Typing six major hepatitis C virus genotypes by polymerase chain reaction using primers derived from nucleotide sequences of the NS5 region

Abstract

Abstract We recently developed a polymerase chain reaction (PCR) based method for determining hepatitis C virus (HCV) genotypes using primers derived from nucleotide sequences of the NS5 region. Using this method, we determined 5 hepatitis C virus (HCV) genotypes (1a, 1b, 2a, 2b and 3b) detected in Japanese patients. Following the recent identification of genotype 3a HCV in a patient from Pakistan currently living in Japan, we improved the typing method used for detecting all of these 6 genotypes. Eighteen of 1100 HCV RNA positive patients who tested negative for genotype by the original method also tested negative by the new method. Type 3a genotype HCV was thus shown to be very rare among Japanese patients with HCV infection. Nucleotide sequence and quantitative analysis of these negative samples revealed that negative results were due to mutations present on the nucleotide sequence where the 3′ end of the primers was situated due to a very low titer (less than 103) of the virus. The new single step genotyping method may be clinically useful as it allows a fast determination of all 6 genotypes present in Japan.