Real-time multiplex PCR assay to quantify hepatitis C virus RNA in peripheral blood mononuclear cells

Ultrasensitive methods to measure very low levels of hepatitis C virus (HCV) RNA in biological samples may have diagnostic and prognostic significance and be useful to evaluate the response to antiviral treatment. A sensitive assay to quantify HCV RNA in peripheral blood mononuclear cells (PBMCs) was developed and validated using the iCycler iQ™ Detection System (Bio-Rad) coupled with TaqMan^® chemistry. HCV was co-amplified with the endogenous control glyceraldehyde-3-phosphate dehydrogenase in a multiplex reaction. Calculated PCR amplification efficiencies for both target and control genes were used in a mathematical model for relative quantitation of HCV RNA. A linear relationship between input RNA and C_T values over 6 log dilutions was observed for both HCV- and GAPDH-specific products (R² ≥ 0.99). As few as 1.5 IU/reaction could be detected, with high accuracy (CV ≤ 3.94%) and reproducibility (CV ≤ 2.20%). Quantitation of HCV RNA levels ranging from 10³ to 10⁷ IU/ml as measured in 47 plasma samples was highly correlated with values obtained by the COBAS Amplicor™ HCV Monitor test, v2.0 (Roche) (R² = 0.977). In conclusion, this assay provides an excellent tool to determine accurately HCV kinetics in PBMCs during antiviral therapy and to assess the long-term significance of different patterns of response to treatment.