Improved Molecular Detection Tools for the Invasive Crop Pest \u3ci\u3eHelicoverpa armigera\u3c/i\u3e (Hübner)

Helicoverpa armigera is a major crop pest native to Europe, Asia, Australia, and Africa. H. armigera has recently invaded South America and has caused billions of dollars in agricultural losses. It is difficult to differentiate H. armigera from H. zea, a closely-related species native to North and South America. A few genetic tests have been previously developed to detect H. armigera DNA in pooled samples of moth legs. In this study, an improved qPCR melt curve assay with higher detection sensitivity and a field-based recombinase polymerase amplification (RPA) assay were developed for specific detection of H. armigera DNA in pooled moth samples. In addition, a crude DNA extraction protocol for whole moths was developed to allow a faster and more cost-effective way of preparing DNA samples for these assays. The qPCR assay was able to detect ≥ 100 fg of purified H. armigera DNA and the crude DNA of one H. armigera sample in a background of 99,999 H. zea equivalents. The RPA with the lateral flow strip assay was able to detect ≥ 10 pg of purified H. armigera DNA and the crude DNA of one H. armigera sample in a background of 999 H. zea equivalents. These newly developed molecular assays to detect H. armigera samples will contribute to a large-scale surveillance of H. armigera, which will in turn improve the biosecurity of the United States which is at risk of H. armigera becoming an invasive species