Colorimetric detection of competitive PCR products for quantification of hepatitis C viremia.
A method based on competitive polymerase chain reaction (PCR) and colorimetric detection of the amplified products was developed to quantify hepatitis C virus (HCV) genomes. Serum samples were obtained from patients who were treated with interferon alpha (IFN-alpha). After reverse transcription of the HCV RNA, the cDNA was coamplified with a serially diluted cloned HCV competitor DNA using nested PCR. The competitor DNA consisted of the amplified region of the wild type HCV cDNA with an internal region substituted with the lac operator (lacO) sequence. The PCR products were quantitated specifically by a colorimetric solid-phase assay. The results suggest that the method is well suited for analysing the kinetics of the anti-HCV effects during IFN-alpha treatment. The quantification assay is simple, reliable and suitable for quantitating HCV genomes in a large number of clinical samples.
Yun
Z
Z
Department of Virology, Central Microbiological Laboratory, Stockholm County Council, Sweden.
Lundeberg
J
J
Johansson
B
B
Hedrum
A
A
Weiland
O
O
Uhlén
M
M
Sönnerborg
A
A
D001483:Base Sequence; D003124:Colorimetry / Q000379:methods; D004279:DNA, Viral / Q000097:blood; D005814:Genes, Viral / Q000235:genetics; D016174:Hepacivirus / Q000302:isolation & purification*; D006526:Hepatitis C / Q000382:microbiology* / Q000628:therapy; D006801:Humans; D016898:Interferon-alpha / Q000627:therapeutic use; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000302:isolation & purification*; D015203:Reproducibility of Results; D014766:Viremia / Q000382:microbiology*
D004279:DNA, Viral; D016898:Interferon-alpha; D012367:RNA, Viral