1. Institute of Physico-Chemical Medicine Ministry of Public Health 2. Institute of Physico-Chemical Medicine Ministry of Public Health, Institute of Biomedical Chemistry RAMS 3. Institute of Biomedical Chemistry RAMS 4. Institute of Epidemiology of Russia Federation Ministry of Public Health
Determination of the hepatitis C virus (HCV) genotype has become the standard procedure in laboratory diagnostics of HCV infection. Genotype elucidation has prognostic value assignment helps in assessing disease prognosis and promotes establishing appropriate duration of treatment. Now 11 major genotypes and more than 70 subtypes of HCV have been identified using the sequence variability within 5' non-coding region (5' NCR). In Russia the most common subtypes are 1a, 1b, 2a, 3a and more rare - 4 and 5 types. While the "gold standard" for testing is nucleic acid sequencing, a variety of other assays, including the line probe assay or type-specific amplification, has been developed to provide more rapid and cheaper forms of testing. The aim of this study was to determine the type-specific single nucleotide polymorphism (SNP) in 5' NCR HCV by the classical three-step minisequencing method with followed MALDI-TOF mass spectrometry detection. The fragments of 5'NCR of HCV genomes were amplified by the nested RT PCR. The removal of excess nucleotides and primers was performed. Three oligonucleotide primers were design to detect two sets of type-specific SNP in 5' NCR HCV. The primer extension reaction was performed using modified thermostable DNA polymerase and in the presence of ddNTP. The molecular weights of primers extension reaction products were analyzed using MALDI-TOF mass spectrometry. The HCV genotype was determined according the presence in analyses sample the molecules with expected molecular weights. The suggested method was used to type HCV from 69 HCV-positive sera. The 1a genotype was determine in 4.5% samples, 1b - 48%, 2a - 4.5%, 3a - 29%, 4 - 1,5%. The mixes of two genotypes were found in 13% samples. All data confirmed by direct nucleic acid sequence. Thus, the new method for HCV typing has been developed using the minisequencing reaction and mass spectrometry for the determination of nucleic acid molecular weight.