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Rapid and sensitive genotyping of hepatitis C virus by
single-strand conformation polymorphism
There is an increasing demand for genotyping hepatitis C virus
(HCV) isolates due to the rapidly expanding list of distinct HCV
genotypes and the mounting evidence of genotype-specific clinical
consequences. We describe an SSCP-based assay for determining
genotypes in HCV infections. HCV RNA extracted from serum was
amplified by a sensitive nested-PCR assay producing a 287 bp
fragment of the conserved 5' non-coding region (NCR) and analysed
by non-denaturing polyacrylamide gel electrophoresis. Following
empirical optimisation of the SSCP assay we identified distinct
conformation polymorphisms (characteristic band patterns)
corresponding to types 1a, 1b, 2a, 2b, 2c, 3 and 4 found in the
Western Australian population. Seventy-three HCV RNA-positive
samples were used to evaluate the SSCP genotyping assay for
accuracy and efficiency by comparison with the previously
established genotyping methods of manual direct sequencing and
dideoxy fingerprinting. SSCP genotyping was in concord with control
methods while performing more rapidly and at a fraction of the
cost. Moreover, SSCP detected two co-infected samples that were not
shown by the control methods. The PCR-SSCP assay provides an
accurate and rapid method for genotyping of HCV RNA-positive
samples at the 5' NCR by type-specific sequence polymorphisms which
is applicable to large-scale screening.
J Virol Methods 64(1): 11-18 (1997) ; Lareu RR, Swanson NR,
Fox SA; University Department of Medicine, Sir Charles Gairdner
Hospital, Perth, WA, Australia. ; PMID: 9029525, MUID:
97181364
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