Genotyping of Hepatitis C Virus by Direct Sequence Analysis of Polymerase Chain Reaction Products

Genotyping of hepatitis C virus (HCV) has become important and clinical interest has increased because of its associations with response to therapy and progless of disease. Furthermore, phylogenetic analysis is useful in epidemiological studies. The aim of this study is to evaluate the routine use of direct sequence analysis of polymerase chain reaction (PCR) products for determining HCV genotypes and phylogenetic analysis. Fifty serum samples found to be HCV RNA positive by Cobas Amplicor test (Roche Molecular Systems, Branchburg NJ) were included in the study. Genotyping was based upon regions identified as discriminative within 184 base pairs of 5’ noncoding region (NCR) of viral genome. Nucleotide sequences were determined by using Big Dye Terminator kit with ABI Prism 310 DNA sequencer (PE Applied Biosystems, Foster City, Calif.) and were aligned using Clustal W program. Phylogenetic tree was constructed by Treecon program. Genotyping could be achieved with 45 serum samples (90%) belonging to 24 chronic hepatitis C, 7 renal transplant and 14 thalasemia patients. Among 45 HCV isolates, 15 were 1a and 30 were 1b. Although nucleic acid sequencing is the gold standard for genotyping, commercial kits are more practical for routine laboratory use. However, sequencing helps to identify the genotypes of the isolates which could not be determined, precisely. 5’NCR of HCV did not seem to be appropriate in our group of samples for detailed phylogenetic analysis such as tracing viral transmission route.