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Updated: Apr 20, 2026
Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection
Published on: November 7, 2018
A novel method for detection of HBVcccDNA in hepatocytes using rolling circle amplification combined with in situ PCR
Yanwei Zhong1, Shuangye Hu2, Chen Xu3
1Institute of Infectious Diseases, Beijing 302 Hospital, Beijing, China. zhongyanwei@126.com.
Insights
A new method combining rolling circle amplification (RCA) with in situ polymerase chain reaction (IS-PCR) sensitively detects hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in liver tissues, aiding antiviral drug development.
Area of Science:
- Hepatology
- Virology
- Molecular Biology
Background:
- Intrahepatic hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is crucial for viral persistence and recurrence.
- Accurate detection of cccDNA is vital for developing new antiviral therapies and understanding HBV infection.
- Existing in situ polymerase chain reaction (IS-PCR) methods for cccDNA detection lack sensitivity and specificity.
Purpose of the Study:
- To develop a novel, highly sensitive, and specific method for detecting HBV cccDNA in liver tissues.
- To evaluate the efficacy of rolling circle amplification (RCA) combined with IS-PCR for HBV cccDNA detection.
Main Methods:
- Developed a novel detection technique by combining rolling circle amplification (RCA) with in situ polymerase chain reaction (IS-PCR).
- Optimized RCA using specific primers for HBV cccDNA amplification in liver tissue sections from 26 HBV-infected patients.
- Employed plasmid-safe ATP-dependent DNase (PSAD) treatment and digoxigenin-labeled primers for enhanced IS-PCR detection.
Main Results:
- The combined RCA-IS-PCR method successfully detected HBV cccDNA in the nuclei of hepatocytes in 73.07% of patients.
- This novel method significantly improved the limit of detection compared to standard IS-PCR.
- Strong positive signals were observed even with low cccDNA copy numbers (2 copies/cell) in hepatocellular carcinoma (HCC) tissues, with no signal in negative controls.
Conclusions:
- RCA combined with IS-PCR is an effective and practical method for sensitive and specific detection of low-copy-number HBV cccDNA.
- This technique can accurately reflect cccDNA expression levels and their relationship with liver tissue pathology.
- The findings support the use of this method in antiviral drug development and understanding HBV pathogenesis.
Background:
Intrahepatic hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is the original template for HBV replication. The persistence of cccDNA is responsible for the recurrence of HBV infection. The detection of cccDNA can help the development of new antiviral drugs against HBV replication links, and reduce the resistance and recurrence as well as to discover extrahepatic HBV infection. In situ polymerase chain reaction (IS-PCR) can be used to determine the distribution and localization of cccDNA in liver tissues, but it is hampered by its low sensitivity and specificity. We developed a novel method to detect HBV cccDNA using rolling circle amplification (RCA) combined with IS-PCR.
Methods:
Biopsy liver tissues were obtained from 26 patients with HBV infection, including 10 chronic hepatitis B (CHB), 6 liver cirrhosis (LC) and 10 hepatocellular carcinoma (HCC) patients. Four pairs of primers were designed to mediating RCA for the first round amplification of HBV cccDNA specifically. The liver tissue sections from patients were treated by plasmid-safe ATP-dependent DNase (PSAD) prior to RCA. After RCA, HBV cccDNA was further amplified by a pair of selective primers labeled digoxigenin that target the gap region between the two direct repeat regions (DR1 and DR2) of the virus via IS-PCR.
Results:
HBVcccDNA was expressed and located in hepatocyte nucleus in 19 patients (73.07%). Compared with the IS-PCR, the introduction of RCA increase the limit of detection. RCA combined with IS-PCR yielded strong positive signals in HCC liver tissue in spite of low copy number cccDNA (2 copies of target sequence per cell), meanwhile, no positive signal was detected via negative control.
Conclusions:
RCA combined with IS-PCR is an effective and practicable method which could detect the presence of low copy number of cccDNA sensitively and specifically, and reflect the relationship between cccDNA expression level and liver tissue pathological characteristics.
