Quantitation of HCV RNA using real-time PCR and fluorimetry.
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.
Komurian-Pradel
F
F
UMR2142 CNRS-bioMérieux, 21 rue Tony Garnier, 69007, Lyon, France.
Paranhos-Baccalà
G
G
Sodoyer
M
M
Chevallier
P
P
Mandrand
B
B
Lotteau
V
V
André
P
P
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
D012367:RNA, Viral