Identifying hepatitis C virus (HCV) genotypes has become increasingly important for determining clinical course and the outcome of antiviral therapy. Here we describe the development of restriction fragment mass polymorphism (RFMP) analysis, a novel matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) assay suitable for high-throughput, sensitive, specific genotyping of multiple HCV species. Methods: The assay is based on PCR amplification and mass

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... nt of oligonucleotides containing genotype-specific motifs in the 5 untranslated region, to which a type IIS restriction endonuclease recognition was introduced by PCR amplification. Enzymatic cleavage of the products led to excision of multiple oligonucleotide fragments representing variable regions whose masses were determined by MALDI-TOF MS. Results: The RFMP assay identified viral genotypes present at concentrations as low as 0.5% and reliably determined their relative abundance. When sera from 318 patients were analyzed, the RFMP assay exhibited 100% concordance with results obtained by clonal sequencing and identified mixed-genotype infections in 22% of the samples, in addition to several subtype variants. Conclusions: The RFMP assay has practical advantages over existing methods, including better quantitative detection of mixed populations and detection of genotype variants without need for population-based cloning, enabling reliable viral genotyping in laboratories and efficient study of the relationship between viral genotypes and clinical outcome.