Quantification of hepatitis C virus RNA by competitive amplification of RNA from denatured serum and hybridization on microtiter plates.
The direct detection of hepatitis C virus (HCV) RNA by PCR is widely used to determine the presence of circulating virions. The most relevant limit of this approach is the lack of quantitative information about the viral titer. We report a technique of competitive amplification allowing the estimation of HCV RNA copy number in biological samples. We constructed a standard competitive RNA template containing only two point mutations compared with its wild-type counterpart. The competitor was added in titrated amounts to the target RNA, and the mixture was then reverse transcribed and amplified in the same reaction tube. The relative amounts of target and competitor were determined by differential hybridization on microtiter plates with nonradioactive probes. The evaluation of HCV RNA titer required a single coamplification with the competitor and could be read from a standard curve. Furthermore, this method proved suitable for amplification of HCV RNA directly from serum, thus avoiding the intrinsic variability of the RNA extraction step.
Ravaggi
A
A
Institute of Chemistry, School of Medicine, University of Brescia, Italy.
Zonaro
A
A
Mazza
C
C
Albertini
A
A
Cariani
E
E
D001483:Base Sequence; D001667:Binding, Competitive; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology; D006521:Hepatitis, Chronic / Q000175:diagnosis / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood* / Q000235:genetics; D014766:Viremia / Q000175:diagnosis / Q000821:virology
D017931:DNA Primers; D012367:RNA, Viral