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 10(5) 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 EC(50) 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.
Zhao
Yongsen
Y
Achillion Pharmaceuticals, 300 George Street, New Haven, CT 06511, USA.
Sanchez
Amy
A
Nie
Xingtie
X
Liu
Dongmei
D
Hou
Xiaohong
X
Fabrycki
Joanne
J
Phadke
Avinash
A
Deshpande
Milind
M
Huang
Mingjun
M
Yang
Wengang
W
D000998:Antiviral Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D008099:Liver / Q000166:cytology / Q000821:virology; D008826:Microbial Sensitivity Tests; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D012093:Replicon; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014779:Virus Replication
D000998:Antiviral Agents; D012367:RNA, Viral