A 2ND-GENERATION METHOD OF GENOTYPING HEPATITIS-C VIRUS BY THE POLYMERASE CHAIN-REACTION WITH SENSE AND ANTISENSE PRIMERS DEDUCED FROM THE CORE GENE

A second-generation method of genotyping hepatitis C virus (HCV) was d eveloped by the polymerase chain reaction (PCR) with sense as well as antisense primers deduced from the core gene. HCV RNA specimens extrac ted from sera were reverse-transcribed and amplified with universal pr imers in the first round of PCR to obtain fragments of 433 base pairs representing nucleotides 319-751. In the second round of PCR, portions of PCR products were amplified separately with sense and antisense pr imers specific for each of the five common genotypes prevailing across the world, i.e,, I/1a, II/1b, III/2a, IV/2b and V/3a. The specificity of the method was verified by a panel of 177 HCV isolates of various genotypes in the genetic groups 1-9. It allowed clear differentiation of genotype I/1a from II/1b which was not always accomplished by the p revious method. When 501 sera from blood donors and hepatitis patients with HCV viremia from various countries were genotyped by the second- generation method, 478 (95.4%) were classified into the five genotypes . HCV RNA samples from 23 (4.6%) sera were not classifiable into any o f the five common genotypes and, by sequence analysis, 22 were found t o be of four genotypes in group 4 and one of genotype Ic in Simmond's classification.