J. Holland et al., HEPATITIS-C GENOTYPING BY DIRECT SEQUENCING OF THE PRODUCT FROM THE ROCHE AMPLICOR TEST - METHODOLOGY AND APPLICATION TO A SOUTH AUSTRALIANPOPULATION, Pathology, 30(2), 1998, pp. 192-195
The Roche AMPLICOR RT-PCR amplifies a 244 nucleotide sequence within t
he 5' non coding region (5'NCR) of the viral genome and is a widely us
ed commercial test for the qualitative determination of hepatitis C RN
A from sera. This paper describes a routine procedure for the purifica
tion of the PCR product, and its use in automated DNA sequencing, for
determining the genotype of hepatitis C virus (HCV) isolates. Direct s
equencing of the purified product was possible for 86% of samples, whi
lst 14% required additional amplification using a nested PCR method in
order to read the resulting electropherogram. This method of genotypi
ng 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 exten
t comparable to commercial HCV typing methods. Using this method on av
ailable laboratory samples and on patients about to commence interfero
n therapy, we found a predominance of genotype 1 (59%) and 3a (31%). A
nalysis 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.