ITS2 PCR Amplification
PCR amplification of the ITS2 region was performed with Hot FirePol DNA Polymerase Kit (Solis Biodyne). Total PCR mix volume per sample was 50 µL with 48 µL premix and 2 µL DNA. The premix contained 36.8 µL H2O, 5 µL 10X Buffer B1, 3 µL 25 mM MgCl2, 1 µL 10 mM Deoxynucleotide (dNTP) Solution Mix (New England BioLabs), 1 µL 10 nM Forward ITS2 Primer, 1 µL 10 nM Reverse ITS2 Primer and 0.2 µL (5 U/µL) Hot FirePol DNA Polymerase. The DNA was either undiluted for extracted midguts and legs or diluted at 1/10 in DNAse/RNAse free water for extracted carcasses.Anopheles ITS2 primer sequences are 5’-TGTGAACTGCAGGACACAT-3’ (Forward) and 5’-TATGCTTAAATTCAGGGGGTAG-3’ (Reverse)[31,39] . PCR reactions were done on a SimpliAmp Thermal Cycler (Applied Biosystems) using the cycle: 95 °C x 15 min, [95 °C x 30 s, 56 °C x 45 s, 72 °C x 40 s] x 35 cycles, 72 °C x 10 min, 4 °C x ∞. Amplified DNA was either used directly after or stored at 4 °C or -20 °C until use.