title: Detection of negative-stranded hepatitis C virus RNA using a novel strand-specific reverse transcription-polymerase chain reaction.
abstract: We developed a novel single-tube reverse transcription-polymerase chain reaction (RT-PCR) for the specific detection of negative-stranded hepatitis C virus (HCV) RNA. By using in vitro synthesized positive- and negative-stranded HCV RNAs, it was demonstrated that as few as 50 copies of negative-stranded HCV RNA could be specifically detected with a set of primers that amplify a 232-base pair sequence unique to the 5'-non-coding region of HCV RNA, while 10(8) copies of positive-stranded HCV RNA were not detected. In addition, we demonstrated that this method allows the detection as few as 100 copies of negative-stranded HCV RNA even with the coexistence of a 100-fold excess of positive-stranded HCV RNA. Furthermore, with this method, negative-stranded HCV RNA was detected in RNAs from liver biopsy specimens obtained from patients with chronic hepatitis C, but not in RNAs from HCV-positive sera.
authors.affiliation: Virology Division, National Cancer Center Research Institute, Tokyo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Mizutani
authors.forename: M
authors.initials: M
authors.lastname: Ikeda
authors.forename: S
authors.initials: S
authors.lastname: Saito
authors.forename: K
authors.initials: K
authors.lastname: Sugiyama
authors.forename: K
authors.initials: K
authors.lastname: Shimotohno
authors.forename: N
authors.initials: N
authors.lastname: Kato
mesh_terms: D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction; D012367:RNA, Viral
chemical_list: D012367:RNA, Viral