A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA

Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain abundant replicative intermediates of HBV DNA that share overlapping sequences but arranged in slightly different forms. HBV cccDNA can be detected by Southern blot or qPCR methods which involve enzymatic digestion. These assays are laborious, have limited sensitivity, or require degradation of cellular DNA (which precludes simple normalization). The method described in this protocol, cccDNA inversion quantitative (cinq)PCR, instead uses a series of restriction enzyme-mediated hydrolysis and ligation reactions that convert cccDNA into an inverted linear amplicon, which is not amplified or detected from other forms of HBV DNA. Importantly, cellular DNA remains quantifiable during sample preparation, allowing normalization and markedly improving precision. Further, a second linear fragment (derived from enzymatic digestion of a separate region of the HBV DNA genome and is present in all forms of HBV DNA) can be used to simultaneously quantify total HBV levels. Graphic abstract: Selective detection of HBV cccDNA and total HBV DNA using cinqPCR (Reproduced from Tu et al., 2020a ).