Rapid real-time PCR genotyping of mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum

Abstract

The resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) is an emerging public health threat. Resistance to these drugs is associated with point mutations in the genes encoding dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). We describe here an assay using real-time PCR and sequence-specific probes that detects these mutations. Using DNA from plasmids, cultured strains, and clinical samples, real-time PCR could distinguish four DHPS polymorphisms (codons 437, 540, 581, and 613) and three DHFR polymorphisms (codons 51, 59, and 108). This assay is rapid and sensitive, with a detection limit of 10 copies in most cases. This assay is amenable to large-scale studies of drug resistance.

FIG. 1.

Real-time PCR detection of P. falciparum genotypes. Plasmid DNA containing wild-type and mutant template DNA (as labeled) were amplified with mixtures of wild-type probe (FAM labeled [solid line]) and mutant probe (VIC labeled [broken line]). Relative fluorescence was calculated by subtracting the minimal fluorescence from each value.

FIG. 2.

Real-time PCR genotyping of a mixed genotype sample. Various mixtures of wild-type and mutant plasmid DNA were amplified. The results for the 51 VIC probe are presented as the averages of three replicates from which the minimal fluorescence has been subtracted. The solid line represents a 100% mutant sample. The gray line represents a 10% mutant and a 90% wild-type sample. The broken line represents a 100% wild-type sample.

FIG. 3.

Real-time PCR genotyping of polymorphism 108 in clinical sample CS1338. The FAM-labeled probe (solid line) is designed to detect the wild-type sequence. The VIC-labeled probe (broken line) is designed to detect the mutant sequence. The change of fluorescence of the two probes was compared to controls run at similar concentrations. The genotype was determined to be mutant (Asn/Thr) with no wild-type component. The relative fluorescence was calculated by subtracting the minimal fluorescence from each value.