Hepatitis C genotyping by direct sequencing of the product from the Roche AMPLICOR test: methodology and application...

The Roche AMPLICOR RT-PCR amplifies a 244 nucleotide sequence within the 5' non coding region (5'NCR) of the viral genome and is a widely used commercial test for the qualitative determination of hepatitis C RNA from sera. This paper describes a routine procedure for the purification of the PCR product, and its use in automated DNA sequencing, for determining the genotype of hepatitis C virus (HCV) isolates. Direct sequencing of the purified product was possible for 86% of samples, whilst 14% required additional amplification using a nested PCR method in order to read the resulting electropherogram. This method of genotyping is considerably less expensive than currently available commercial kits, and is convenient for the increasing number of laboratories that have access to automated DNA sequencers. The highly conserved nature of the 5'NCR limited differentiation of types and subtypes to an extent comparable to commercial HCV typing methods. Using this method on available laboratory samples and on patients about to commence interferon therapy, we found a predominance of genotype 1 (59%) and 3a (31%). Analysis of data on the interferon patients showed the median length of time from first exposure to diagnosis to be significantly longer for patients with genotype 1 than genotype 3a.