Quantification of intrahepatic total HBV DNA in liver biopsies of HBV-infected patients by a modified version of COBAS® Ampliprep/COBAS®TaqMan HBV test v2.0

Abstract

Intrahepatic total HBV DNA (it-HBV DNA) level might reflect the size of virus reservoir and correlate with the histological status of the liver. To quantitate it-HBV DNA in a series of 70 liver biopsies obtained from hepatitis B chronic patients, a modified version of the COBASAmpliprep/COBASTaqMan HBV test v2.0 was used for this purpose. The linearity and reproducibility of the modified protocol was tested by quantifying serial dilutions of a full-length HBV containing plasmid and it-HBV DNA from a reference patient. A good linear trend between the expected values and those generated by the assay was observed at different concentrations of both plasmid and reference patient (R = 0.994 and 0.962, respectively). Differences between the values obtained in two independent runs were ≤0.3 log IU for the plasmid and ≤0.6 log IU/mg for the reference patient, showing a high inter-run reproducibility. In the 70 liver biopsies, it-HBV DNA level ranged from 1.4 to 5.4 log IU/mg, with a good linearity and reproducibility between the values obtained in two runs [R = 0.981; median (IQR) difference of it-HBV DNA 0.05 (0.02-0.09) IU/mg]. The modified COBASAmpliprep/COBASTaqMan HBV test v2.0 allows an accurate quantitation of it-HBV DNA. Its determination may have prognostic value and may be a useful tool for the new therapeutic strategies aimed at eradicating the HBV infection.

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“…To perform PCR according to the manufacturer's protocol, HBV DNA was extracted from 600 µl of serum or cell-cultured supernatant using a DNA Extraction Reagent of HBV Fluorescence Quantitative Polymerase Chain Reaction kit (Fosun Pharma Diagnostics, China). Furthermore, viral and genomic DNA was extracted from 50 mg of BC tissue (stored in liquid nitrogen) using the DNeasy Blood & Tissue kit (Qiagen) as previously described (20). Extracted DNA (20 µl) was used as a template and was mixed with 30 µl PCR Master Mix (Fosun Pharma Diagnostics), containing tricine buffer, potassium hydroxide, dNTPs, HBV-specific primers and fluorescent-labeled oligonucleotide probes.…”