title: Real-time polymerase chain reaction assay based on high-resolution melting analysis for the determination of the rs12979860 polymorphism involved in hepatitis C treatment response.
abstract: Treatment of chronic hepatitis C is associated with varying success rates, substantial medical costs and serious side effects. Several host polymorphisms have been identified near the IL28B gene, of which the homozygous rs12979860 CC was found to be associated with significantly favourable treatment outcome. To determine accurately the presence of this variant, a real-time PCR assay with detection based on post-PCR high-resolution melting analysis (HRM) was developed. The assay, performed on a Roche LightCycler 480, was able to differentiate among all three rs12979860 variants (CC, TT, CT) across a dynamic range of four orders of magnitude (10(3)-10(7)). The sensitivity of the assay was determined at a 95% detection level to be 44.6 copies/reaction for the CC variant, 57.1 copies/reaction for the TT variant and 47.4 copies/reaction for the CT variant. Input DNA amount above 10(3) copies/reaction is recommended for clinical samples to ensure optimal performance. Clinical performance was assessed on 60 clinical samples by comparative testing using the assay and Sanger sequencing. Concordant results were obtained for all 60 samples, showing high specificity of the assay. The novel assay can be easily added to the testing repertoire of virological laboratories, providing additional information for physicians treating chronic hepatitis C patients.
authors.affiliation: Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia. anja.kovanda@mf.uni-lj.si
authors.forename: Anja
authors.initials: A
authors.lastname: Kovanda
authors.forename: Mario
authors.initials: M
authors.lastname: Poljak
mesh_terms: D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000188:drug therapy / Q000235:genetics*; D006801:Humans; D007372:Interferons; D007378:Interleukins / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D011110:Polymorphism, Genetic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017423:Sequence Analysis, RNA; D044366:Transition Temperature; D016896:Treatment Outcome; D000095022:Interferon Lambda
chemical_list: D007372:Interferons; D007378:Interleukins; C000624353:interferon-lambda, human; D000095022:Interferon Lambda