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Updated: May 31, 2026
Real-Time Polymerase Chain Reaction-Based Detection and Quantification of Hepatitis B Virus DNA
Published on: December 15, 2023
Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed
Chun-Hai Xu1, Zhao-Shen Li, Jun-Ying Dai
1Department of Infectious Diseases, Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150086, China.
Insights
A new nested real-time PCR assay effectively detects hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in patient cells. This method aids in monitoring treatment efficacy by quantifying HBV cccDNA levels, crucial for complete viral clearance.
Area of Science:
- Hepatology
- Virology
- Molecular Biology
Background:
- Complete clearance of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is essential for successful treatment.
- Detecting HBV cccDNA in patients remains a significant clinical challenge.
- HBV cccDNA serves as the template for viral DNA replication.
Purpose of the Study:
- To develop a nested real-time quantitative polymerase chain reaction (PCR) assay.
- To detect HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs).
Main Methods:
- Developed a nested real-time PCR assay utilizing specific primers and a TaqMan probe.
- Incubated samples with mung bean nuclease to eliminate relaxed circular DNA (rcDNA).
- Quantified HBV cccDNA levels using a positive standard after amplification.
Main Results:
- The assay demonstrated a successful linear range from 3.0 × 10^2 to 3.9 × 10^8 copies/ml.
- HBV cccDNA was detected in 9 out of 25 PBMC samples and 3 out of 7 MMNC samples from patients.
- All PBMC samples from healthy controls (21 samples) tested negative for HBV cccDNA.
Conclusions:
- The developed nested real-time quantitative PCR is a viable tool for detecting HBV cccDNA.
- This assay can be important for monitoring hepatitis B patients.
- Accurate detection of cccDNA is crucial for assessing treatment outcomes.
Background:
Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus (HBV) DNA replication, the covalently closed circular HBV DNA (cccDNA) can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction (PCR) assay for detecting HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs).
Methods:
Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA (rcDNA), two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard.
Results:
The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0 × 10(2) copies/ml to 3.9 × 10(8) copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative.
Conclusion:
The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.
