Development of Magnetic Capture Hybridization and Quantitative Polymerase Chain Reaction for Hepatitis B Virus Covalently Closed Circular DNA

BACKGROUND Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) served as a vital role in the life cycle of the virus and persistent infection. However, specific and quantitative methods for cccDNA detection have not been available. OBJECTIVES Our aim was to develop and primarily evaluate a quantitative method for HBV cccDNA based on magnetic capture hybridization and quantitative PCR technology. MATERIALS AND METHODS The functionalized-nanoparticles specifically to capture HBV cccDNA, located on both sides of relaxed circle DNA (rcDNA) gap, were designed. Then, magnetic capture hybridization and quantitative PCR (MCH-qPCR) assay were developed and its performance was primarily evaluated with cccDNA standards and serum samples of patients with chronic hepatitis B. RESULTS Specific nanoparticles of cccDNA capture were prepared and a magnetic capture hybridization and quantitative assay method for cccDNA was developed successfully. The limit of detection was 90 IU/mL, and a good linear relationship in the range of 10(2)-10(6) IU/mL was revealed (r(2) = 0.994) with the MCH-qPCR. Compared with directly real-time PCR, a high content of HBV DNA did not affect the detection of cccDNA for the MCH-qPCR method, and there was no cross-reactivity between cccDNA and rcDNA. CONCLUSIONS The novel MCH-qPCR method has good sensitivity and specificity. It could meet the requirement of clinical routine detection.