ddOTs: A multiplexed quantitative ddPCR approach for resolving overlapping hepatitis B virus transcripts to decipher...

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.