Rapid detection of Single Nucleotide Polymorphisms (SNPs) associated with drug-resistant malaria using an isothermal amplification approach.

Despite the effort to reduce malaria transmission worldwide, no major advances have been made during the past decade. Drug resistance is recognized as one of the major obstacles to malaria control, with key drivers of resistance associated with specific single nucleotide polymorphisms (SNPs). A lack of rapid, costeffective, and simple diagnostic technologies for detecting antimalarial drug resistance is one of the main causes for the spread of drug resistant parasites in endemic countries.
We aim to develop a fast, reliable, and portable biosensor using isothermal amplification approach for the detection of SNPs associated with resistance against antifolates (sulfadoxine-pyrimethamine, SP) used in Intermittent Preventive Treatment of Malaria in Pregnancy (IPTp).
The assay exploits thiolated primers which have the same sequences with the exception of the 3′-terminal base. Target DNA containing the SNP site hybridizes to the immobilized probes, with primer extension only taking place from the primer containing the terminal base that is complementary to the SNP under
interrogation(1).
In a first attempt, the optimization assays will be performed using synthetic DNA templates and appropriate control DNA of parasite clones with known sequences; afterwards field-collected isolates(2) will be analyzed.
With this purpose:
Phase I - Biorecognition Elements: 1) selection of SNPs in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes associated with SP resistance; 2) design of synthetic DNA templates and primers; and 3) preparation of DNA templates from well-characterized P. falciparum clones with known sequences from in vitro cultures;
Phase II - Assay Optimization: optimization the isothermal primer elongation for SNPs detection.
A total of five SPNs were selected, two located in the dhps gene (A437G+K540E) (associated with sulfadoxine resistance) and three in the dhfr gene (N51I+C59R+S108N) (associated with pyrimethamine resistance). Primers, probes and synthetic DNA templates were designed and synthetized. DNA from in vitro cultures of P. falciparum clones were prepared.
The duration of primer extension was optimized to the identification of one SNP (N51I). Ongoing work is focused on optimization of percentage of probe coating and biotinylated dNTPs. Optimized conditions will be applied to the remaining SNPs detection.
By the end of the project, we anticipate to have a proof-of-concept for all SNPs aiming at adapting the isothermal amplification detection of malaria resistance genes to a portable device.