Single-step reverse transcription-polymerase chain reaction for the detection of hepatitis C virus RNA.
We developed a novel single-step reverse transcription-polymerase chain reaction (RT-PCR), which is equal in sensitivity and specificity to RT-nested PCR, based on both reverse transcriptase and Taq DNA polymerase working efficiently under single buffer reaction conditions. Using in vitro synthesized hepatitis C virus (HCV) RNA, it was demonstrated that 10-100 copies of HCV RNA could be detected with a set of primers that amplify a 144 base-pair sequence unique to the 5'-noncoding region of HCV RNA. Furthermore, this method was successfully performed on serum and liver biopsy specimens obtained from patients with chronic hepatitis C. In addition, HCV RNA from in vitro HCV-infected MT-2C cells, which supported HCV replication, was also detected by this method. The method is anticipated to improve the detection of small amounts of RNA, such as that of HCV, promoting both labor savings and the prevention of carry-over contamination.
Virology Division, National Cancer Center Research Institute, Tokyo, Japan.
T
T
Mizutani
M
M
Ikeda
S
S
Saito
K
K
Sugiyama
K
K
Shimotohno
N
N
Kato
D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification*; D019698:Hepatitis C, Chronic / Q000821:virology*; D006801:Humans; D008099:Liver / Q000821:virology; D012367:RNA, Viral / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012367:RNA, Viral