Simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR assay on the lightcycler instrument.
BACKGROUND
Clinically, both viral load and genotypes have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C and they are, under normal circumstances, performed as separate assays.


METHODS
In order to improve the diagnostic strategy and subsequently reduce the reagent costs we have developed and established the simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR on the LightCycler Instrument (Roche Diagnostics).


RESULTS
The quantification assay was calibrated against WHO Standard 96/790. The detection limit was 30 IU/ml, the dynamic range up to 500,000,000 IU/ml. Intra- and interassay imprecisions were 1.2% and 1.9% (n = 10), respectively. The HCV RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Cobas Amplicor HCV monitor test (r = 0.992; p < 0.001).


CONCLUSIONS
The genotyping was performed by means of the melting temperature analysis. The concordance between our new genotyping method and the Trugene HCV 5'NC Kit was at the level of genotypes 100%. This rapid (3 h) and convenient assay is suitable for HCV genotyping, HCV detection and disease monitoring.
Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart Katharinenhospital, Stuttgart, Germany. m.abe@klinikum-stuttgart.de
Manabu
M
Abe
Corinne
C
Klett
Eberhard
E
Wieland
Sascha
S
Gille
Olfert
O
Landt
D001483:Base Sequence; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D044366:Transition Temperature; D019562:Viral Load
D012367:RNA, Viral