Quantitation of HCV RNA using real-time PCR and fluorimetry

J Virol Methods. 2001 Jun;95(1-2):111-9. doi: 10.1016/s0166-0934(01)00300-7.


Real-time PCR technology may provide an accurate and sensitive method to quantify hepatitis C virus (HCV) RNA. So far, studies have been carried out using the Taqman technology with the ABI Prism 7700 sequence detector. An alternative and simple real-time PCR assay is described with no probe requirement, based on the SYBR Green I dye and LightCycler fluorimeter. Amplicon synthesis was monitored continuously by SYBR Green I dye binding to double stranded DNA during PCR of the 5' HCV non-coding (NC) region. Specificity was verified by amplicon melting temperatures. An external standard curve was constructed with serial 10 fold dilutions of a modified synthetic HCV 5' NC RNA. A wide range linear relationship (up to 3.7x10(9) copies/ml) was observed between number of PCR cycle needed to detect a fluorescent signal and number of RNA copy. Intra- and inter-assay coefficients of variation were 0.7 to 2.1 and 3.7% respectively, indicating good reproducibility of the method. Thirty-three HCV positive sera of different genotypes were quantified by this method and gave similar but more sensitive results compared to the branched DNA (bDNA) technology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorometry / methods
  • Hepacivirus / genetics*
  • Humans
  • Polymerase Chain Reaction / methods
  • RNA, Viral / analysis*
  • Reproducibility of Results


  • RNA, Viral