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Rapid and reproducible quantification of hepatitis C virus cDNA by fluorescence correlation spectroscopy
1Department of Cell Biology, Max Planck Institute for Biochemistry, Martinsried, Germany.
Background/Aims:
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.
Conclusion:
FCS may become a useful tool for rapid and reproducible HCV RNA quantification in the future.