Genotyping of hepatitis C virus isolates using CLIP sequencing

Determination of hepatitis C virus (HCV) genotypes and subtypes has become increasingly important for the clinical management and prognosis of HCV inf ections. The aim of the present study was to assess the specificity and rel iability of a newly developed, commercially available HCV genotyping kit (T RUGENE HCV 5'NC genotyping kit). This technique utilizes PCR fragments prev iously generated by the diagnostic Roche AMPLICOR HCV test, which are subse quently subjected to simultaneous PCR amplification and direct sequencing ( CLIP sequencing) of the 5' noncoding region (5'NCR). HCV isolates from 100 randomly chosen patients were genotyped by both the TRUGENE HCT 5'NC genoty ping kit and DNA enzyme immunoassay (DEW). Typing results obtained by both methods were in complete concordance in 91% of the cases. HCV RNA from the samples with discordant genotype assignment in both assays was additionally amplified with primers from the HCV core and NS5B regions. Phylogenetic an alysis of the obtained sequences supported the results obtained from DEIA i n six cases and CLIP sequencing in two cases. In the former six cases, the TRUGENE HCV 5'NC genotyping kit could not correctly differentiate between s ubtypes of genotypes 1 and 2 due to the high conservation of the 5'NCR, How ever, since there was not any misclassification between HCV genotypes 1 and non-1 types, the results obtained with this system are, in general, reliab le and can be used in clinical practice. The TRUGENE HCV 5'NC genotyping ki t in our hands proved to be a fast and convenient technique that might be a n attractive option for HCV genotyping in laboratories already using the Ro che AMPLICOR HCV test for diagnostic reverse transcription-PCR.