Comparative evaluation of four genotyping methods for hepatitis C virus.
Four most widely accepted genotyping methods for hepatitis C virus (HCV) were applied to 40 HCV RNA isolates obtained from Slovenian patients in order to determine the concordance and applicability of various genotyping systems. The four methods are: (i) amplification of the core region with genotype-specific primers; (ii) nested polymerase chain reaction (PCR) in the core region followed by hybridization to HCV type-specific probes; (iii) reverse hybridization with the line probe assay Inno LiPA (Innogenetics, Gent, Belgium) using type-specific probes for the 5' non-coding region (NCR); and (iv) restriction fragment length polymorphism analysis of DNA amplified from the 5' NCR. Additionally, in isolates with discordant results nucleotide sequence analysis of a part of the NS-5 region was performed. Both genotyping methods based on the analysis of the 5' NCR were found more sensitive than those methods based on the analysis of the HCV core region. None of the four genotyping methods correctly classified all Slovenian HCV RNA isolates. PCR with genotype-specific primers was identified as entirely unsuitable for genotyping of Slovenian HCV RNA isolates. The remaining genotyping methods could clearly differentiate between HCV genotypes, but were not entirely reliable for HCV subtyping. The specificity of genotyping methods, which are based on the 5' NCR or the core region, was occasionally hampered, due to a lack or excess of sequence variation in their respective target regions.
Seme
K
K
Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, Slovenia. seme@ibmi.mf.uni-lj.si
Poljak
M
M
Dovc
P
P
Koren
S
S
D005838:Genotype; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics*; D018936:Hepatitis C Antigens / Q000235:genetics; D006801:Humans; D007124:Immunoenzyme Techniques; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012367:RNA, Viral / Q000032:analysis; D014758:Viral Core Proteins / Q000235:genetics
D018936:Hepatitis C Antigens; D012367:RNA, Viral; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus