Mosquito collections
Mosquitoes were collected at monthly intervals from March 2001 to February 2002 in seven sites throughout Uganda (Okello, unpublished data). Three houses per site were chosen and human landing collections were done during six consecutive nights from 20.00 hr until 06.00 hr. The mosquitoes were morphologically identified using the key developed by Gillies and Coetzee [8] and were stored on silica gel.
DNA extraction and molecular identification of species
The protocol for DNA extraction is adapted from Vythilingam et al. [9]. Since collections were made in the frame of a transmission study (Okello, unpublished data), heads and thoraxes of individual mosquitoes were homogenized in 150 μl blocking buffer [10] and 50 μl of blocking buffer/Nonidet P-40 (blocking buffer containing 0.5 % Nonidet P-40). These enzyme linked immunosorbent assay (ELISA) homogenates were used for mosquito DNA extraction, by adding 200 μl of a 20% Chelex solution (Biorad, Hercules, USA) to 20 μl of the mosquito homogenate. Samples were placed in a thermo-mixer at 56°C and 1,400 rpm for 30 minutes, vortexed at high speed for six seconds and placed in a thermo-mixer at 95°C and 1,400 rpm for 10 minutes. After incubation, the samples were centrifuged twice at 13,400 g for five minutes at 4°C. The supernatants containing the DNA was transferred and stored at -20°C.
The protocol used for molecular identification of the members of the An. gambiae complex was adapted from Scott et al. [11]. Genomic DNA was mixed with the primers AR (specific for An. arabiensis), AG (specific for An. gambiae s.s.) and UN (common for both species) in a 25 μl reaction. Amplification reactions contained 1 μL of DNA, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.1% Triton X-100, 200 μM of dNTP's (Amersham, Buckinghamshire, United Kingdom), 80 nM of primers UN and AR, 40 nM of primer GA and 0.25 U of Silverstar DNA polymerase (Eurogentec, Seraing, Belgium). The PCR was carried out as described in Scott et al. [11]. The amplified products were checked on a 2% agarose gel, stained with ethidium bromide, and visualized on the Image Master VDS (Amersham Pharmacia, Uppsala, Sweden).
AS-PCR for detection of the L1014S and L1014F kdr alleles
The protocol used for the detection of the L1014S or L1014F kdr alleles was adapted from the protocols developed by Martinez-Torres et al. [3] and Ranson et al. [4]. Primers Agd1 (5'-atagattccccgaccatg-3'), Agd2 (5'-agacaaggatgatgaacc-3'), Agd3 (5'-aatttgcattacttacgaca-3') and Agd4 (5'-ctgtagtgataggaaattta-3') were used to detect the L1014F allele (AS-PCR Agd3), whereas primers Agd1, Agd2, Agd4 and Agd5 (5'-tttgcattacttacgactg-3') were used to detect the L1014S allele (AS-PCR Agd5) (Fig 1). Amplification was performed in a 50 μl reaction containing 1 μl of template DNA, 1 × Qiagen PCR buffer, 0.5 mM MgCl2, 100 nM of each primer, 200 μM of dNTP's, and 1 U of Taq DNA polymerase (Taq PCR core kit, Qiagen, Hilden, Germany). The cycling conditions were: initial 94°C denaturation for five minutes, 10 cycles of one minute denaturation at 94°C, 30 seconds annealing at 54°C and 30 seconds extension at 72°C, followed by 30 cycles of one minute denaturation at 94°C, 30 seconds annealing at 47°C and 30 seconds extension at 72°C, and a final extension at 72°C for 10 minutes. Amplification products were checked on a 2% agarose gel and visualized after ethidium bromide staining.
FRET/Melt Curve analysis
Primers and probes were designed using the Meltcalc software [12]. The forward primer AgdF-ROX (5'- tggccactgttgtgaTagg-3') is labelled with ROX, 4 nucleotides from the 3'-end (Capital T), while the probe KDR-FAM (5'-tacttacgactaaatttcctat-3') is labelled with FAM, at its 3'-end. The probe complements the wild type antisense strand of the PCR product. Two non-specific g's were added to the 5'-end of the reversed primer AgdR (5'-ggtgacaaaagcaaggctaag-3') to increase the melting temperature and the GC-content of the primer.
A primary PCR was performed with a 50 μl reaction mix containing 1 μl of DNA, 1 × Qiagen PCR buffer, 1 mM MgCl2, 200 μM of each dNTP, 100 nM of the primers Agd1 and Agd2 and 1 U Taq DNA polymerase (Taq PCR core kit, Qiagen, Hilden, Germany). Amplification conditions were as follows: initial denaturation at 94°C for three minutes, forty cycles of one minute denaturation at 94°C, 30 seconds annealing at 47°C and 30 seconds extension at 72°C followed by a final extension of 10 minutes at 72°C (Figure 2A).
The secondary PCR assay and the MCA were performed on an iCycler with a 490/20X FAM excitation filter and a 620/30M ROX emission filter (Bio-rad, Hercules, USA). The secondary PCR was performed in a 96-well plate. Amplification reactions (50 μl) contained 1 × iQ supermix (Bio-Rad, Hercules, USA), 1 mM MgCl2, 500 nM of AgdF-ROX, 100 nM of AgdR and 2 μl of a 10-fold dilution of the primary PCR product resulting from amplification with Agd1 and Agd2 (Figure 2B). Cycling conditions were as follows: initial denaturation at 95°C for four minutes, forty cycles of one minute denaturation at 95°C, 30 seconds annealing at 52°C and 30 seconds extension at 72°C followed by a final extension at 72°C for eight minutes. During this asymmetric PCR, the target strand to which the FAM-labelled probe binds, was produced in excess. After amplification, the probe was added in a final concentration of 200 nM, and a melt curve was performed, consisting of 95°C for one minute, cooling to 40°C for one minute and 80 repeats heating for 20 seconds, starting at 40°C and with 0.5°C increments. During this melt curve, the cooling to 40°C will allow the FAM-labelled probe to anneal adjacent to the ROX-fluorophore of the PCR product (Figure 2C). The temperature is subsequently slowly increased, while the ROX-fluorescence resulting from FRET, is continually monitored. When the melting temperature of the probe-amplicon hybrid is reached, FRET can no longer occur, and the ROX-fluorescence will decrease (Figure 2D). Changes in the ROX-fluorescence appear as a peak on the plot of the first negative derivative of the fluorescence versus temperature function. All data were analysed with the iCycler™ iQ Optical system software version 3.0a (Bio-rad, Hercules, USA). The experiments were performed in triplicate to verify reproducibility.
Three plasmids, containing as inserts the wild type, the L1014S or the L1014F kdr allele, were used on each plate as positive controls. Two lines, a susceptible line and a homozygote resistant line for the West African kdr allele L1014F, were obtained from laboratory colonies of the LIN/IRD and were used to construct the L1014L (wild type) and the L1014F plasmid, respectively. The L1014S plasmid originated from a sequenced homozygous L1014S field specimen of Uganda. The plasmids were constructed by ligation and transformation of the 293 bp PCR product resulting from amplification with primers Agd1 and Agd2, by using the Original TA cloning kit according to the manufacturer's instructions (Invitrogen, Carlsbad, California). Three millilitres of each clone was purified on a column (QIAprep Spin Miniprep kit, Qiagen, Hilden, Germany) and DNA was resuspended in 50 μl water. Two microlitres of a 100-fold dilution of the plasmid was used directly in the secondary, asymmetric PCR of the FRET/MCA assay instead of the template DNA. Plasmid and direct PCR sequencing were performed by the VIB genetic service facility (University of Antwerp, Belgium).