Computational protocol: Knockdown resistance in Anopheles vagus, An. sinensis, An. paraliae and An. peditaeniatus populations of the Mekong region

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Protocol publication

[…] Primers Agd1 and Agd2 [] were used to amplify the DIIS6 region (kdr- region or transmembrane segment 6 of the domain II) of the para-type sodium channel gene for An. vagus, whereas primers Agd1Mi and Agd2H (Figure ) amplified the DIIS6 region for An. sinensis, An. paraliae and An. peditaeniatus. Amplification was performed in a 50 μl reaction containing 1 μl of template DNA, 1 × Qiagen PCR buffer, 1 mM MgCl2, 200 μM of each dNTP, 100 nM of each primer and 1 unit Taq DNA polymerase (Taq PCR core kit, Qiagen, Hilden, Germany).The cycling conditions were as follows: initial denaturation at 94°C for 3 min, 40 cycles of 1 min denaturation at 94°C, 30 s annealing at 47°C and 30 s extension at 72°C followed by a final extension of 10 min at 72°C. Amplification products were checked on a 2% agarose gel, stained with ethidium bromide and visualised on the Syngene Ingenius LHR (Westburg, Leusden, The Netherlands). The resulting PCR product was cloned by use of the Original TA cloning kit according to the manufacturer's instructions (Invitrogen, Carlsbad, California). Plasmid and direct PCR sequencing were done by the VIB genetic service facility (University of Antwerp, Belgium) and aligned with ClustalW version 1.3 [].The DIIS6 sequences were used to develop primers for four allele-specific PCR assays (AS-PCRs) to assess the kdr frequencies in the different Anopheles populations. An additional A was added to 3'-end of the primer Vaguss in order to minimize self-complementarity and additional mismatch bases were introduced in the Sinensiss and Sinensisr primers at the 4th nucleotide from the 3'-end (A was replaced by T) to obtain more specific results (Figure ). The AS-PCR assays were optimized by running genomic DNA templates that had been previously genotyped by DNA sequencing. All AS-PCR assays were performed in a 50 μl reaction mixture containing: 1 × Qiagen PCR buffer, 1 × Q solution, 0.5 mM MgCl2, 200 μM dNTP's, 400 nM of the outer forward and reverse primer, 500 nM of the inner resistant and sensitive primer (Figure ), 1 unit Taq DNA polymerase (Qiagen, Hilden, Germany) and 1 μl template. The cycling conditions described above for the amplification of the DIIS6 region were used. The amplification products were electrophoresed on a 3% mixed agarose gel (1.5% agarose and 1.5% small fragment agarose) and visualised under UV light after ethidium bromide staining. In each assay, a sequenced heterozygote resistant mosquito was run with the AS-PCR as positive control.The kdr genotype frequencies of mosquitoes exposed to WHO bioassay were compared for dead and surviving using the exact tests for population differentiation in Genepop (version 3.4) []. […]

Pipeline specifications

Software tools Clustal W, Genepop
Application Population genetic analysis
Diseases Malaria
Chemicals DDT, Pyrethrins, Sodium