title: 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.
authors.affiliation: Research Centre for Genetic Engineering and Biotechnology "Georgi D Efremov", Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000 Skopje, Republic of Macedonia. Electronic address: katarina@manu.edu.mk.
authors.forename: Katarina
authors.initials: K
authors.lastname: Davalieva
authors.affiliation: Research Centre for Genetic Engineering and Biotechnology "Georgi D Efremov", Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000 Skopje, Republic of Macedonia.
authors.forename: Sanja
authors.initials: S
authors.lastname: Kiprijanovska
authors.affiliation: Research Centre for Genetic Engineering and Biotechnology "Georgi D Efremov", Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000 Skopje, Republic of Macedonia.
authors.forename: Dijana
authors.initials: D
authors.lastname: Plaseska-Karanfilska
mesh_terms: D057205:Automation, Laboratory / Q000379:methods; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D019562:Viral Load
keywords: HCV detection; HCV genotyping; HCV quantification; Real-time RT-PCR; Type-specific RT-PCR