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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Editorial Board Member: Gillery, Philippe / Kazmierczak, Steven / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Schlattmann, Peter / Whitfield, John B.

13 Issues per year

IMPACT FACTOR 2013: 2.955
Rank 5 out of 29 in category Medical Laboratory Technology in the 2013 Thomson Reuters Journal Citation Report/Science Edition

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Issues

Development, validation and evaluation of a homogenous one-step reverse transcriptase-initiated PCR assay with competitive internal control for the detection of hepatitis C virus RNA

Jens Mueller1 / Matthias Gessner2 / Anja Remberg3 / Jochen Hoch4 / Gerold Zerlauth5 / Peter Hanfland6

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Corresponding author: Jens Mueller, Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany Phone: +49-228-2876327, Fax: +49-228-2874762,

Citation Information: Clinical Chemical Laboratory Medicine. Volume 43, Issue 8, Pages 827–833, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2005.139, August 2005

Publication History

Received:
April 17, 2005
Accepted:
June 17, 2005

Abstract

Nucleic acid amplification testing for hepatitis C virus (HCV) RNA has become an essential tool for the prevention and clinical management of hepatitis C. We describe the development, validation and evaluation of a homogenous reverse transcriptase-initiated HCV-PCR assay with competitive internal control that is applicable to both the quantitative detection of HCV genomes in single patient samples and the screening of blood donations by mini-pool testing. For the implementation of a positive run control, a HCV RNA-positive plasma sample was calibrated against an international HCV RNA standard preparation. For quantification purposes, an in vitro-transcribed RNA calibrator sequence was used. The detection limit of the assay (95% positive cut-off) was determined by probit analysis and was calculated as 114 IU/mL. Comparable sensitivity to different HCV template sequences was verified for HCV genotypes 1–5. Quantitative test results correlated well with viral loads that had been previously determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay (n=53, R=0.943, p<0.001). During more than 5 years of blood donation testing, the specificity of the assay was found to be 99.51%. All assay components showed constant performance during this time period. In conclusion, we introduce a well-proven method that allows fast and reliable quantification of HCV genomes.

Keywords: hepatitis C virus (HCV); HCV RNA; internal control; real-time RT-PCR

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