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

J Virol Methods. 1995 Jan;51(1):75-88. doi: 10.1016/0166-0934(94)00144-6.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA Primers / genetics
  • Evaluation Studies as Topic
  • Hepacivirus / genetics*
  • Hepacivirus / isolation & purification*
  • Hepatitis C / virology
  • Hepatitis, Chronic / virology
  • Humans
  • Luminescent Measurements
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards
  • Polymerase Chain Reaction / statistics & numerical data
  • RNA, Viral / analysis*
  • RNA, Viral / genetics*
  • Reference Standards
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Viremia / virology
  • Virology / methods
  • Virology / standards
  • Virology / statistics & numerical data

Substances

  • DNA Primers
  • RNA, Viral