A MUTATION-SPECIFIC PCR SYSTEM TO DETECT SEQUENCE VARIATION IN THE DIHYDROPTEROATE SYNTHETASE GENE OF PLASMODIUM-FALCIPARUM

Sulphur-based antimalarial drugs targeted at dihydropteroate synthetas e (DHPS) are frequently used in synergistic combination with inhibitor s of dihydrofolate reductase (DHFR) to combat chloroquine-resistant ma laria. We have previously shown that lines of Plasmodium falciparum re sistant to the most commonly used sulpha drug, sulphadoxine, carry poi nt mutations in the DHPS coding region, relative to the sequence of se nsitive strains (Brooks et al., fur. J. Biochem. 224 (1994) 397-405). We have now developed PCR diagnostic assays based on allele-specific a mplification that are able to detect such mutations. The four tests de scribed can reliably discriminate all of the mutations observed to alt er codons 436, 581 and 613, yielding allele-specific amplification pro ducts 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 simul taneously on one thermocycler. These assays should prove invaluable in further assessing the contribution of specific base changes in the DH PS gene of the parasite to the sulphadoxine resistance phenotype and t o the clinical failure of the sulphadoxine/pyrimethamine combination F ansidar.