Genotyping of hepatitis C virus by Taqman real-time PCR.
BACKGROUND
Genotype of hepatitis C virus (HCV) is of major importance for the outcome of treatment. The response rate is considerably lower for genotype 1, the predominant genotype in western countries.


OBJECTIVE
To develop and evaluate a new, simple method for genotyping of HCV based on real-time polymerase chain reaction (PCR) and Taqman probes targeting the 5' non-coding region.


METHODS
The method was compared with Innolipa on 220 serum samples representing genotypes 1-4, and was applied on a further 614 clinical samples.


RESULTS
Taqman typing of the 220 samples showed genotype 1 in 69, genotype 2 in 58, genotype 3 in 57 and genotype 4 in 19, while 17 were non-reactive. There was a complete concordance with Innolipa with the exception of seven samples, which were of genotype 1 by Taqman, but genotype 4 by Innolipa. Sequencing of these samples showed a subtype 4 variant which differed at two positions compared with subtypes 4b/c/d, which are targeted by the probe. By adding a modified probe, these genotype 4 variants could also be identified. Out of 614 consecutive clinical samples, 524 could be typed by the Taqman assay; 45.2% were genotype 1, 19.3% genotype 2, 33.8% genotype 3 and 1.7%, genotype 4.


CONCLUSIONS
The method was overall accurate and provides an attractive alternative for genotyping because processing time and costs are significantly reduced. Inclusion of probes targeting genotypes 5 and 6 is required for the method to be useful in areas where these genotypes are present.
Lindh
Magnus
M
Department of Clinical Virology, Guldhedsgatan 10 B, 413 46 Göteborg, Sweden. magnus.lindh@microbio.gu.se
Hannoun
Charles
C
D020121:5' Untranslated Regions / Q000235:genetics; D017931:DNA Primers; D005838:Genotype; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics*; D006801:Humans; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D020121:5' Untranslated Regions; D017931:DNA Primers; D012333:RNA, Messenger; D012367:RNA, Viral