A reliable internally controlled RT-nested PCR method for the detection of hepatitis 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 Renilla luciferase (Rluc) RNA as an internal control. Using this method, the 5'-noncoding region of HCV RNA (144 nucleotides) and Rluc RNA (276 nucleotides) were efficiently amplified in a single tube, and the sensitivity and specificity of this method were comparable to standard RT-nested PCR. This method was successfully performed on RNA specimens obtained from in vitro HCV-infected human hepatocyte PH5CH8 cells, which support HCV replication. In addition, we demonstrated that this method was useful for the evaluation of antiviral reagents by confirming the anti-HCV activity of bovine lactoferrin, which we previously found to be a new inhibitor of HCV infection. Therefore, this method may be useful for the studies of not only HCV but also of other viruses.
Nozaki
A
A
Department of Molecular Biology, Institute of Cellular and Molecular Biology, Okayama University Medical School, Japan.
Naganuma
A
A
Nakamura
T
T
Tanaka
K
K
Sekihara
H
H
Kato
N
N
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D001517:Coleoptera / Q000201:enzymology; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics*; D022781:Hepatocytes / Q000737:chemistry / Q000821:virology; D006801:Humans; D007781:Lactoferrin / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D012313:RNA; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014779:Virus Replication
D000998:Antiviral Agents; D012367:RNA, Viral; D012313:RNA; D008156:Luciferases; D007781:Lactoferrin