Design of a simple PCR assay to detect the 4.3kb SV and analysis of its distribution and association with pyrethroid resistance.
A PCR was designed to discriminate between mosquito samples with the 4.3kb structural variant and those without, consisting of 3 primers. Two primers (4.3kb_INSL_F: GGG GCG CTT TAG TTG AGA T and 4.3kb_INSR_R: CAC GTT TCA AGT GCA GGT GA) form a pair flanking the insertion but, due to the size of the insertion, amplify only for samples lacking the insertion, to produce a 281bp amplicon. A third primer (4.3kb_INS_R: CAT ACG CCT CTC CAG CAT GGA) binding within the structural variant forms a pair with 4.3kb_INSL_F to give a 780bp product only from samples containing the insertion. PCR amplification was done using the Kapa Taq PCR kit (Kapa Biosystems) with a 15µl reaction mix composed of 10x buffer A, 0.75µl of 25mM MgCl2, 0.12µl of 25mM dNTPs, 0.51µl of each primer, 0.12µl of Kapa Taq enzyme, 10.49µl of deionised water and 1ul of genomic DNA. Thermocycler conditions were: pre-denaturation at 95 °C for 5 minutes; 35 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute; a final extension at 72°C for 5 minutes. PCR products were separated by (1.5%) agarose gel electrophoresis, stained with Midori Green Advance DNA Stain (Nippon Genetics Europe GmbH) and visualised on a UV transilluminator. After optimisation, these assays were used to investigate any possible association between this structural variant and pyrethroid resistance using field F1 and laboratory mosquitoes by genotyping mosquitoes dead and alive after insecticide bioassays.
The spatio-temporal distribution of this structural variant in An. funestus s.s. populations collected in different location across Africa (Ghana, Cameroon, Kenya, Uganda, Tanzania, and Mozambique) was investigated. Genomic DNA samples from previous collections from across Africa (Weedall et al., 2020) were also used.