High throughput genotyping of single nucleotide polymorphisms in the Plasmodium falciparum dhfr and dhps genes by asymmetric PCR and melt-curve analysis

  • Author / Creator
    Cruz, Rochelle
  • Mutations within Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes contribute to resistance to antimalarials such as sulfadoxine-pyrimethamine (SP). Single nucleotide polymorphisms (SNPs) within codons 51, 59, 108 and 164 in the pfdhfr gene and codons 436, 437, 540, 581, and 613 in the pfdhps gene are associated with SP treatment failure. This study focused on the development of a genotyping assay based on asymmetric real-time PCR and melt-curve analysis. Probes specific to each SNP hybridize differentially to mutant and wild-type sequences, generating distinct melting curves. The analytical sensitivity and specificity were determined on the LightCycler® platform using reference strains of malaria. The assay was validated with clinical samples from patients with malaria and compared to a gold standard test. This assay was also tested for its application to in-gel PCR technology for point-of-care testing in resource-limited areas. Genotyping is possible with further enhancements to this technology.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.