Validation of a molecular workflow for Cochliomyia hominivorax (New World screwworm) identification in field samples

Cochliomyia hominivorax, New world Screwworm (NWS), has become a reemerging veterinary concern in the United States due to the recent northward expansion of fly detections as far as northern Mexico. Rapid, accurate and validated detection pipelines need to be developed in the case of an incursion into the United States. Confirmatory cases are evaluated by morphological identification with no paired test to verify identifications. With the frequency of submissions of non-ideal samples, particularly from fly traps, a molecular tool would be necessary for species identification. In this manuscript, we develop and assess a pipeline including three real-time PCR assays targeting the ribosomal RNA and five sets of Sanger primers targeting the mitochondrial genome that would be used as a paired tool with morphological identification. Two of the assessed real-time PCR assays are highly specific, sensitive and repeatable requiring <1 copy per reaction for detection. Four of the five Sanger primer sets were assessed, optimized and results evaluated for potential use in preliminary geographic analysis of specimens. This workflow will expedite screening of samples, provide a method to verify results using different tools and help understand genetic variations within the mitochondria for NWS outbreaks.