Detection of mock mixed infections using the three different methods. Mock mixed infections were prepared as per Table 1. One microliter of each of the mock mixed infection was amplified according to the different protocols resulting in the final parasite concentrations given by Additional file 1. Representative gels are shown for the nested (Snounou) (A), multiplex (Padley) (B), and semi-nested (Rubio) (C). Lane numbers correspond to the lanes as shown in Additional file 1. F: P. falciparum, M: P. malariae, O: P. ovale, V: P. vivax, L: 100 bp molecular weight marker, SL: Species Ladder created by mixing equal amounts of individually amplified species DNA using Rubio primers.