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Clinical Chemistry and Laboratory Medicine (CCLM)

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

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Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

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Volume 43, Issue 8 (Aug 2005)

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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 Mueller
  • Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Bonn, Germany
/ Matthias Gessner
  • Molecular Biology, Baxter AG, Vienna, Austria
/ Anja Remberg
  • Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Bonn, Germany
/ Jochen Hoch
  • Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Bonn, Germany
/ Gerold Zerlauth
  • Molecular Biology, Baxter AG, Vienna, Austria
/ Peter Hanfland
  • Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Bonn, Germany
Published Online: 2011-09-21 | DOI: https://doi.org/10.1515/CCLM.2005.139

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 114IU/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 5years 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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About the article

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,


Received: 2005-04-17

Accepted: 2005-06-17

Published Online: 2011-09-21

Published in Print: 2005-08-01



Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/CCLM.2005.139. Export Citation

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