A novel fully automated system for quantification of Hepatitis B virus DNA using magnetic bead-based method combined with real-time PCR

Abstract

Background: Hepatitis B virus (HBV) infection is a major global health problem and causes liver damage as cirrhosis of the liver or liver cancer. Development of an accurate, sensitive and reproducible detection method for detecting and monitoring HBV DNA is very necessary and urgent.

Objectives: The aims were to evaluate the analytical performances of the fully automated Pre-NAT system comparing to domestic assay, and to explore the role of highly sensitive quantification of HBV DNA in the management of chronic HBV infection.

Study design: Pseudo-viral particles at high HBV DNA concentration were serially diluted to assess linear range. Accuracy and lower limit of detection were assessed by determining a panel of HBV standard substance. HBV DNA positive clinical specimen and internal quality control were measured 20 times to evaluate precision and reproducibility. 20 non HBV-infected specimens were used for the specificity assay. 96 chronic hepatitis B samples were quantified for HBV DNA to evaluating the correlation between the new test and Da-an assay. HBV serological markers were detected using ELISA method.

Results: Pre-NAT quantitated HBV DNA levels covered a wide dynamic range (10 logs) with a close correlation between expected and observed values (r=0.999, P<0.05), satisfactory precision and higher specificity. The lower detection limit was 20IU/mL. Comparability assay showed Pre-NAT had a good agreement with but more sensitive than Da-an assay (t=0.149, P>0.05). HBV DNA level was partially correlated to but more reliable and sensitive than serological evidence in reflecting the viral level.

Conclusion: This novel fully-automated real-time PCR assay exhibits good analytical and clinical performances for highly sensitive detection of HBV DNA. It is well suited for monitoring antiviral responses and making treatment strategies according to current clinical practice guidelines for the management of chronic HBV infection.

Keywords: Automated; Hepatitis B virus; High sensitivity; Quantification; Real-time polymerase chain reaction.