Molecular screening for insecticide resistance

DNA was extracted from control and alive mosquitoes following insecticide exposure using the LIVAK buffer method [23 (link)]. Specimens were identified to species and molecular form by the SINE-PCR protocol [2 (link)]. L1014F, L1014S and N1575Y were screened using TaqMan assays as previously described [6 (link), 24 (link)]. Forward and reverse primers and three minor groove binding (MGB) probes (Applied Biosystems) were designed using the Primer Express™ Software Version 2.0. Primers kdr-Forward (5' CATTTTTCTTGGCCACTGTAGTGAT-3'), and kdr-Reverse (5'-CGATCTTGGTCCATGTTAATTTGCA-3') were standard oligonucleotides with no modification. The probe WT (5'-CTTACGACTAAATTTC-3') was labelled with VIC at the 5' end for the detection of the wild type allele, the probes kdrW (5'-ACGACAAAATTTC-3') and kdrE (5'-ACGACTGAATTTC- 3') were labelled with 6-FAM for detection of the kdr-w and kdr-e alleles respectively. For the N1575Y, the primers F3’TGGATCGCTAGAAATGTTCATGACA-5’ R3’CGAGGAATTGCCTTTAGAGGTTTCT-5’were used [6 (link)].