A novel highly specific qPCR assay for distinguishing HBV cccDNA from rcDNA following bisulfite pretreatment

ABSTRACT The persistence of covalently closed circular DNA (cccDNA) underlies chronic hepatitis B virus (HBV) infection and remains as the major barrier to achieving a functional cure. An accurate and sensitive quantification of cccDNA is critical for evaluating therapeutic strategies. The conventional quantitative PCR (qPCR) lacks specificity to distinguish cccDNA from viral relaxed circular DNA (rcDNA) and other replicative intermediates, while nuclease pretreatment (e.g., plasmid-safe ATP-dependent nuclease, exonucleases I/III, or T5 exonuclease) may either incompletely digest rcDNA and/or risk over-digesting cccDNA. Here, we evaluated a novel patented bisulfite conversion-based cccDNA (BSC-cccDNA) qPCR platform that employs bisulfite treatment to disrupt rcDNA complementarity, followed by qPCR with primer sets targeting the rcDNA gap region, to enable selective amplification of cccDNA. Using synthetic HBV DNA controls and DNA samples extracted from HBV stable cell line and HBV-infected cells, we demonstrate that the BSC-cccDNA qPCR assay achieves high specificity, with tolerance of up to 10 9 rcDNA copies and sensitivity to 10 cccDNA copies per reaction. Comparative analyses with sensitive digital PCR (dPCR), cross-gap qPCR, and Southern blot further validated the sensitivity and accuracy of the BSC-cccDNA assay. These findings establish the BSC-cccDNA qPCR assay as a reliable tool for standardized cccDNA measurement and highlight its potential for both basic research and clinical applications. IMPORTANCE Accurate quantification of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is critical for understanding viral persistence and evaluating curative therapeutic strategies. However, current PCR-based assays lack sufficient specificity to distinguish cccDNA from relaxed circular DNA (rcDNA). Enzymatic pretreatment approaches to remove rcDNA introduce variability, risk incomplete digestion, and potential cccDNA loss, thereby compromising quantitative accuracy. We evaluated a novel bisulfite conversion-based cccDNA (BSC-cccDNA) qPCR assay that chemically disrupts rcDNA complementarity, enabling highly specific cross-gap amplification without reliance on nuclease digestion. Using synthetic substrates, cell-derived HBV DNA, and infected hepatocyte models, we demonstrate that this assay tolerates up to 10⁹ rcDNA copies while detecting as few as 10 cccDNA copies, with performance validated against Southern blot and dPCR. By improving both specificity and quantitative reliability, the BSC-cccDNA platform provides a potential standardized tool for accurate cccDNA measurement in basic research and therapeutic development.