A new method for detection of pfmdr1 mutations in Plasmodium falciparum DNA using real-time PCR

Abstract

Background: Surveillance for drug-resistant Plasmodium falciparum should be a component of malaria control programmes. Real-time PCR methods for the detection of parasite single-nucleotide polymorphisms (SNPs) and gene amplification could be useful survellance tools.

Methods: A real-time PCR assay has been developed that identifies single nucleotide polymorphisms (SNPs) at amino acids 86, 184, 1034 and 1042 in the P. falciparum multi-drug resistant (pfmdr 1) gene that may be associated with anti-malarial drug resistance.

Results: This assay has a sensitivity and specificity of 94% and 100% when compared to traditional PCR methods for genotyping. Only 54 of 68 (79%) paired pre- and post-culture DNA samples were concordant at all four loci.

Conclusion: Real-time PCR is a sensitive and specific method to detect SNP's in pfmdr 1. Genotypes of parasites after in vitro culture may not reflect that seen in vivo.

Figure 1

Fluorescent intensity (Rn) of the FAM reporter dye (blue line) compared to VIC reporter dye (green line) as PCR amplification cycles proceed (x axes). Fluorescent intensity is measured as Rn (y-axes), the absolute fluorescence of the reporter dye divided by that of the passive reference dye, Rox. An increase in FAM indicates a wild type population [86(e), 184(f), 1034(g), 1042(h)] while an increase in VIC indicates a mutant population [86(a), 184(b), 1034(c), 1042(d)]. Polyclonal infections may be identified by an increase in fluorescent intensity of both reporter dyes (not shown). Mutant 1034 DNA (c) was obtained from a previous study [6] because none of the samples in this study contained that mutation.