Analysis of 5' non-coding region in hepatitis C virus by single-strand conformation polymorphism and low-stringency...

Single-strand conformation polymorphism (SSCP) and low-stringency single specific primer (LSSP)-PCR in hepatitis C virus (HCV) genotyping were examined for informativeness and reliability. The analysis of HCV isolates included seven type 1 isolates, two type 2 isolates, and two type 3 isolates. We also analyzed five isolates that presented as mixed infections determined by type-specific PCR. Among mixed isolates, one isolate was 1a/1b and four isolates were 1b/3a. SSCP and LSSP-PCR were applied to the analysis of 5' non-coding region of HCV (-289 to -5) that contains genotype-specific sequences. Direct cycle sequencing of this region determined sequence divergences that define genotype and sequence alterations within the same genotype. Optimized conditions for the SSCP analysis clearly distinguished between genotypes 1, 2 and 3. In addition, the SSCP analysis detected sequence variants within the same genotype. However, the SSCP analysis and DNA sequencing did not confirm the presence of mixed infections. LSSP analysis, not previously employed in HCV genotyping, enabled clear distinction between genotypes 1, 2 and 3, however, this method did not differentiate between sequence variants within a genotype. Importantly, the LSSP profile demonstrated distinction between mixed infection isolates.