Quantitative detection of hepatitis B virus DNA in serum by a new rapid real‐time fluorescence PCR assay

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Abstract
A sensitive and accurate HBV DNA quantification assay is essential for monitoring hepatitis B virus (HBV) replication. This study evaluated a real‐time PCR method performed in the LightCyclerTM analyser for quantitative HBV DNA assay. HBV DNA results with this method were compared with those obtained using a branched‐chain DNA (bDNA) solution hybridization assay. Real‐time PCR was performed using two adjacent fluorescently labelled probes and primers corresponding to the HBV core gene. The same standard employed in the bDNA assay was used for calibration. Serum samples came from 193 HBV surface antigen (HBsAg)‐positive patients (34 HBV e antigen (HBeAg)‐positive and 93 with antibody to HBeAg (anti‐HBe)), and 66 asymptomatic HBV carriers. In addition, we analysed serum samples from 8 anti‐HBe‐positive patients who had been receiving lamivudine treatment for more than three years. A linear standard curve was seen in the range from 103 to 108 copies/mL. In the reproducibility analysis, intra‐assay coefficient of variation (CVs) at two known HBV DNA concentrations were 4% and 2% and interassay CVs were 6% and 4%. The median of serum HBV DNA by real‐time PCR was 9.2 × 108 copies/mL in HBeAg‐positive patients with persistently elevated alanine aminotransferase (ALT) levels, 1.3 × 107 copies/mL in anti‐HBe‐positive cases with persistently elevated ALT levels, 3.7 × 104 copies/mL in anti‐HBe‐positive patients with fluctuating ALT levels and 104 copies/mL in asymptomatic HBV carriers. The differences in HBV DNA levels among the various groups studied were statistically significant (P < 0.05). The cut‐off between chronic hepatitis patients and asymptomatic carriers was found to be at a serum HBV DNA concentration of 5 × 104 copies/mL. Of the 109 serum samples with a viral load < 7.5 × 105 (negative by bDNA assay) 44 (40%) were positive by real‐time PCR: 24 (56%) chronic hepatitis and 20 (33%) asymptomatic carriers. There was a positive association between HBV DNA levels determined by real‐time PCR and ALT levels (P < 0.05), which was not observed with the bDNA assay for HBV DNA quantification. At 12 months of lamivudine treatment, 6 patients (75%) showed HBV DNA levels < 5 × 104 copies/mL (range < 103–2 × 103), significantly lower than at baseline. At 36 months, 2 of 8 (25%) showed HBV DNA levels persistently lower than 5 × 104 copies/mL (1.7 × 103, 6 × 103). The LightCycler quantitative real‐time PCR is a practical, sensitive, reproducible single‐tube assay with a wide dynamic range of detection. The assay is automatic except for DNA extraction and the running time is only 70 min. The LightCycler real‐time PCR is useful for identifying different states of HBV infection and for evaluating the efficacy of viral therapy.