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

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 allele-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 sulphadoxine resistance phenotype and to the clinical failure of the sulphadoxine/pyrimethamine combination Fansidar.