A genotype independent, full-genome reverse-transcription protocol for HCV genotyping and resistance testing

•Most hepatitis C virus (HCV) direct acting antivirals (DAA) are genotype dependent.•New approved DAA against HCV require resistance testing prior therapy.•Genotyping and genotype specific resistance testing are done in separate reactions.•A protocol for full-length, genotype independent reverse transcription was established.•This cDNA can be used as template for genotyping as well as resistance testing.

BackgroundHCV treatment options and cure rates have tremendously increased in the last decade. Although a pan-genotype HCV treatment has recently been approved, most DAA therapies are still genotype specific. Resistance-associated variants (RAVs) can limit the efficacy of DAA therapy and are associated with increased risk for therapy failure. With the approval of DAA regimens that recommend resistance testing prior to therapy, correct assessment of the genotype and testing for viruses with RAVs is clinically relevant. However, genotyping and resistance testing is generally done in costly and laborious separate reactions.ObjectiveThe aim of the study was to establish a genotype-independent full-genome reverse transcription protocol to generate a template for both genotyping and resistance testing and to implement it into our routine diagnostic setup.Study designThe complete HCV genome was reverse transcribed with a pan-genotype primer binding at the 3′end of the viral RNA. This cDNA served as template for transcription of the genotyping amplicon in the core region as well as for the resistance testing of NS3, NS5A, and NS5B.ResultsWith the established RT-protocol the HCV core region was successfully amplified and genotyped from 124 out of 125 (99.2%) HCV-positive samples. The amplification efficiency of RAV containing regions in NS3, NS5A, NS5B was 96.2%, 96.6% and 94.4%, respectively.ConclusionsWe developed a method for HCV full-genome cDNA synthesis and implemented it into a routine diagnostic setup. This cDNA can be used as template for genotyping amplicons covering the core or NS5B region as well as for resistance testing amplicons in NS3, NS5A and NS5B.