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

Citation
Rw. Chen et al., Real-time PCR for detection and quantitation of hepatitis B virus DNA, J MED VIROL, 65(2), 2001, pp. 250-256
Citations number
30
Categorie Soggetti
Clinical Immunolgy & Infectious Disease",Microbiology
Journal title
JOURNAL OF MEDICAL VIROLOGY
ISSN journal
01466615 → ACNP
Volume
65
Issue
2
Year of publication
2001
Pages
250 - 256
Database
ISI
SICI code
0146-6615(200110)65:2<250:RPFDAQ>2.0.ZU;2-C
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, consist ing of 22 patients with an acute infection, 20 patients with a previous his tory of hepatitis B infection, and 34 patients with a chronic hepatitis B. Four patients with a chronic infection were treated with either an IFN-alph a monotherapy or a combination of IFN-alpha and lamivudine. Twenty-nine ser a 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 x 10(7) gen ome equivalents per ml. The detection limit for the real-time PCR was 3.73 x 10(2) genome equivalents per ml (geq/ml), while it was 3.73 x 10(3) geq/m l 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 neg ative controls by either of these assays. In conclusion, real-time PCR is a sensitive, specific, and a reproducible approach for the detection and qua ntitation of HBV DNA in clinical serum samples, useful also for monitoring the efficacy of antiviral treatment. (C) 2001 Wiley-Liss, Inc.