Browsing by Subject "5'UTR"

Hepatitis C virus (HCV) is a globally significant blood-borne agent causing liver diseases. HCV has infected over 170 million people worldwide and it has become a significant causative agent of chronic liver inflammation also in Finland. HCV is a very diverse group of viruses that is divided into genotypes 1–7 as well as subtypes. HCV infection can be treated with antiviral drugs, and the drug of choice as well as treatment success are determined by the HCV genotype that the patient is carrying. The aim of this study was to develop a new, sequencing based HCV genotyping method for the Laboratory of the Hospital District of Helsinki and Uusimaa (HUSLAB), at the Department of Virology and Immunology. The focus of the study was to establish a steady and robust genotyping method that would be suitable for the workflow in clinical diagnostics. The samples used in this study had been previously analysed in regular HCV genotyping diagnostics at HUSLAB. The genomic regions chosen for amplification with several different primer options were 5’UTR, core/E1 and NS5B. 5’UTR turned out to be the only suitable option for the diagnostic workflow. The amplification products were sequenced using Sanger method. Amplification of the whole HCV genome in several different reaction conditions for 454 deep sequencing was also attempted to obtain information about possible mixed infections and drug resistance changes in the genome. In this study, a new HCV genotyping method based on Sanger sequencing of the 5’UTR region was successfully established. The method is robust, stable and suitable for the use in clinical diagnostics. The established HCV genotyping method is the first entirely sequencing-based method in clinical viral diagnostics in Finland. The secondary aim, amplification of the whole HCV genome using a method suitable for the workflow of clinical diagnostics was not achieved. Given the demands and restrictions of the workflow of clinical diagnostics we can conclude that routine HCV genotyping with deep sequencing methods is for the present not yet possible.