Whitby, Kevin Peter; (1999) The development and application of quantitative PCR-based assays for the detection of hepatitis C and related viruses. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

PCR has previously been used to measure hepatitis C virus (HCV) levels during antiviral chemotherapy, in viral cultures and in diverse clinical samples. The use of this technique led to a rapid expansion of our understanding of this clinically important virus. However, the majority of methods previously used to quantify HCV were laborious and relatively insensitive. This thesis describes the development of PCR-based quantitative methods for HCV RNA and their application in a number of research settings. Significant advances in assay speed and throughput are demonstrated, as well as important improvements in sensitivity. The aim of the project was to develop and optimise quantitative PCR methods which could conveniently be applied to serum or plasma and also to cells and cell culture supernatants from in vitro culture experiments. The PCR method developed was shown to be effective in providing quantitative viral monitoring data from several trials of antiviral chemotherapy. Viral RNA levels were also monitored in the serum of a patient infected during pregnancy, in a cultured hepatocyte line and the method was also applied to the study of a potential small primate model. The quantity of virus in cultured hepatocytes and in plasma pools prior to fractionation was demonstrated to be very low. In addition a novel single tube 'hot-start' RT-PCR method (RT-HS-PCR) is described. This method was shown to increase the sensitivity of RT-PCR and to provide quantitative data. The discovery of a new flavivirus (GBV-C), closely related to HCV, provided an opportunity to demonstrate the flexibility of this quantitative PCR method. The technique has now been applied to a wide range of DNA and RNA viruses in several laboratories within the UK.

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