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.