Detection of viremia by a one step polymerase chain reaction method in hepatitis C virus infection.
A simple, sensitive, and specific one step polymerase chain reaction (PCR) method for the detection of hepatitis C virus (HCV) RNA in infected patients' serum or plasma samples is described. We performed the one step PCR amplification in combination with the initial step of reverse transcription by using oligonucleotide primers derived from the conserved 5'-untranslated region (5'-UTR) of the HCV genome. By utilizing this strategy, there was no need for nested or second stage PCR amplification. The PCR products (cDNAs) were easily visualized by agarose gel electrophoresis and ethidium bromide staining. Furthermore, the PCR products were characterized by Southern blot hybridization and DNA sequencing. We then used the one step PCR amplification method to detect the presence of HCV RNA in several infected patients' samples with acute and chronic infections. There was a 100% concordance between the results of PCR and second generation recombinant immunoblot assay (RIBA II). In addition, this method was found to be useful in determining viremia in HCV infected patients with indeterminate RIBA II results. The 5'-UTR of the HCV genome, being the most conserved region among different viral isolates, could be amplified by PCR for the detection of HCV RNA, as shown here, as well as serving as a potential target for antiviral agents.
Ahmad
N
N
Division of Molecular Virology, James N. Gamble Institute of Medical Research, Cincinnati, OH 45219.
Schiff
G M
GM
Baroudy
B M
BM
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005069:Evaluation Studies as Topic; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification*; D006526:Hepatitis C / Q000175:diagnosis* / Q000382:microbiology; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012367:RNA, Viral / Q000097:blood / Q000235:genetics; D012680:Sensitivity and Specificity; D014766:Viremia / Q000175:diagnosis* / Q000382:microbiology
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral