molecular and structural basis of HBV-resistance to nucleos(t)ide analogs. J Clin Transl Hepatol 2014;2:202-211 View Article
  11. Alacam S, Karabulut N, Yolcu A, Onel M, Atasoy A, Kaymakoglu S, et al. Evaluation of drug resistance mutations in patients with chronic hepatitis B. Folia Microbiol (Praha). 2019;64:237-243 View Article
  12. Tacke F, Kroy DC. Treatment for hepatitis B in patients with drug resistance. Ann Transl Med 2016;4:334 View Article
  13. Tong S, Revill P. Overview of hepatitis B viral replication and genetic variability. J Hepatol 2016;64:S4-S16 View Article
  14. Zoulim F, Locarnini S. Hepatitis B virus resistance to nucleos(t)ide analogues. Gastroenterology 2009;137:1593-1608.e1-2 View Article
  15. Tang LSY, Covert E, Wilson E, Kottilil S. Chronic hepatitis B infection: A review. JAMA 2018;319:1802-1813 View Article
  16. Chan HL, Wang H, Niu J, Chim AM, Sung JJ. Two-year lamivudine treatment for hepatitis B e antigen-negative chronic hepatitis B: a double-blind, placebo-controlled trial. Antivir Ther 2007;12:345-353
  17. Lai CL, Gane E, Liaw YF, Thongsawat S, Wang YM, Chen Y, et al. Telbivudine (LdT) vs. lmivudine for chronic Hepatitis B: First-year results from the international phase III GLOBE trial. Hepatology 2005;42:748A
  18. Tenney DJ, Levine SM, Rose RE, Walsh AW, Weinheimer SP, Discotto L, et al. Clinical emergence of entecavir-resistant hepatitis B virus requires additional substitutions in virus already resistant to Lamivudine. Antimicrob Agents Chemother 2004;48:3498-3507 View Article
  19. Kim HJ, Cho YK, Jeon WK, Kim BI. Clinical characteristics of patients with chronic hepatitis B who developed genotypic resistance to entecavir: Real-life experience. Clin Mol Hepatol 2017;23:323-330 View Article
  20. Liu Y, Zhou Y, Li X, Niu M, Chen R, Shao J, et al. Hepatitis B virus mutation pattern rtL180M+A181C+M204V may contribute to entecavir resistance in clinical practice. Emerg Microbes Infect 2019;8:354-365 View Article
  21. Shaw T, Bartholomeusz A, Locarnini S. HBV drug resistance: mechanisms, detection and interpretation. J Hepatol 2006;44:593-606 View Article
  22. Guo X, Wu J, Wei F, Ouyang Y, Li Q, Liu K, et al. Trends in hepatitis B virus resistance to nucleoside/nucleotide analogues in North China from 2009-2016: A retrospective study. Int J Antimicrob Agents 2018;52:201-209 View Article
  23. Jiang D, Wang J, Zhao X, Li Y, Zhang Q, Song C, et al. Entecavir resistance mutations rtL180M/T184L/M204V combined with rtA200V lead to tenofovir resistance. Liver Int 2020;40:83-91 View Article
  24. Bachman J. Reverse-transcription PCR (RT-PCR). Methods Enzymol 2013;530:67-74 View Article
  25. Bustin SA, Benes V, Nolan T, Pfaffl MW. Quantitative real-time RT-PCR—a perspective. J Mol Endocrinol 2005;34:597-601 View Article
  26. Green MR, Sambrook J. Quantification of RNA by real-time reverse transcription-polymerase chain reaction (RT-PCR). Cold Spring Harb Protoc 2018 View Article
  27. Singh C, Roy-Chowdhuri S. Quantitative real-time PCR: Recent advances. Methods Mol Biol 2016;1392:161-176 View Article
  28. Freeman WM, Walker SJ, Vrana KE. Quantitative RT-PCR: pitfalls and potential. BioTechniques 1999;26(1):112-125 View Article
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Detection of Hepatitis B Virus M204V Mutation Quantitatively via Real-time PCR

Jingjing Liang, Xinmiao Liang, Hong Ma, Leng Nie, Ying Tian, Guang Chen, Yu Wang
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