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

Abstract

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

Rapid, Field-Deployable Method for Genotyping and Discovery of Single-Nucleotide Polymorphisms Associated with Drug Resistance in Plasmodium falciparum

“…falciparum (1,7,14); however, the techniques presented here show clear advances over the previous works. We have implemented a probe-based technique that improves the sensitivity of these assays over the whole-amplicon methodology presented in the work of Andriantsoanirina et al and similar to the technology presented by both Gan and Loh and Cruz et al…”

A rapid and cost‐effective quantitative microsatellite genotyping protocol to estimate intraspecific competition in protist microcosm experiments

“…) has largely been replaced by other methods to estimate genotype frequencies from mixed populations of cells, including qPCR melt curve analysis (Cruz et al . ), SNP‐based pyrosequencing (Wasson et al . ) and FREQ‐Seq (Chubiz et al .…”

Prevalence of Plasmodium falciparum Resistance Markers to Sulfadoxine-Pyrimethamine among Pregnant Women Receiving Intermittent Preventive Treatment for Malaria in Uganda

“…Asymmetric PCR was performed in the presence of an unlabeled probe specific to the SNP of interest, followed by melting curve analysis to identify the wild-type or mutant form. This methodology has been described to effectively genotype using a closed-tube system and has many advantages, including minimizing cross-contamination and high-throughput capacity (30,32,33). Parasite DNA was successfully extracted from 413 P. falciparum-positive samples.…”

“…A molecular assay was used for genotyping SP-resistant strains of P. falciparum that identifies single-nucleotide polymorphisms (SNP) within the Pfdhfr and Pfdhps genes that are associated with resistance to SP (30). The assay targets three SNPs within the Pfdhfr gene at codons 51I, 59R, and 108N that confer resistance to pyrimethamine and four SNPs within the Pfdhps gene at codons 437G, 540E, 581G, and 613G that confer resistance to sulfadoxine (8,30). This method is based on high-resolution melting curve analysis using a LightCycler (Roche), as previously described (31).…”