Evaluation of an automated, highly sensitive, real-time PCR-based assay (COBAS Ampliprep™/COBAS TaqMan™) for quantification of HCV RNA

BackgroundDiagnosis of hepatitis C virus (HCV) infection and its therapy is based on qualitative and quantitative measurement of HCV RNA.ObjectivesA new assay that employs automated specimen extraction and real-time RT-PCR (COBAS Ampliprep™/COBAS TaqMan™, “CAP/CTM”, Roche Diagnostics, Pleasanton, USA) was designed for linear quantification and highly sensitive detection of HCV RNA.Study designThe performance characteristics of CAP/CTM were compared to standard RT-PCR-based COBAS Amplicor Monitor 2.0 (CAM) assay in a multicenter study.ResultsThe limit of detection of CAP/CTM was 7.4 IU/ml (95% CI 6.2–10.6) and clinical specificity was 99%. The linear range of HCV RNA quantification by CAP/CTM was between 28 and 1.4 × 107 IU/ml, with a correlation coefficient between expected and observed results of >0.99. A fivefold dilution of serum- or plasma-samples showed a linear correlation of HCV RNA levels in undiluted and diluted samples. Analyses of the mean intra- and inter-assay imprecision within the linear range of quantification showed a coefficient of variation of 3% and 3%, respectively. HCV genotypes 1a/b, 2b, 3a, 4, 5 and 6 were equally quantified by the CAP/CTM and CAM assay with mean deviations ranging from −0.29 log10 to 0.32 log10 IU/ml. HCV RNA quantification by CAP/CTM and CAM was highly concordant (correlation coefficient of 0.96).ConclusionsThe CAP/CTM assay is a reliable and robust assay for highly sensitive detection and quantification of HCV RNA within a broad linear range.