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.
Lareu
R R
RR
University Department of Medicine, Sir Charles Gairdner Hospital, Perth, WA, Australia.
Swanson
N R
NR
Fox
S A
SA
D004279:DNA, Viral / Q000032:analysis*; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity
D004279:DNA, Viral