Methods in Anopheles Research - MR4

Chapter 8 : Field Techniques8.5 Real-Time PCR Assays8.5.1 Vector Population Monitoring Tool using Real-Time PCR8.5.1.3 Knockdown resistance (kdr) assaysPage 5 of 6made assigning genotypes more difficult. Difficulty in scoring genotypes is often associated with very lowsignals due to the poor or low yield of DNA obtained.In our experience, distinguishing between Rw/Rw and S/Rw genotypes can seem less obvious thanscoring other genotypes because a Rw/Rw genotype shows a very low level signal in the VIC channelrather than a completely flat line (as with a no-template control). In addition the signal strength from theFAM and VIC probe can be different. Running controls of the two genotypes is very helpful and allows fora direct comparison with unknown samples. Further examples of RwRw and S/Rw genotypes (without theautoscaling) illustrating these points are included below in Figure 8.5.1.3.4.10080Cycling of FAM probe(phenylalanine)Fluorescence6040Rw/RwS/Rw2005 10 15 20 25 30 35 40Cycle10080Cycling of VIC probe (leucine)Fluorescence6040S/Rw20Rw/Rw05 10 15 20 25 30 35 40CycleFigure 8.5.1.3.4. Real-time TaqMan detection of kdr-w (L1014F). Blue trace, R/Rw: Resistant allele,Green trace S/Rw, Black trace, blank. Note the autoscale function was not used in this example and thesignal from the VIC probe is lower than the FAM probe. In addition note that the Rw/Rw signal in the VICchannel is above that of the blank shown in black.Use of Assay on single legsIn our experiments to date when we have run the assay on DNA extracted from a single leg we haveobtained mixed results (possibly due to the low quality/concentration of DNA extracted) and therefore wecannot recommend the TaqMan kdr assays for this approach.ReferencesBass C et al. (2007) Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: acomparison of two new high-throughput assays with existing methods. Malar J 6:111