Pathogen genotyping in polyclonal infections: application of a fluorogenic polymerase-chain-reaction assay in malaria

Abstract

Pathogen genotyping of polyclonal infections is limited by 2 major drawbacks: (1). how to establish whether multiple mutations detected in 1 gene belong to the same clone and (2). how to evaluate the proportion of different genotypes in a given sample. For drug-resistance genotyping in Plasmodium falciparum malaria, we address these problems by using a fluorogenic assay that combines fluorescence-resonance energy transfer, between fluorophores present on a probe and a polymerase-chain-reaction primer, and a melt-curve analysis. We demonstrate that this tool allows a more accurate insight into the P. falciparum populations present in complex biological samples.