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Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control

Garson, JA; Grant, PR; Ayliffe, U; Ferns, RB; Tedder, RS; (2005) Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control. J VIROL METHODS , 126 (1-2) 207 - 213. 10.1016/j.jviromet.2005.03.001. Green open access

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Abstract

Quantitation of circulating hepatitis B virus (HBV) DNA is important for monitoring disease progression and for assessing the response to antiviral therapy. Several commercial and 'in house' assays for HBV DNA quantitation have been described but many of these have limitations of relatively low sensitivity and limited dynamic range. This study describes the development and evaluation of a FRET-based real-time PCR assay designed to overcome these limitations and to provide accurate quantitation of DNA from all eight genotypes of HBV (A-H). The assay employs a fully automated nucleic acid extraction system permitting high-sample throughput with minimal 'hands-on' time and incorporates a murine cytomegalovirus (mCMV) internal control to prevent false negative results and under-reporting due to unrecognised problems with viral lysis, DNA purification or PCR amplification. Sensitivity, assessed by Probit analysis at the 95% detection level, was 24.4 IU/ml, associated with an extremely wide dynamic range (similar to 9 log(10)). Coefficients of variation were low for both intra-assay and inter-assay variability (CV%, 7-11%) and quantitative data correlated well (R-2 = 0.97) with the Digene hybrid capture assay. This assay provides an ideal system for therapeutic monitoring and for studying the relationship between HBV viral load and stage of disease. (c) 2005 Elsevier B.V. All rights reserved.

Type: Article
Title: Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.jviromet.2005.03.001
Keywords: hepatitis B virus, HBV DNA, chronic hepatitis, real-time PCR, automation, therapeutic monitoring, SERUM HBV DNA, RAPID QUANTIFICATION, ASSAY, INFECTION, EXTRACTION, STANDARD, CAPTURE, ANTIGEN, RNA
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Infection and Immunity
URI: http://discovery.ucl.ac.uk/id/eprint/385
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