Real-time PCR for detection and quantitation of hepatitis B virus DNA

✍ Scribed by Ren Wei Chen; Heli Piiparinen; Mikko Seppänen; Pentti Koskela; Seppo Sarna; Maija Lappalainen

• Publisher: John Wiley and Sons
• Year: 2001
• Tongue: English
• Weight: 185 KB
• Volume: 65
• Category: Article
• ISSN: 0146-6615
• DOI: 10.1002/jmv.2027

✦ Synopsis

Abstract

A sensitive and reproducible real‐time PCR assay based on TaqMan technology was developed for the detection and quantitation of hepatitis B virus (HBV) DNA in serum, and compared with an “in‐house” qualitative PCR assay. HBV DNA was measured in 125 serum samples from 76 hepatitis B patients, consisting of 22 patients with an acute infection, 20 patients with a previous history of hepatitis B infection, and 34 patients with a chronic hepatitis B. Four patients with a chronic infection were treated with either an IFN‐alpha monotherapy or a combination of IFN‐alpha and lamivudine. Twenty‐nine sera from healthy individuals and non‐hepatitis B patients served as negative controls. The assay was validated by using a 10‐fold dilution series of the World Virological Quality Control (VQC) sample containing 3.73 × 10^7^ genome equivalents per ml. The detection limit for the real‐time PCR was 3.73 × 10^2^ genome equivalents per ml (geq/ml), while it was 3.73 × 10^3^ geq/ml for the in‐house PCR. The real‐time PCR assay had an 8‐logarithm dynamic range spanning from 10^2^ to 10^10^ geq/ml. In clinical serum samples, the real‐time PCR and the in‐house PCR detected HBV DNA in 81% (101/125) and 66% (83/125) of samples, respectively. HBV DNA was not detected among the negative controls by either of these assays. In conclusion, real‐time PCR is a sensitive, specific, and a reproducible approach for the detection and quantitation of HBV DNA in clinical serum samples, useful also for monitoring the efficacy of antiviral treatment. J. Med. Virol. 65:250–256, 2001. © 2001 Wiley‐Liss, Inc.