The three-in-one (3io) method for total and cccDNA quantification in small-size samples from HBV infected patients

Aims: Hepatitis B virus (HBV) is a member of Hepadnaviridae family comprising viruses replicating via particular reverse transcription mechanism. The first step in HBV replication is filling the single stranded fragments of the viral genome into the completely double stranded form and formation of covalently closed circular DNA (cccDNA). cccDNA exists in the liver as an independent episomal molecule serving as a long-lasting reservoir of the virus. It acts as a template for all genomes of viral progeny. It is an excellent though evasive parameter for monitoring the course of liver disease and treatment efficiency.

Method: We developed a new approach for serum and intracellular HBV-DNA quantification based on the ROCHE LightCycler480 II® platform. A novel plasmid containing an HBV fragment and β-actin gene (ACTB) was cloned as a standard and TaqMan® probes labeled with different fluorescent dyes were designed for the detection of target sequences.

Results: In one reaction, total (genomic) HBV-DNA, cccDNA and ACTB gene can be successfully quantified with a lower limit of quantification of 48 copies per 5ul extracted DNA per 20µl PCR reaction for all sequences. The correlation coefficient (R) is 0.96 (<0.0001), 0.94 (<0.0001) and 0.95 (<0.0001) for total HBV, cccDNA and ACTB, respectively.

The method was tested on 10 DNA samples from FFPE preserved liver tissue of HBV infected patients. All tissue samples were positive for genomic HBV-DNA and 8 of them gave signals in cccDNA quantification. There was a strong correlation between the concentration of total HBV-DNA and cccDNA (R=0.86, p=0.0059) consistent with previously published data. The costs for a 96-well plate are 95.50 Euro or approximately 1/3 of the cost of the conventional method.

Conclusion: The three-in-one method allows the quantification of viral DNA in serum and nucleic acid from fresh and FFPE preserved liver tissue in a single step. Therefore less liver tissue, faster data acquisition, a lowered error and significantly lower costs are the advantages of the single-step qPCR quantification.