Multiplex Real-Time PCR Assay for Simultaneous Detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri

Amplification curves from cultured trophozoites, serially diluted in parasite-free CSF. Negative samples were unspiked CSF. Each sample was amplified in parallel in the single-target assay with just one set of primers and probe (dashed lines) and in the triplex assay containing oligonucleotides for detection of all three variants of amebas (solid lines). Only the fluorescence signal from the relevant probe in the triplex reactions is displayed in each figure (the signals from the other two probes remained below threshold for all samples throughout all runs). All experiments were repeated three times; the average results are displayed. dRn, baseline subtracted fluorescence reading normalized to a reference dye.