Detecting Sporozoites in Anopheles coluzzii

93 blood fed female An. coluzzii collected indoor using PSC, and 37 females which laid eggs (collected with aspirators) were used to detect sporozoite with a TaqMan genotyping approach previously described^{24 (link)}. Real-time PCR MX 3005 (Agilent, Santa Clara, USA) was used for the amplification. 1 μl of gDNA extracted from each female head/thorax was used as a template for PCR, with an initial denaturation at 95 °C for 10 min, followed by 40 cycles each of 15 sec at 95 °C and 1 min at 60 °C. Primers described by Bass^{24 (link)} were used together with two probes labelled with fluorophores, FAM to detect Plasmodium falciparum, and HEX to detect combination of P. ovale, P. vivax and P. malariae. Positive controls (known FAM+ and OVM+) were used in addition to a negative control in which 1 μl of dH₂0 was added. To validate findings of the TaqMan assay a nested PCR of Snounou and colleagues^{25 (link)} was carried out using all the TaqMan-positive samples. Sporozoite rate was calculated as percentage of mosquitoes with sporozoites relative to the total number of the females examined²⁰ and entomological inoculation rate was estimated from the sporozoite rate and human-biting rate, as previously described²⁶ .