OPTIMIZATION 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 a re captured on streptavidin coated microtitre plates, denatured with s odium hydroxide and hybridised with an alkaline phosphatase-labelled o ligonucleotide probe. Quantification is achieved by measuring the inte nsity 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 log(10) d ynamic range sufficient to cover the 10(3)-10(8) genomes/ml viraemia l evels typically seen in patient samples, (iii) specificity and reprodu cibility suitable for application in a clinical context, and (iv) a ra pid non-nested assay format with the ability to handle large throughpu ts and with a potential for automation. The feasibility of using the a ssay to monitor viraemia level changes in patients undergoing interfer on therapy for chronic HCV infection has been demonstrated.