Genotyping of Hepatitis C Virus Types 1, 2, 3, and 4 by a One-Step LightCycler Method Using Three Different Pairs of Hybridization Probes

Abstract

ABSTRACT Determination of hepatitis C virus (HCV) genotypes has become increasingly important during the last years for prediction of the clinical course and the outcome of antiviral therapy. Therefore, numerous different methods have been developed to enable HCV genotyping. However, many of them are very laborious and expensive, leading to limited usage in daily routine diagnostics. We have established a method which combines the speed of the new LightCycler technology with the use of amplification products generated for diagnostic quantitative HCV RNA determination. Differentiation of HCV genotypes is performed with these amplicons in a single step by using fluorophore-labeled hybridization probes. Although currently only two different acceptor fluorophores are available for the LightCycler, types 1, 2, 3, and 4, which are by far the prevailing HCV genotypes in Europe and the United States, can be distinguished. Genotypes of specimens from 190 chronically HCV-infected patients were determined by the LightCycler method and compared with the results of nucleotide sequencing. Concordant results were obtained for all samples. This new method offers a fast and convenient possibility to determine the quantitative HCV RNA load and the genotype in large-scale settings within about 4 h.