Mgb probe

RNA was extracted using RNeasy columns (Qiagen) and converted to cDNA using the High-capacity cDNA archive Kit (Applied Biosystems). qPCR was performed using Taqman Universal PCR Master Mix and MGB probes (Applied Biosystems) and amplified using an ABI Prism 7700 System. The expression levels of target genes were normalized to four housekeeping genes: β-actin (NM_03114), HPRT1 (Rn01527840_m1), rplp2 (Rn01479927_g1) and psmc5 (Rn00579821_m1). Differences between samples were analyzed using the comparative Ct method (ΔΔCt). Gene specific arrays were obtained from Thermo-Fisher Scientific. Details are available on request.

Primers and probes for IS481, IS1001, and IS1002 were based on Roorda et al. (2011) [19 (link)], those for recA were based on Guthrie et al. (2010) [18 (link)], and those used for internal control were based on van Doornum et al. (2003) [21 (link)] (sequences in Table 1). Primers and probes were manufactured by Eurogentec (Liège, Belgium), MGB probes were manufactured by Applied Biosystems (Warrington, UK).
Two different multiplex PCR’s were performed, one for the detection of IS481 and IS1001, and one for the detection of recA and IS1002. A 50 μl reaction mixture consisting of 0.3 μM of each of the primers, 0.2 μM of each of the probes, iQ Multiplex Powermix (Bio-Rad Laboratories, Temse, Belgium), and 5.0 μl of the extracted NA was used in each PCR. Amplification was performed on the LightCycler 480 II PCR system (Roche Diagnostics, Mannheim, Germany). The PCR thermal profile consisted of a 3-minute cycle at 95°C, followed by 45 cycles of 15 seconds at 95°C and 1 minute at 60°C. Acquisition of the fluorescence signal was set during each cycle. A final cooling step of 30 seconds at 40°C was included. The crossing point (Cp) values were determined automatically using the “second derivative maximum” method present in the Roche LightCycler 480 Software, version 1.5. An algorithm with cut-off values was developed and used for the interpretation of the real-time PCR results (Fig 1).

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)].