Rapid and cost-effective screening of genetic markers associated with pyrethroid resistance in Musca domestica using RNAse H2 PCR (rhPCR)

Pyrethroid resistance, particularly knockdown resistance ( kdr ), is widespread in insect pest populations, but rarely has kdr been associated with field-level pest control failure. The prevailing understanding is that kdr contributes to a resistant phenotype, but this knowledge has remained largely an academic pursuit and has not translated to tools and strategies needed by agricultural producers to make rapid decisions for effective resistance management. As a first step in providing these operational tools, we developed robust assays using the high specificity of rhPCR to reduce kdr assessment time by approximately 80% and costs ~ 75% from the traditional Sanger based method used for Musca domestica . An important consideration for the use of an operational tool is the ability to get an accurate result on the first attempt, so we used Nanopore sequencing to confirm genotypes in a subset of samples and found the first pass genotyping accuracy of rhPCR method to be 75.0%, versus 41.2% with the traditional Sanger method. To demonstrate the broad applicability and comparability of screening for kdr SNPs using rhPCR, we conducted the largest assessment of kdr genotypes of M. domestica in United States dairy operations and found similar kdr patterns to other recent studies using traditional methods.