Fast, reliable and low cost user-developed protocol for detection, quantification and genotyping of hepatitis C virus

Abstract

Early detection and genotyping of HCV infection is important for disease management. It is important to develop fast and cost-effective semi-automated techniques allowing an accurate and reproducible detection, quantification and genotyping of HCV. The proposed protocol includes a real-time RT-PCR assay for HCV detection/quantification and a type-specific one-tube RT-PCR assay for genotyping. Both assays detect genotypes 1-4 as intended. The limit of detection was 112IU/ml for the real-time assay and 600±278IU/ml (mean±SD) for the genotyping assay. Concordance between the real-time assay and AMPLICOR HCV v2.0 test was 100%. The real-time assay has wide linear dynamic range of detection and quantification and excellent reproducibility with 2% and 0.75% coefficients of variations, for inter- and intra-assays, respectively. The observed correlation with AMPLICOR HCV Monitor v2.0 kit was linear with the correlation coefficient of 0.988. The diagnostic specificity and sensitivity of the genotyping assay, tested on 102 samples, was 100% and 95%, respectively. The overall procedure of HCV diagnosis is completed within 6h in a closed system with minor contamination risk. In addition to being fast and cost-effective, this approach is reproducible and avoids post-PCR enzymatic and hybridization steps while detecting and genotyping HCV with high clinical sensitivity.

Keywords: HCV detection; HCV genotyping; HCV quantification; Real-time RT-PCR; Type-specific RT-PCR.