Optimisation and evaluation of a quantitative cherniluminescent polymerase chain reaction assay for hepatitis C virus RNA

• Published in: Journal of virological methods 1995, Vol.51 (1), p.75-88
• Main Authors: Whitby, K., Garson, J.A.
• Format: Article
• Language: English
• Subjects: Chemiluminescence; Hepatitis C virus; Quantitative PCR
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/0166-0934(94)00144-6

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 cherniluminescent substrate. The chief advantages of the assay are: (i) extreme sensitivity with the ability to detect single molecules of HCV cDNA, (ii) a 5 log 10 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.