Detection of negative-stranded hepatitis C virus RNA using a novel strand-specific reverse transcription-polymerase chain reaction.
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.
Mizutani
T
T
Virology Division, National Cancer Center Research Institute, Tokyo, Japan.
Ikeda
M
M
Saito
S
S
Sugiyama
K
K
Shimotohno
K
K
Kato
N
N
D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction; D012367:RNA, Viral
D012367:RNA, Viral