Related Experiment Video
Updated: Aug 5, 2026
Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection
Published on: November 7, 2018
ddOTs: A multiplexed quantitative ddPCR approach for resolving overlapping hepatitis B virus transcripts to decipher
Nazim Sarica1, Oceane Lopez1, João Diogo Dias1
1Institut de Génétique Humaine. Laboratoire de Virologie Moléculaire, Université de Montpellier, CNRS, Montpellier, France.
Insights
Hepatitis B virus (HBV) infection is a global health issue. A new ddPCR for Overlapping Transcripts (ddOTs) assay precisely quantifies HBV RNA, aiding research into viral transcription and drug development.
Area of Science:
- Hepatology
- Virology
- Molecular Biology
Background:
- Chronic Hepatitis B virus (HBV) infection is a major global health concern, leading to severe liver diseases like hepatocellular carcinoma.
- The viral minichromosome, covalently closed circular DNA (cccDNA), is crucial for HBV persistence and transcription.
- Quantifying HBV RNA is challenging due to overlapping viral open reading frames, hindering the understanding of transcriptional regulation.
Purpose of the Study:
- To develop a novel method for precise quantification of all major HBV RNA species, including splice variants.
- To overcome the limitations of existing methods in deconvoluting overlapping transcripts.
- To enable detailed analysis of cccDNA-driven transcription and facilitate antiviral drug development.
Main Methods:
- Development of a multiplexed assay, ddPCR for Overlapping Transcripts (ddOTs).
- Utilizing the high specificity and sensitivity of droplet digital PCR (ddPCR).
- Simultaneous quantification of diverse HBV RNA species from biological samples.
Main Results:
- The ddOTs assay successfully quantifies all major HBV RNA species with high specificity and sensitivity.
- The method effectively deconvolutes overlapping transcripts, overcoming previous quantification challenges.
- Demonstrated ability to interrogate individual promoter/enhancer activities and RNA stability.
Conclusions:
- The ddOTs assay provides a cost-effective and scalable solution for precise HBV RNA profiling.
- This breakthrough tool advances the study of cccDNA biology and HBV transcriptional regulation.
- Enables accelerated development of novel antiviral therapies for chronic HBV infection.
Abstract:
Chronic hepatitis B virus infection persists as a global health crisis, driving life-threatening liver pathologies such as hepatocellular carcinoma. Central to HBV's resilience is the covalently closed circular DNA (cccDNA), a viral minichromosome that orchestrates viral transcription and sustains infection. Dissecting cccDNA-driven transcription remains a formidable challenge due to the dense overlap of viral open reading frames, which generates RNA transcripts with shared sequences, complicating precise quantification and hindering efforts to unravel HBV's transcriptional regulation. To address this bottleneck, we developed a multiplexed assay named ddPCR for Overlapping Transcripts (ddOTs) capable of simultaneously quantifying all major HBV RNA species, including splice variants, with unprecedented specificity and sensitivity. This method overcomes current limitations by leveraging the high-resolution power of ddPCR to deconvolute overlapping transcripts, enabling direct interrogation of individual promoter/enhancer activities and RNA stability. By offering a cost-effective, scalable solution for precise RNA profiling in rare biological samples, this breakthrough tool unlocks new avenues for exploring cccDNA biology and accelerating antiviral drug development.
