Hepatitis C Genotype Determination by Melting Curve Analysis with a Single Set of Fluorescence Resonance Energy Transfer Probes

Abstract

The genotype of hepatitis C virus (HCV) is a predictor of antiviral therapeutic response. We describe an approach for HCV genotype determination by real-time PCR and melting curve analysis. After automated nucleic acid extraction, we used reverse transcription-PCR in a block cycler to amplify nucleotides 6-329 of the 5'-untranslated region of HCV. The product was further amplified by single-tube real-time seminested PCR in a LightCycler instrument (Roche). The final product was analyzed by melting curves with the use of fluorescence resonance energy transfer (FRET) probes. The FRET sensor probe was directed at nucleotides 151-170 of type 1 HCV and was designed to distinguish types 1a/b, 2a/c, 2b, 3a, and 4, with melting temperatures (T(m)s) predicted to differ by 1 degrees C. Genotypes were compared in a blinded fashion with those of the INNO-LiPA(TM) test (Bayer Diagnostics) on 111 serum samples. In preliminary experiments, the Mg(2+) concentration was found to be critical in allowing clear separation of melting points, with the best separation at a Mg(2+) concentration of 2 mmol/L. The results for 111 samples clustered at expected T(m)s for genotypes 1a/b (n = 78), 2a/c (n = 2), 2b (n = 11), 3a (n = 14), and 4 (n = 2). Of the 111 samples, results for 110 were concordant with the comparison method at the level of type 1, 2, 3, or 4. Subtyping results were discordant for two samples, both of type 2. For 108 samples concordant with INNO-LiPA at the genotype and subtype levels, the mean T(m)s were 64.1, 59.5, 54.2, 52.6, and 50.1 degrees C for types 1a/b, 2a/c, 4, 2b, and 3a, respectively, with SDs of 0.2, 0.3, 0.3, 0.2, and 0.3 degrees C. All 78 samples identified as type 1 were concordant with results of the comparison method. Melting analysis with a single pair of FRET probes can rapidly provide information about HCV genotypes and identifies type 1 samples with high specificity.