Validation of a bulk real-time PCR assay for detection of Helicoverpa armigera (Lepidoptera: Noctuidae)

Helicoverpa armigera (Hübner) is an agricultural pest of great economic and environmental importance. Although this species is native to the Old World, it is now established in South America and the Caribbean. Due to its ability to migrate long distances, it is likely to disperse to the United States in coming years. As such, methods for high throughput detection of H. armigera are needed to aide in preventing establishment of this species. An optimized assay was recently developed for detection of H. armigera in bulk samples across both ddPCR and real-time PCR methods. While this study showed that real-time PCR could be used to process bulk samples, further validation and optimization of this assay is needed to ensure that consistent results are produced across a range of sample types. Previous results indicate that increased salt concentrations in extraction buffers produced lower C_q values, and that paramagnetic bead purification improved sensitivity for real-time PCR calls. In this study, we validate the previously described real-time PCR assay for detection of H. armigera in bulk samples through optimization of DNA extraction and purification by replicating previous work across a larger set of samples as well as testing modifications to the extraction buffer.