A novel real‐time PCR for New World screwworm fly ( Cochliomyia hominivorax ) and its application in a non‐destructive multiplex for efficient detection of screwworm flies

Surveillance and diagnostics are critical for the early detection, containment and eradication of exotic pests. For the screwworm fly, this is usually via targeted surveillance and exclusion testing of trap‐caught flies, as well as the identification of larvae associated with myiasis wounds. We present a specific and sensitive real‐time polymerase chain reaction (PCR) assay for the detection of the New World screwworm fly, Cochliomyia hominivorax Coquerel (Diptera: Calliphoridae). The assay targets the cytochrome oxidase subunit 1 ( cox1 ) gene from adult flies or larvae and retains high analytical sensitivity when multiplexed with an existing assay for the Old World screwworm fly, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), achieving a limit of detection of less than 1 copy per microlitre of reaction. To assess its utility for surveillance and diagnostics, a novel non‐destructive DNA extraction method was performed on spiked trap catches of field‐caught flies, and on boiled and unboiled specimens of larval instars. The multiplexed assay detected 95% of spiked flies, and all screwworm flies from positive samples were retrieved and morphologically identified. Results from larval instars confirmed that the assay can be used for larvae, with higher sensitivity observed for unboiled larval instars. This molecular assay enables the simultaneous detection of Co. hominivorax and Ch. bezziana , offering a reliable alternative to existing single‐target and destructive methods of bulk fly testing. It also holds potential for broader application in the identification of larval stages.