Development and Validation of a Real-Time PCR Assay for Identification of a Common Agrotis Species: Agrotis segetum (Lepidoptera: Noctuidae)

The common cutworm, Agrotis segetum, is a serious soil pest of many vegetable and field crops all over the world. Morphological identification of Agrotis species is predominantly performed on adults due to the deficiency of adequate identification keys for immature stages. In international trade, the immature life stages are frequently being intercepted at point of inspection, challenging the possibilities of morphological identification. To realize a rapid and reliable identification for all stages of A. segetum, a TaqMan real-time Polymerase Chain Reaction (PCR) was developed based on the mitochondrial Cytochrome Oxidase I (COI) gene. All specimens of A. segetum (including various life stages) were detected and no cross-reactivity was observed with 5 non-target Agrotis species in the specificity tests. The tests showed to be repeatable, reproducible, and robust. The assay performed equally well with crushed insects and purified DNA, so, the efficiency was added by removing DNA extraction step. The method has proven to be suitable tools for routine identification of all life stages of A. segetum considering the speed, specificity, as well as sensitivity of the assay.