Optimisation and evaluation of a quantitative chemiluminescent polymerase chain reaction assay for hepatitis C virus RNA.
A quantitative non-isotopic assay for measuring hepatitis C virus (HCV) RNA has been developed and evaluated. Viral RNA extracted from serum is reverse transcribed and amplified by the polymerase chain reaction (PCR) using biotinylated 5' non-coding region primers. PCR products are captured on streptavidin coated microtitre plates, denatured with sodium hydroxide and hybridised with an alkaline phosphatase-labelled oligonucleotide probe. Quantification is achieved by measuring the intensity of light emitted by a dioxetane-based chemiluminescent substrate. The chief advantages of the assay are: (i) extreme sensitivity with the ability to detect single molecules of HCV cDNA, (ii) a 5 log10 dynamic range sufficient to cover the 10(3)-10(8) genomes/ml viraemia levels typically seen in patient samples, (iii) specificity and reproducibility suitable for application in a clinical context, and (iv) a rapid non-nested assay format with the ability to handle large throughputs and with a potential for automation. The feasibility of using the assay to monitor viraemia level changes in patients undergoing interferon therapy for chronic HCV infection has been demonstrated.
Whitby
K
K
Department of Medical Microbiology, University College London Medical School, UK.
Garson
J A
JA
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification*; D006526:Hepatitis C / Q000821:virology; D006521:Hepatitis, Chronic / Q000821:virology; D006801:Humans; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014766:Viremia / Q000821:virology; D014773:Virology / Q000379:methods / Q000592:standards / Q000706:statistics & numerical data
D017931:DNA Primers; D012367:RNA, Viral