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.