Immiscible phase filter extraction and equivalent amplification of genotypes 1-6 of hepatitis C RNA: The building blocks for point-of-care diagnosis

•A novel HCV RT-qPCR assay that equivalently detects genotypes 1-6 was developed.•RNA was isolated by automated immiscible phase filter extraction.•The limit of quantification was 30 IU/ml with 200 μl plasma sample volume.•Laboratory validation of 61 specimens demonstrated 100% sensitivity and specificity.

The lack of hepatitis C virus (HCV) diagnostic tests designed for use in decentralized settings is a major obstacle for providing access to treatment and prevention services particularly in low and middle income countries. Here we describe the development and validation of two building blocks of the HCV Quant Assay, a test in development for point-of-care use: 1) an RT-qPCR assay with noncompetitive internal control that equivalently detects the 6 major HCV genotypes and 2) an automated sample prep method using immiscible phase filter technology. This novel assay has wide dynamic range of HCV quantification and a limit of detection of 30 IU/ml with 200 μl specimen volume. In a preliminary study of 61 clinical specimens, the HCV Quant Assay demonstrated 100% sensitivity and specificity and gave comparable viral load results across 4 logs of IU/ml when compared to the Abbott RealTime HCV Assay.