Rapid and cost-effective screening of pyrethroid resistance markers in Musca domestica using RNAse H2 PCR

Abstract Insecticide resistance is widespread in insect pest populations and has been linked to reductions in the efficacy of chemical control as well as negative consequences for public health and agricultural production. Monitoring resistance is therefore an important element of effective integrated pest management and is a recommended practice. Genetic mutation of target sites is often an important resistance mechanism, and, for some species, particular target site mutations or combinations of mutations are high value markers of resistance to specific classes of pesticides. Implementing new assessment technologies can allow more rapid and cost-effective assessment of these important mutations. We developed assays using the high specificity of RNAse H2 PCR (rhPCR) to assess the presence of five well characterized knockdown resistance ( kdr ) mutations in Musca domestica , an important pest in dairy operations with a complicated kdr phenotype. Using a common sample set, we assessed reaction accuracy and overall kdr genotyping success of four methods: (1) the traditional gold standard Sanger method using purified DNA template as the PCR template, (2) a Sanger method using a leg homogenate as the PCR template, (3) an rhPCR method using leg homogenate and (4) a Nanopore-based method using leg homogenate. Results showed that individual rhPCR reactions were accurate and that overall kdr genotyping success was, on the first attempt, comparable to the gold standard Sanger method. Cost and time to genotype by rhPCR were reduced by 75% or more when compared to Sanger based genotyping. To demonstrate the broad applicability and comparability of screening for kdr single nucleotide polymorphisms using the rhPCR method we conducted an assessment of kdr genotypes in United States dairy operations and found similar kdr patterns to other recent studies using the traditional methods but at significant reduction in cost.