Application of a high-resolution melting technique for the rapid detection of partial replacement of HCV-1b by HCV-1a after PEG-IFN alpha/RBV therapy

摘要

A modified method which can be used for the rapid screening of mutations in the protein kinase R-binding domain (PKR-BD) region and the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) is described. This method is based on a high-resolution melting (HRM) technique used for genotyping single nucleotide polymorphisms and allows the detection of single nucleotide substitutions in the DNA sequence by measuring its T-m. The modified method, in addition to precisely measuring the T-m, allows the recording of the melting curve of the investigated cDNA fragment, which can provide provisional information about the number of different quasi-species present in the sample. The HRM analysis of the amplified cDNAs encoding the PKR-BD and HVR1 allowed the detection of partial replacement of HCV-1b by HCV-1a subspecies in one of our patients, as well as evaluation of the effectiveness of pegylated interferon alpha/ribavirin (PEG-IFN alpha/RBV) therapy. The HRM technique has never been used for the rapid screening of sequence variations in these regions and may be used for a similar purpose in any viral genome.