A highly sensitive and reproducible HCV RNA hybridization method valuable for antiviral drug discovery

Real-time RT-PCR and Northern blot are employed for the measurement of HCV RNA but suffer from multiple purification steps, high cost, and relatively large variability. In this study, a hybridization method for HCV RNA detection is described. This method does not need RNA purification, and is sensitive enough to detect HCV RNA present in replicon cellular lysates harvested from a single well of a 96-well plate. Fixation of RNA by UV cross-linking is crucial for this sensitivity. A linear relationship exists between hybridization signal and cell density ranging from 105 to as few as 300 cells per well. The signal-to-background ratio is greater than 40 and the Z factor is above 0.7. Using several known anti-HCV agents, dose–response curves and EC50 values generated from hybridization were similar to those obtained from a luciferase assay. This method has been successfully applied to replicons of different HCV subtypes and hepatitis B virus in our laboratory. In summary, this hybridization assay is sensitive, highly reproducible, easy to handle, and a valuable tool for antiviral drug discovery.