Absolute quantitation of different genotypes of hepatitis C virus RNA in clinical samples by a modified real-time PCR method

Background: Our aim was to develop a quantitative real-time polymerase chain reaction (qPCR) assay for the quantitation of hepatitis C virus (HCV) RNA samples from patients infected with different HCV genotypes. Methods: A standard curve was generated by amplification of serial dilutions of HCV-plasmid (pFKI389-NS3-3') harboring genotype 1b HCV-subgenomic replicon. Samples from 15 HCV-infected patients (genotypes 1, 2, and 3) were analyzed to quantify HCV-RNA by qPCR with primers and probes specific for the 5'-UTR viral region. Results: The HCV qPCR assay had a sensitivity of 100 copies/reaction with a dynamic range of detection between 10 2-20 � 10 6 HCV copies. The assay was highly reproducible with a low coefficient of variation. We observed that the HCV genotypes included could be identified by our method. Conclusions: Our results showed that this modified qPCR assay provides a valid platform for quantifying HCV-RNA, combining good analytical sensitivity with a wide dynamic range and high reproducibility