Use of duplex mutation primers for real-time PCR quantification of hepatitis C virus RNA in serum

Background: The duplex mutation primers offer many advantages over other multi-labeled probes for real-time detection of amplification products. Objectives: To develop and validate a novel real-time PCR for quantification of HCV RNA based on the duplex mutation primers technology. Materials and Methods: The duplex mutation primers were selected in the highly conservative 5' non-coding region (5'NCR) of the HCV RNA. The assay was validated with the Viral Quality Control panel, which also includes Chinese HCV RNA standards. Results: The detection limit was 57 IU/mL, and a good linear correlation in the range of 102-108 IU/mL was revealed (r2 = 0.999) with the novel method. This assay has a dynamic range of at least 8 log10 without the need for specimen dilution, good clinical intra- and inter-run precision, and excellent correlation with a commercially available assay(r2 = 0.95). Conclusions: The high sensitivity, wide linear range, and good reproducibility, combined with low cost, make this novel quantitative HCV real-time PCR assay particularly well suited for application to clinical and epidemiological studies.