Analysis of additional samples by RFMP and direct sequencing may identify additional patterns and thus reduce the number of nontyped samples.
(12) have developed an intriguing new method (RFMP) for genotyping HCV specimens, based on MALDI-TOF MS analysis of PCR product from the 5'UTR.
To assess the sensitivity and validity of the RFMP assay for detecting mixed genotypes in a viral population, we performed the assay with samples composed of defined ratios of 5' UTR sequences of HCV genotype 1b and 2a/c in RNAs.
The RFMP assay thus could detect mixed genotypes in a viral population and determine their relative abundances.
HCV GENOTYPE DISTRIBUTION IN PATIENTS DETERMINED BY RFMP ANALYSIS
We determined the genotype distribution of the 318 samples from chronically infected patients by RFMP and its representative spectra (Table 1 and Fig.
In this study, we established an RFMP assay for HCV genotyping that exploits differences among HCV genotypes in the molecular masses of oligonucleotides comprising the viral 5' UTR.
This research should be facilitated by the high sensitivity of the RFMP assay for detecting mixed infections and the capacity of the RFMP assay to determine relative abundantes among viral species.
The resolution achieved by the RFMP assays reported here should be achievable with MALDI-TOF MS instruments with specifications and settings comparable to or beyond those used here.
In conclusion, the RFMP genotyping method uses the mass difference of oligonucleotides, requires the simple steps of single PCR amplification and restriction enzyme digestion, and is amenable to high-throughput analyses.
 Nonstandard abbreviations: HCV, hepatitis C virus; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; RFMP, restriction fragment mass polymorphism; UTR, untranslated region; and TEAA, triethylammonium acetate.