Non-radioactive hybridization with hepatitis C virus-specific probes created during polymerase chain reaction: a fast and simple procedure to verify hepatitis C virus infection

Abstract

All routinely available assays to detect hepatitis C virus (HCV) infection are based on the specific reaction of serum antibodies to corresponding viral antigens. The only method to detect viral nucleic acids in serum or liver is the enzymatic amplification of reversely transcribed HCV-RNA by polymerase chain reaction. Here, we describe a fast and simple method to confirm the HCV specificity of amplified fragments by hybridization with a non-radioactive cDNA probe created during polymerase chain reaction. A sequenced 521 bps HCV fragment derived from serum of an anti-HCV/EIAII-positive patient A with post-transfusion hepatitis C was used to verify HCV infection diagnosed by nested polymerase chain reaction in an anti-HCV/EIAII-seronegative patient B who did not produce anti-HCV-specific antibodies during the observation period of 1 year. The method is applicable to RNAs derived from serum or liver tissue and combines the sensitivity of nested polymerase chain reaction with the specificity of Southern blot hybridization. The procedure is independent of cloned viral sequences. In clinical use it permits fast and accurate confirmation of HCV infection, especially when antibodies detectable with conventional methods are absent.