High-Throughput Genotyping of Single Nucleotide Polymorphisms in the Plasmodium falciparum dhfr Gene by Asymmetric PCR and Melt-Curve Analysis

摘要: Mutations within the Plasmodium falciparum dihydrofolate reductase gene (Pfdhfr) contribute to resistance antimalarials such as sulfadoxine-pyrimethamine (SP). Of particular importance are single nucleotide polymorphisms (SNPs) codons 51, 59, 108, and 164 in Pfdhfr that associated with SP treatment failure. Given traditional genotyping methods time-consuming laborious, we developed an assay provides rapid, high-throughput analysis of parasite DNA isolated from clinical samples. This is based on asymmetric real-time PCR melt-curve (MCA) performed LightCycler platform. Unlabeled probes specific each SNP included reaction mixture hybridize differentially mutant wild-type sequences amplicon, generating distinct melting curves. Since probe present throughout MCA, proceeds seamlessly no further addition reagents. was validated for analytical sensitivity specificity using plasmids, purified genomic reference strains, cultures. For all four SNPs, correct genotypes were identified 100 copies template. The performance evaluated a blind panel isolates travelers low-level parasitemia. concordance between our sequencing ranged 84 100% depending SNP. We also directly compared MCA published TaqMan major issues probes. Our number technical improvements facilitate screening patient samples identify SP-resistant malaria.