A mutation-specific PCR system to detect sequence variation in the dihydropteroate synthetase gene of Plasmodium falciparum

Ping Wang, Darren R. Brooks, P. F G Sims, John E. Hyde

    Research output: Contribution to journalArticlepeer-review

    Abstract

    Sulphur-based antimalarial drugs targeted at dihydropteroate synthetase (DHPS) are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (DHFR) to combat chloroquine-resistant malaria. We have previously shown that lines of Plasmodium falciparum resistant to the most commonly used sulpha drug, sulphadoxine, carry point mutations in the DHPS coding region, relative to the sequence of sensitive strains (Brooks et al., Eur. J. Biochem. 224 (1994) 397-405). We have now developed PCR diagnostic assays based on allere-specific amplification that are able to detect such mutations. The four tests described can reliably discriminate all of the mutations observed to alter codons 436, 581 and 613, yielding allele-specific amplification products of different sizes in each case. Moreover, by careful adjustment of primer length and the degree of mismatch to target and non-target alleles, we were able to standardise all four tests to a single set of PCR conditions, allowing all possible mutations to be monitored simultaneously on one thermocycler. These assays should prove invaluable in further assessing the contribution of specific base changes in the DHPS gene of the parasite to the suIphadoxine resistance phenotype and to the clinical failure of the sulphadoxine/pyrimethamine combination Fansidar.
    Original languageEnglish
    Pages (from-to)115-125
    Number of pages10
    JournalMolecular and biochemical parasitology
    Volume71
    Issue number1
    DOIs
    Publication statusPublished - 1995

    Keywords

    • Dihydropteroate synthetase
    • Fansidar resistance
    • Malaria
    • Mutation-specific PCR
    • Plasmodium falciparum
    • Sulphadoxine resistance

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