title: Quantitation of HCV RNA using real-time PCR and fluorimetry.
abstract: 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.
authors.affiliation: UMR2142 CNRS-bioMérieux, 21 rue Tony Garnier, 69007, Lyon, France.
authors.forename: F
authors.initials: F
authors.lastname: Komurian-Pradel
authors.forename: G
authors.initials: G
authors.lastname: Paranhos-Baccalà
authors.forename: M
authors.initials: M
authors.lastname: Sodoyer
authors.forename: P
authors.initials: P
authors.lastname: Chevallier
authors.forename: B
authors.initials: B
authors.lastname: Mandrand
authors.forename: V
authors.initials: V
authors.lastname: Lotteau
authors.forename: P
authors.initials: P
authors.lastname: André
mesh_terms: D005470:Fluorometry / Q000379:methods; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000032:analysis*; D015203:Reproducibility of Results
chemical_list: D012367:RNA, Viral