A reliable internally controlled RT-nested PCR method for the detection ofhepatitis C virus RNA

We have developed a reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus (HCV) RNA using in vitro synthesized Re nilla luciferase (Rluc) RNA as an internal control. Using this method, the 5'-noncoding region of HCV RNA (144 nucleotides) and Rluc RNA (276 nucleoti des) were efficiently amplified in a single tube, and the sensitivity and s pecificity of this method were comparable to standard RT-nested PCR. This m ethod was successfully performed on RNA specimens obtained from in vitro HC V-infected human hepatocyte PH5CH8 cells, which support HCV replication. in addition, we demonstrated that this method was useful for the evaluation o f antiviral reagents by confirming the anti-HCV activity of bovine lactofer rin, which we previously found to be a new inhibitor of HCV infection. Ther efore, this method may be useful for the studies of not only HCV but also o f other viruses.