A novel qPCR assay to detect the presence of Anopheles gambiae complex mosquitoes

Abstract Background Anopheles mosquitoes vector pathogens responsible for more than 600,000 human deaths annually. Ecological studies of these insects are important to guide effective vector-control campaigns and to understand their broader ecological consequences. Molecular ecology methods, particularly qPCR, provide a valuable tool in such studies. By detecting trace DNA of a taxon of interest within mixed or environmental samples, qPCR can facilitate identification of prey taxa of interest in the diets of consumers. However, no protocol for the detection of An. gambiae complex mosquitoes in dietary samples has been available. Methods We introduce a new set of qPCR primers (Agam_CO1_F1 and Agam_CO1_R1) and a probe-based assay for detection of Anopheles gambiae -complex mosquitoes, even with short reads common in dietary and environmental samples. The primers were tested in vitro for their specificity and sensitivity, and in silico using Primer-BLAST to assess potential off-target amplification. Results The qPCR primers amplified An. gambiae DNA even at low starting concentrations (5 copies µl - 1 ). The primers did not amplify any non-target DNA in either the in vitro or in silico tests, but consistently amplified An. gambiae complex DNA. The primers can therefore provide reliable tests for the presence or absence of An. gambiae complex in dietary or eDNA samples. Conclusions The new qPCR primers should allow advances in research into mosquito ecology by allowing detection of even trace amounts of An. gambiae DNA in dietary and environmental samples. Graphical abstract