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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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