Limits of detection of the three different methods at detecting the four Plasmodium species. 10-fold serial dilutions of all four species were prepared starting from 40,000p/μl stock. Lane 1 = 40, 000p/μL, 2 = 4,000p/μL, 3 = 400p/μL, 4 = 40p/μL, 5 = 4p/μL 6 = 0.4p/μL and 7 = 0.04p/μL. One microliter of each of these dilutions was amplified as per the different protocols. Representative gels are shown for the nested (Snounou) (A-D), multiplex (Padley) (E-H), and semi-nested (Rubio) (I-L) results. The nested PCR was able to amplify up to 0.4p/μL for each of the species. The multiplex method amplified up to 0.4 p/μL for P. falciparum, 4 p/μL of P. ovale and 40p/μL of both P. malariae and P. vivax while the semi-nested PCR amplified up to 0.04p/μL for P. malariae, 0.4p/μL for both P. falciparum and P. vivax and 4p/μL for P. ovale. L: 100 bp molecular weight marker.