Genotyping of hepatitis C virus by nucleotide sequencing: A robust method for a diagnostic laboratory

Abstract

Hepatitis C virus (HCV) is a globally significant blood-borne agent causing liver diseases, and it has infected over 170 million people worldwide. HCV is a diverse group of RNA viruses currently divided into genotypes 1–7 as well as subtypes. HCV infection can be treated with antiviral drugs, but the HCV genotype has to be determined for optimal selection of treatment strategy. The aim of this study was to set up a sequencing-based HCV genotyping method suitable for the workflow of a diagnostic laboratory. The established method is robust and stable, and it utilizes a one-step reverse transcription and PCR amplification of the 5′ untranslated region (5′UTR) and partial Core region of the HCV genome. Amplification products are sequenced using the standard Sanger method, and the genotype is determined by using a freely accessible web-based genotyping tool. The method was validated at the Helsinki University Hospital Laboratory using 238 previously genotyped serum samples. • A new one-step RT-PCR method for the amplification of the 5′ untranslated region and partial Core region of hepatitis C virus was established. • HCV genotype is determined using Sanger sequencing and a freely accessible, easy-to-use web-based genotyping tool. • The method is robust, reproducible and suitable for diagnostic laboratory workflow, and it requires no costly instrumentation or specialized sequence analysis skills. Method name: HCV genotyping, Keywords: HCV, Genotype, PCR, Amplification, Sanger, 5’UTR