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ISSN 1001-5256 (Print)
ISSN 2097-3497 (Online)
CN 22-1108/R
Volume 37 Issue 8
Aug.  2021
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Article Navigation >  Journal of Clinical Hepatology  > 2021  >  37(8): 1806-1810

Establishment of a droplet digital PCR method for the detection of hepatitis B virus covalently closed circular DNA

DOI: 10.3969/j.issn.1001-5256.2021.08.013
  • 1.

    Beijing YouAn Hospital, Capital Medical University, Beijing Institute of Hepatology, Beijing 100069, China

  • 2.

    Fourth Department of Liver Disease, Beijing YouAn Hospital, Capital Medical University, Beijing Municipal Key Laboratory of Liver Failure and Artificial Liver Treatment Research, Beijing 100069, China

Research funding:

National Natural Science Foundation of China (81770611);

National Natural Science Foundation of China (82002243);

Key Projects of the Beijing Municipal Education Commission's Science and Technology Plan (KZ202010025035);

Key Public Relations Project of Capital Health Development Scientific Research Project (2020-1-1151);

Demonstrating Application and Research of Clinical Diagnosis and Treatment Technology in Beijing (Z191100006619096);

Demonstrating Application and Research of Clinical Diagnosis and Treatment Technology in Beijing (Z191100006619097);

National Science and Technology Key Project on Infectious Diseases (2018ZX10301407-005-002);

National Science and Technology Key Project on Infectious Diseases (2018ZX10302205-004-004);

Beijing Talents Foundation (2018000021469G289);

Beijing Hospitals Authority Youth Programme (QML20201702)

  • Received Date: 2021-01-19
  • Accepted Date: 2021-03-19
  • Published Date: 2021-08-20
    Abstract
  •   Objective  To establish a droplet digital PCR (ddPCR) method for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA).  Methods  HBV cccDNA standard substance was constructed, and HBV cccDNA primers and probes were designed based on the structural differences between HBV cccDNA and relaxed circular DNA (rcDNA). HBV plasmid was amplified to obtain HBV cccDNA standard substance, and a ddPCR detection method was established with the standard substance after gradient dilution as the template for HBV cccDNA detection; the limit of detection and repeatability of this method were analyzed. Liver tissue samples were collected from 20 patients who attended Beijing YouAn Hospital, Capital Medical University, from June 2017 to October 2020, all of whom were diagnosed with HBV infection, and DNA of the samples was extracted and digested with plasmid-safe ATP-dependent DNA enzyme to obtain HBV cccDNA template; the ddPCR detection method was evaluated in clinical samples and was compared with the quantitative real-time PCR (qPCR) detection method. The chi-square test was used for comparison of categorical data between the two groups.  Results  The HBV cccDNA detection method based on ddPCR was established, which accurately detected HBV cccDNA in standard substance after gradient dilution, with a limit of detection of 1 copy/μl, and the coefficients of variation of 1×103, 1×102, and 1×101 copies/μl standard substances were 4.41%, 3.98%, and 5.09%, respectively. HBV cccDNA was detected in the samples of 20 patients with HBV infection; the ddPCR detection method detected HBV cccDNA in 17 patients, with a positive rate of 85%, while the qPCR detection method detected HBV cccDNA in 11 patients, with a positive rate of 55%, and there was a significant difference between the two methods (χ2=4.286, P=0.038).  Conclusion  The established ddPCR method for detecting HBV cccDNA has a low limit of detection and good repeatability, which provides an effective tool for further clinical detection.

     

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    • References(21)
  • [1]
    LIU JJ, BIAN ZQ. Advances in genome wide association of HBV related liver diseases[J]. Int J Virol, 2019, 26(2): 135-139. DOI: 10.3760/cma.j.issn.1673-4092.2019.02.018.

    刘娟娟, 边中启. HBV相关肝病全基因组关联研究进展[J]. 国际病毒学杂志, 2019, 26(2): 135-139. DOI: 10.3760/cma.j.issn.1673-4092.2019.02.018.
    [2]
    RAZAVI-SHEARER D, GAMKRELIDZE I, NGUYEN MH, et al. Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: A modelling study[J]. Lancet Gastroenterol Hepatol, 2018, 3(6): 383-403. DOI: 10.1016/S2468-1253(18)30056-6.
    [3]
    ALLWEISS L, VOLZ T, GIERSCH K, et al. Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo[J]. Gut, 2018, 67(3): 542-552. DOI: 10.1136/gutjnl-2016-312162.
    [4]
    LAI CL, WONG D, IP P, et al. Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B[J]. J Hepatol, 2017, 66(2): 275-281. DOI: 10.1016/j.jhep.2016.08.022.
    [5]
    NEWBOLD JE, XIN H, TENCZA M, et al. The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes[J]. J Virol, 1995, 69(6): 3350-3357. DOI: 10.1128/JVI.69.6.3350-3357.1995.
    [6]
    SEEGER C, MASON WS. Molecular biology of hepatitis B virus infection[J]. Virology, 2015, 479-480: 672-686. DOI: 10.1016/j.virol.2015.02.031.
    [7]
    ZHANG XM, FENG RF. Correctly understand and use analytical sensitivity and limit of detection[J]. Chin J Lab Med, 2014, 37(9): 669-672. DOI: 10.3760/cma.j.issn.1009-9158.2014.09.008.

