Background: Metabolic resistance of the major malaria vector Anopheles gambiae (s.l.) to insecticides is operationally significant, particularly in combination with target site resistance. However, detection of metabolic resistance is not trivial and relies on laborious bioassays, unspecific biochemical methods, or sophisticated and expensive molecular approaches using transcriptomics.
Methods: Rapid one-step multiplex TaqMan-probe based RT-qPCR assays were developed and optimised to measure the expression levels of genes associated with metabolic insecticide resistance in An. gambiae (s.l.). Primers and probes were designed to target the mRNA of cytochrome P450-dependent monooxygenases CYP6P3, CYP6M2, CYP9K1, CYP6P4 and CYP6Z1, and the glutathione-S-transferase GSTE2. The novel assays were validated versus gold standard methods with a range of phenotyped mosquito specimens. The assays were also tested directly on lysates of RNAlater®-preserved mosquitoes without an RNA extraction step.
Results: The novel assays are efficient (reaction efficiencies = 95-109%), sensitive (covering a > 10.0 Ct range with R2 values > 0.99), specific (TaqMan chemistry), reproducible (%CV = 4.46-12.07%), as well as readily expandable to capture additional loci as they evolve or to cover additional species. The assays were successfully validated in terms of expression levels against standard two-step singleplex qPCR assays (overall % difference = -17.6%, 95% CI = -38.7-3.43%) and microarrays, using laboratory strains and field-caught samples. The assays can also be applied directly on lysates of mosquito specimens, without RNA extraction or DNase treatment.
Conclusions: The novel multiplex assays for monitoring the levels of major detoxification genes and metabolic resistance in An. gambiae (s.l.) are simple to perform, robust and rapid. They may complement current diagnostic assays to provide evidence-based and operationally relevant information for insecticide resistance management.
Keywords: Detoxification; GSTEs; Gene expression; Insecticide resistance; Metabolic resistance; Multiplex TaqMan assays; P450s.