Figure 2

Molecular diagnostic assays for the detection of diflubenzuron resistance mutations in Culex pipiens. (A) Diagnostic PCR-RFLP for the mutation I1043M. Upper part: Schematic representation of the assay. The position and sequence of primers used to amplify the 124 bp fragment are presented. The position of the NlaIII restriction site created by the mutation (CATC → CATG) is depicted as a pair of scissors at the middle (64 bp) of the PCR product. Below is shown a representative agarose gel (3%) were the NlaIII digested PCR products are shown. M: HyperLadder V 25pb (Bioline,UK), Lanes 1, 2, 3 individuals homozygous for the wild type allele (II), Lanes 4, 5, 6 individuals heterozygous for the mutated allele (IM), Lanes 7, 8, 9 individuals homozygous for the mutated allele (MM), M2 100 bp DNA ladder. (B) Allele specific PCR for the mutation I1043L.Upper part: Schematic representation of the assay. The position of the four primers used is shown. External_F and External_R primers pair and produce a common 352 bp band. External_F and ATC_ R pair and produce a susceptible (Ile) specific 135 bp band and CTC_F and External_R pair and produce a resistant (Leu) specific 260 bp band. Lower part: Representative agarose gel (2%) with obtained PCR products. M 100 bp ladder, Lane 1, 2 products from homozygous susceptible individuals (II), Lanes 3, 4 products from heterozygous individuals (IL) and Lane 5 product from a homozygous mutated individual (LL). White line denotes the part of the gel that has been cropped and re-grouped. Full-length gels are presented in Supplementary Fig. S1.