title: A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology.
abstract: Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine. However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA)-based technology. Biotinylated PCR products of dhfr, dhps, or Pfcrt were captured on streptavidin-coated microtiter plates and sequence-specific oligonucleotide probes (SSOPs) were hybridized with the PCR products. A stringent washing procedure enabled detection of remaining bound SSOPs and distinguished between the SNPs of dhfr, dhps, and Pfcrt with high specificity. The SSOP-ELISA compared well with a standard PCR-restriction fragment length polymorphism procedure, and gave identical positive results in more than 90% of the P. falciparum slide-positive samples tested. The SSOP-ELISA of all dhfr, dhps, or Pfcrt SNPs on 88 samples can be performed in a single day and provides quick and reproducible results. The system can potentially be modified to detect SNPs in other genes.
authors.affiliation: Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark. Alifrangis@cmp.dk
authors.forename: Michael
authors.initials: M
authors.lastname: Alifrangis
authors.forename: Sonia
authors.initials: S
authors.lastname: Enosse
authors.forename: Richard
authors.initials: R
authors.lastname: Pearce
authors.forename: Chris
authors.initials: C
authors.lastname: Drakeley
authors.forename: Cally
authors.initials: C
authors.lastname: Roper
authors.forename: Insaf F
authors.initials: IF
authors.lastname: Khalil
authors.forename: Watoky Mmm
authors.initials: WM
authors.lastname: Nkya
authors.forename: Anita M
authors.initials: AM
authors.lastname: Rønn
authors.forename: Thor G
authors.initials: TG
authors.lastname: Theander
authors.forename: Ib C
authors.initials: IC
authors.lastname: Bygbjerg
mesh_terms: D000818:Animals; D000962:Antimalarials; D002738:Chloroquine; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis; D004094:Dihydropteroate Synthase / Q000235:genetics*; D004338:Drug Combinations; D004351:Drug Resistance / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000453:epidemiology / Q000209:etiology; D010963:Plasmodium falciparum / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D011739:Pyrimethamine; D013413:Sulfadoxine; D013636:Tanzania / Q000453:epidemiology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
chemical_list: D000962:Antimalarials; D017931:DNA Primers; D016054:DNA, Protozoan; D004338:Drug Combinations; C001205:fanasil, pyrimethamine drug combination; D013413:Sulfadoxine; D002738:Chloroquine; D013762:Tetrahydrofolate Dehydrogenase; D004094:Dihydropteroate Synthase; D011739:Pyrimethamine