Rapid and reproducible quantification of hepatitis C virus cDNA by fluorescence correlation spectroscopy.
OBJECTIVE
Standard methods for hepatitis C virus (HCV) RNA quantification are time-consuming and often hampered by low sensitivity. Therefore, we aimed to test whether fluorescence correlation spectroscopy (FCS) could be used to read out HCV polymerase chain reactions (PCR).


METHODS
A single-step reverse transcriptase (RT) PCR system was adjusted to the clinically relevant range of 1 x 10(3) to 5 x 10(6) HCV cDNA copies/ml serum. Unpurified amplification mixtures were analyzed by FCS and controlled by HPLC analysis.


RESULTS
The outcome of HCV RNA quantitation was nearly identical no matter whether FCS or HPLC techniques were used. FCS-generated standard curves displayed sufficient linearity to allow reproducible determinations. The intraserial variation of cDNA quantification after PCR amplification was +/-3.2%, the interserial variation +/-4.3%. Repeated quantifications of HCV genotype 1b RNA from the sera of 8 patients revealed titers from 1 x 10(4)-5 x 10(6) genome equivalents/ml. The results correlated significantly (r = 0.755; p = 0.03) with a widely used commercially available assay.


CONCLUSIONS
FCS may become a useful tool for rapid and reproducible HCV RNA quantification in the future.
Weiner
O H
OH
Department of Cell Biology, Max Planck Institute for Biochemistry, Martinsried, Germany.
Alt
M
M
Dürr
R
R
Noegel
A A
AA
Caselmann
W H
WH
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000032:analysis*; D005260:Female; D016174:Hepacivirus / Q000302:isolation & purification*; D019698:Hepatitis C, Chronic / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D018076:DNA, Complementary