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The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus

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Abstract

Real-time polymerase chain reaction (PCR) represents a favourable option for the detection of hepatitis C virus (HCV). A real-time reverse transcriptase PCR (RT-PCR) assay was developed as a qualitative diagnostic screening method for the detection of HCV using the ABI PRISM® 7500 Sequence Detection System. The primers and probe were designed to target the 5′-untranslated region of the hepatitis C viral genome. A second heterologous probe assay was developed for the detection of the haemagglutinin gene of phocine distemper virus (PDV) and was used as an internal control. A semi-automated HCV extraction method was also implemented using the ABI PRISM™ 6100 Nucleic Acid PrepStation. The HCV assay was optimised as a qualitative singleplex RT-PCR assay with parallel testing of the target and internal control. The assay results (n = 200) were compared to the COBAS AMPLICOR™ HCV Test v2.0 assay. The assay demonstrated a high rate of sensitivity (99%), specificity (100%) and an acceptable limit of detection (LOD) of 100 IU/ml. The development of a qualitative multiplex assay for the simultaneous detection of HCV and internal control indicates the same high rates of sensitivity and specificity. This sensitive real-time assay may prove to be a valuable method for the detection of HCV.

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Acknowledgements

Thanks go to Mr. Martin Lawrence from the Department of Clinical Microbiology, St. James’s Hospital, Dublin 8, Ireland, for his help with the statistics in this paper.

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Authors and Affiliations

  1. Department of Clinical Microbiology, St. James’s Hospital, Dublin 8, Ireland

    A. Clancy & B. Crowley

  2. Department of Medical Microbiology, Section Virology, UMCG, University Medical Center Groningen, De Brug, Hanzeplein 1, P.O. Box 30.001, 9700 RB, Groningen, The Netherlands

    H. Niesters

  3. School of Biological Sciences, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland

    C. Herra

Authors
  1. A. Clancy
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  2. B. Crowley
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  3. H. Niesters
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  4. C. Herra
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Correspondence to A. Clancy.

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Clancy, A., Crowley, B., Niesters, H. et al. The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus. Eur J Clin Microbiol Infect Dis 27, 1177–1182 (2008). https://doi.org/10.1007/s10096-008-0556-9

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  • DOI: https://doi.org/10.1007/s10096-008-0556-9

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