Identifying kdr Mutations in Aedes Mosquitoes

To identify potential kdr mutations, a fragment of the coding region of the VGSC gene spanning exon 19 to exon 31 (covering the 989, 1011, 1016 and 1534 coding positions) was amplified from cDNA samples and directly sequenced. RNA was extracted from pools of three batches of 10 mosquitoes (not exposed to any insecticide for Ae. aegypti or from DDT resistant for Ae. albopictus) from all the four locations using Picopure kit (Arcturus). cDNA were synthesised using the Superscript III kit (Invitrogen) with oligo-dT20 and RNase H as previously described [21 (link),22 (link)]. The PCR was carried out using 10 pmol of each primers (Additional file 1: Table S1) and 20 ng of cDNA as template in 15 μl reactions containing 1X HF buffer A, 0.2 mM dNTPs, 1.5 mM MgCl₂, 1U Phusion Taq. The cycle conditions were 98°C for 1 min and 35 cycles of 98°C for 10 s, 63°C (60 for Ae. albopictus) for 30 s and 72°C for 1 min and 30 s, followed by a final extension step of 72°C for 10 min. The samples were purified using the Qiaquick PCR purification kit (Qiagen) and sequenced directly. The sequences were aligned and analysed as indicated above.