Journal of Virological Methods 135 (2006) 83–90 Quantitation of hepatitis B virus DNA by real-time PCR using internal amplification control and dual TaqMan MGB probes Kavita S. Lole, Vidya A. Arankalle ∗ Hepatitis Division, National Institute of Virology, Pune, India Received 13 July 2005; received in revised form 7 February 2006; accepted 9 February 2006 Available online 23 March 2006 Abstract Hepatitis B virus (HBV) DNA quantitation is used extensively for monitoring of antiviral treatment of HBV infection. A real-time PCR assay was developed using a TaqMan minor groove binder probe and primers corresponding to HBV pre-core region for HBV DNA quantitation. A 228bp fragment from this genomic region of HBV was cloned and serial dilutions of plasmid DNA were used as an external DNA standard. Comparison of the real-time PCR quantitation results from 35 clinical serum samples with those obtained by COBAS Amplicor HBV DNA monitor kit (Roche Diagnostics) revealed a significant correlation (r = 0.92) for all the samples. The assay showed wide dynamic linear range between 2.5 × 10 2 and 2.5 × 10 10 copies/ml serum. Sera from 25 healthy individuals tested negative indicating the high specificity of the assay. The median coefficients of variation for both intra- and inter-experimental variability were 4.9% and 10.6%, respectively, which indicated remarkable reproducibility. An internal amplification control (IC) was developed to detect the presence of PCR inhibitors in the samples to avoid false negative results. The IC had the same primer binding sites but different internal sequence and it competed with the virus-derived target. The optimum concentration of IC was found to be 100 copies/reaction. The assay was validated by testing serial dilutions of the WHO international HBV DNA standard. Since conserved regions were considered during primer and probe design, the assay should be applicable to all HBV genotypes. The real-time assay will be useful for monitoring HBV-infected patients in routine diagnostic laboratories and in clinical practice enabling analysis of a wide dynamic range of HBV DNA in a single, undiluted sample. © 2006 Elsevier B.V. All rights reserved. Keywords: HBV DNA quantitation; Real-time PCR; Internal control 1. Introduction Hepatitis B virus (HBV) can either cause acute self-resolving hepatitis or asymptomatic chronic state or may lead to severe illness such as fulminant hepatitis, chronic hepatitis, liver cir- rhosis, and hepatocellular carcinoma (HCC) (Beasley, 1988). Hepatitis B is an important global problem and it is estimated that worldwide there are about 400 million people with chronic HBV infection. In India, nearly a third of the patients with acute viral hepatitis, two thirds of the cases of chronic liver disease and hepatocellular carcinoma are caused by HBV and the overall estimated carrier rate is 4.7% (Thyagarajan et al., 1996). HBV was shown to be an important contributing factor to the etiology ∗ Corresponding author at: Hepatitis Division, National Institute of Virology, 20-A, Dr. Ambedkar Road, Pune 411001, India. Tel.: +91 20 2612 7301; fax: +91 20 2612 2669. E-mail address: varankalle@yahoo.com (V.A. Arankalle). of HCC in southern and northern India (Jayshree et al., 2003; Sarin et al., 2001). Multiple HBV genotypes (A, C and D) circu- late in India (Arankalle et al., 2003; Gandhe et al., 2003; Thakur et al., 2002; Vivekanandan et al., 2004). Interferon alfa, lamivudine or combinations of both drugs are the therapeutic options available for the management of HBV infected patients (Dienstag et al., 1995; Janssen et al., 2005; Wong et al., 1993). Quantitative and sensitive determina- tion of viral DNA can provide indirect evidence on the level of viral replication, the degree of infectivity and changes of viral DNA during the course of infection. The efficacy of antiviral therapy is evaluated in terms of changes in the levels of HBV DNA (Mommeja-Marin et al., 2003). HBV DNA in serum can be monitored by several commercially available assays such as the Abbott solution hybridization assay, the Digene first and second generation hybrid capture assays, the Chiron quan- tiplex bDNA assay, the COBAS Amplicor HBA DNA monitor assay (Roche diagnostics), the VERSANT HBV DNA 1.0 assay (bDNA) (Bayer Health care diagnostics). However, these assays 0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.02.004