    张秀明, 冯仁丰. 正确理解和使用分析灵敏度及检出限[J]. 中华检验医学杂志, 2014, 37(9): 669-672. DOI: 10.3760/cma.j.issn.1009-9158.2014.09.008.
    [8]
    WERLE-LAPOSTOLLE B, BOWDEN S, LOCARNINI S, et al. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy[J]. Gastroenterology, 2004, 126(7): 1750-1758. DOI: 10.1053/j.gastro.2004.03.018.
    [9]
    SI LL, LI XD, LI L, et al. Inhibitory effect of Suduxing extracts on covalently closed circular DNA of hepatitis B virus[J/CD]. Chin J Exp Clin Infect Dis(Electronic Edition), 2020, 14(4): 265-271. DOI: 10.3877/cma.j.issn.1674-1358.2020.04.001.

    思兰兰, 李晓东, 李乐, 等. 复方肃毒星提取物抑制乙型肝炎病毒cccDNA的作用[J/CD]. 中华实验和临床感染病杂志(电子版), 2020, 14(4): 265-271. DOI: 10.3877/cma.j.issn.1674-1358.2020.04.001.
    [10]
    TUTTLEMAN JS, POURCEL C, SUMMERS J. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells[J]. Cell, 1986, 47(3): 451-460. DOI: 10.1016/0092-8674(86)90602-1.
    [11]
    ZHONG Y, HAN J, ZOU Z, et al. Quantitation of HBV covalently closed circular DNA in micro formalin fixed paraffin-embedded liver tissue using rolling circle amplification in combination with real-time PCR[J]. Clin Chim Acta, 2011, 412(21-22): 1905-1911. DOI: 10.1016/j.cca.2011.06.031.
    [12]
    XU CH, LI ZS, DAI JY, et al. Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA[J]. Chin Med J (Engl), 2011, 124(10): 1513-1516.
    [13]
    GUO Y, SHENG S, NIE B, et al. Development of magnetic capture hybridization and quantitative polymerase chain reaction for hepatitis B virus covalently closed circular DNA[J]. Hepat Mon, 2015, 15(1): e23729. DOI: 10.5812/hepatmon.23729.
    [14]
    WHITE RA 3rd, QUAKE SR, CURR K. Digital PCR provides absolute quantitation of viral load for an occult RNA virus[J]. J Virol Methods, 2012, 179(1): 45-50. DOI: 10.1016/j.jviromet.2011.09.017.
    [15]
    HINDSON BJ, NESS KD, MASQUELIER DA, et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number[J]. Anal Chem, 2011, 83(22): 8604-8610. DOI: 10.1021/ac202028g.
    [16]
    VOGELSTEIN B, KINZLER KW. Digital PCR[J]. Proc Natl Acad Sci U S A, 1999, 96(16): 9236-9241. DOI: 10.1073/pnas.96.16.9236.
    [17]
    JAHNE MA, BRINKMAN NE, KEELY SP, et al. Droplet digital PCR quantification of norovirus and adenovirus in decentralized wastewater and graywater collections: Implications for onsite reuse[J]. Water Res, 2020, 169: 115213. DOI: 10.1016/j.watres.2019.115213.
    [18]
    PAN Y, MA T, MENG Q, et al. Droplet digital PCR enabled by microfluidic impact printing for absolute gene quantification[J]. Talanta, 2020, 211: 120680. DOI: 10.1016/j.talanta.2019.120680.
    [19]
    YU F, YAN L, WANG N, et al. Quantitative detection and viral load analysis of SARS-CoV-2 in infected patients[J]. Clin Infect Dis, 2020, 71(15): 793-798. DOI: 10.1093/cid/ciaa345.
    [20]
    PROFAIZER T, SLEV P. A multiplex, droplet digital pcr assay for the detection of T-cell receptor excision circles and kappa-deleting recombination excision circles[J]. Clin Chem, 2020, 66(1): 229-238. DOI: 10.1373/clinchem.2019.308171.
    [21]
    CAVIGLIA GP, ABATE ML, TANDOIF, et al. Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: A new tool to detect occult infection[J]. J Hepatol, 2018, 69(2): 301-307. DOI: 10.1016/j.jhep.2018.03.021.
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