Abstract
Despite efforts to reduce malaria transmission worldwide, no major progress has been made in 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). The lack of rapid, cost-effective, and simple diagnostic technologies to detect antimalarial drug
resistance is a major cause of the spread of drug-resistant parasites in endemic countries.
We aim to develop a rapid, reliable, and portable biosensor using an isothermal amplification approach for the detection of SNPs associated with resistance of Plasmodium falciparum parasites to antifolates (sulfadoxine-pyrimethamine) used in various chemoprevention regimens for children and pregnant women in endemic areas. The test is based on the optical detection of the isothermal
extension of primers in the solid phase. 5'-end thiolated primers containing the SNP of interest at the 3′-end nucleotide hybridize to target DNA. Primer extension with peroxidase-labeled dinucleotides occurs only from the primer containing the 3’-end nucleotide complementary to the SNP of interest.
To achieve the objective, the following procedures have been performed: i) selection of SNPs associated with pyrimethamine (N51I+C59R+S108N) and sulfadoxine (A437G+K540E) resistance located in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) P. falciparum genes, respectively, ii) design of synthetic DNA templates and primers, iii) preparation
of DNA templates from in vitro cultures of P. falciparum clones containing the SNPs of interest, iv) optimization assays using synthetic DNA templates (ii) and appropriate control DNA from parasite clones (viii). The methodology will then be validated using DNA templates from field-collected P. falciparum isolates from Equatorial Guinea2. At the end of the project, we expect to have a proof of-
concept for all SNPs with the aim of adapting the isothermal amplification detection of drug resistance in malaria parasites to a portable device.
resistance is a major cause of the spread of drug-resistant parasites in endemic countries.
We aim to develop a rapid, reliable, and portable biosensor using an isothermal amplification approach for the detection of SNPs associated with resistance of Plasmodium falciparum parasites to antifolates (sulfadoxine-pyrimethamine) used in various chemoprevention regimens for children and pregnant women in endemic areas. The test is based on the optical detection of the isothermal
extension of primers in the solid phase. 5'-end thiolated primers containing the SNP of interest at the 3′-end nucleotide hybridize to target DNA. Primer extension with peroxidase-labeled dinucleotides occurs only from the primer containing the 3’-end nucleotide complementary to the SNP of interest.
To achieve the objective, the following procedures have been performed: i) selection of SNPs associated with pyrimethamine (N51I+C59R+S108N) and sulfadoxine (A437G+K540E) resistance located in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) P. falciparum genes, respectively, ii) design of synthetic DNA templates and primers, iii) preparation
of DNA templates from in vitro cultures of P. falciparum clones containing the SNPs of interest, iv) optimization assays using synthetic DNA templates (ii) and appropriate control DNA from parasite clones (viii). The methodology will then be validated using DNA templates from field-collected P. falciparum isolates from Equatorial Guinea2. At the end of the project, we expect to have a proof of-
concept for all SNPs with the aim of adapting the isothermal amplification detection of drug resistance in malaria parasites to a portable device.
| Original language | English |
|---|---|
| Pages | P3.38 |
| Publication status | Published - Dec 2023 |
| Event | Congress of Microbiology and Biotechnology 2023 - Covilhã, Portugal Duration: 7 Dec 2023 → 9 Dec 2023 https://microbiotec23.organideia.com/ |
Conference
| Conference | Congress of Microbiology and Biotechnology 2023 |
|---|---|
| Abbreviated title | Microbiotec'23 |
| Country/Territory | Portugal |
| City | Covilhã |
| Period | 7/12/23 → 9/12/23 |
| Internet address |
Keywords
- malaria
- diagnostic
- drug resistance
- isothermal-amplification
- biosensors
