Detection of mutations in the Plasmodium falciparum dihydrofolate reductase (dhfr) gene by dot-blot hybridization

Abstract

There is a need for a specific, sensitive, robust, and large-scale method for diagnosis of drug resistance genes in natural Plasmodium falciparum infections. Established polymerase chain reaction (PCR)-based methods may be compromised by the multiplicity of P. falciparum genotypes in natural infections. Here we adopt a dot-blot method to detect point mutations at nucleotide 323 (residue 108) in the P. falciparum dihydrofolate reductase (dhfr) gene using allele-specific oligonucleotide probes. Serine (Ser) or threonine (Thr) at this position are associated with sensitivity to pyrimethamine while asparagine (Asn) is associated with resistance. The method combines PCR amplification and hybridization of amplified products with radiolabeled allele-specific probes. This technique is specific and sensitive; it detects parasitemia of less than 100 parasites/microl of blood, and can identify a minority parasite genotype down to 1% in a mixture. Analysis of P. falciparum isolates from Sudan, of known response to pyrimethamine, has demonstrated the sensitivity and specificity of the method and its ability to detect multiple genotypes in single infections. Furthermore, it has confirmed the association between pyrimethamine responses and dhfr alleles. The method has been successfully extended for analysis of other point mutations in dhfr at residues 51 and 59, which are associated with a high level of pyrimethamine resistance.