3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Development of a real-time PCR incorporating high resolution melting analysis to screen HIV-1 samples for resistance-related codons(2011-02-01) Sacks, DavidIntroduction High resolution melting analysis (HRMA) accurately, rapidly and cost effectively detects single nucleotide polymorphisms by monitoring DNA dissociation kinetics. This technology was applied to HIV samples to assess whether it could be used to detect clinically relevant drug resistance mutations. Methods HRMA-PCR assays incorporating unlabeled probes were designed to genotype 12 mutation codons in the HIV-1 p66/p51 of engineered plasmids and 116 HIV-1 samples. Results HRMA correctly genotyped 63%-88% of the K103N, Y181C, M184V, Q151M and G190A mutations. Each assay had a 1.7%-3.4% discordance, most of which was due to the increased analytical sensitivity of HRMA (~5-20%). Only mutant K65R and V106M were correctly identified while the 41, 67, 70, 215 and 225 codons could not be genotyped. Assay modifications had some success in masking the affects of polymorphisms. Conclusion These assays can be used for genotyping selected major HIV-1 resistance mutations and should be further developed as a resistance surveillance tool.Item Molecular characterisation of Hepatitis B virus vaccine escape mutants in South Africa(2006-11-17T12:51:16Z) Crowther, PennySince the introduction of vaccination against hepatitis B virus (HBV) infection in South Africa, at least one case of infection despite vaccination has occurred. The purpose of this study was to determine whether this infection was the result of mutations within the region of the surface (S) gene encoding the a determinant epitopes of the hepatitis B surface antigen, which permitted viral vaccine-escape. HBV DNA was extracted from the serum and liver tissue of the patient and amplified within the complete 3 215 bp genome and S gene, respectively. Following cloning, sequencing revealed a minor population displaying unique or uncommon S gene mutations that resulted in C138R, C139R, K141R, P142L, T143A, N146D, and T148A amino acid substitutions in the clones from the serum, and C139Y and D144N in the clones from the liver. Such isolates may represent South African HBV vaccine-escape mutants that caused chronic infection in the host prior to their reversion to wild-type.