MSc (Med), Virology, Faculty of Health Sciences, University of the Witwatersrand / Introduction
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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/8993 |
| Date | 01 February 2011 |
| Creators | Sacks, David |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf, application/pdf |
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