DNA from EDTA-anticoagulated whole blood is extracted and tested using real-time PCR on the LightCycler 2.0 instrument (Roche Applied Science) with primers and fluorescence resonance energy transfer (FRET) probes. A genus-specific primer set corresponding to 18S rRNA is used to amplify target sequence. One pair of FRET hybridization probes was designed for Plasmodium falciparum over a region containing base pair mismatches allowing for differentiation of other Plasmodium species by use of melting curve analysis, while a second probe set is specific for Plasmodium knowlesi (Babady NE, Sloan LM, Rosenblatt JE, Pritt BS: Detection of Plasmodium knowlesi by Real-Time Polymerase Chain Reaction. Am J Trop Med Hyg 2009 Sept;81(3):516-518).
Slides are used to determine percentage of parasitemia if PCR is positive.