Hepatitis C virus (HCV) is the main cause of viral hepatitis in the UK and leads to chronic liver disease in many infected individuals. There is a substantial burden on diagnostic laboratories to provide rapid, cost-effective tests for monitoring HCV infection. Commercial assays are expensive and so the development of validated in-house methods is beneficial. This thesis describes the development and implementation of rapid and inexpensive real-time PCR assays for HCV quantitation and genotyping to support clinical practice. Additionally, the development of methods for defining HCV isolates at the subtype level, important in epidemiological and transmission studies, is described. These assays were utilised in a study on spontaneous HCV clearance, the results of which suggest that HCV type 1 infection and younger age at infection are factors which are associated with spontaneous viral clearance. Chronic HCV infection is linked to oxidative stress with numerous deleterious cellular effects. Mitochondrial DNA (mtDNA) is more susceptible to oxidative damage than nuclear DNA making it an ideal marker to assess the overall level of cellular DNA damage - including deletions and mutations. This thesis illustrates the development of real-time PCR assays to detect and quantify two major mtDNA deletions. The D-Ioop region of mtDNA is particularly prone to damage - with two well recognised 'hotspots of mutation'. The creation of an RSCA (heteroduplexbased) method using capillary electrophoresis, to detect and quantify damage in this region, is described. To evaluate the clinical utility of these assays, a study of mtDNA damage in patients with liver disease was undertaken. The aim of this study was to identify whether chronic HCV infection results in increased levels of mtDNA damage compared to other liver pathologies. Low levels of mtDNA deletions were detected in the majority of liver biopsy specimens and there was no correlation between liver aetiology and quantity of deletions. The RSCA method identified numerous D-Ioop mtDNA species within the liver tissue of several individuals. There was no correlation between liver aetiology and the presence of multiple D-Ioop species.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:495578 |
| Date | January 2008 |
| Creators | Rolfe, Kathryn Jane |
| Publisher | Open University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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