Assay Optimization Can Equalize the Sensitivity of Real-Time PCR with ddPCR for Detection of Helicoverpa armigera (Lepidoptera: Noctuidae) in Bulk Samples

SIMPLE SUMMARY: Invasive species are a constant threat to agriculture throughout the world against which early detection is one of the primary defenses. The Old World bollworm is one of the most important invasive agricultural pests in the world. While historically absent from the Americas, this species was first found in South America in 2013 and poses an ongoing threat of spreading into North America. Surveys are conducted each year, which result in hundreds or thousands of traps that must be screened for this species. Unfortunately, the most common non-target is the native corn earworm, which is nearly identical morphologically to the Old World bollworm and cannot be easily separated. Molecular methods have been developed to screen these trap samples, but the required equipment is expensive and not commonly available. This study details improvements to current molecular methods that will allow for screening of bulk trap samples using standard laboratory instruments and protocols. The ability to perform these methods in nearly any molecular biology lab will greatly enhance our ability to detect and exclude this important pest. ABSTRACT: Helicoverpa armigera (Hübner) is one of the most important agricultural pests in the world. This historically Old World species was first reported in Brazil in 2013 and has since spread throughout much of South America and into the Caribbean. Throughout North America, H. armigera surveys are ongoing to detect any incursions. Each trap is capable of capturing hundreds of native Helicoverpa zea (Boddie). The two species cannot be separated without genitalic dissection or molecular methods. A ddPCR assay is currently used to screen large trap samples, but this equipment is relatively uncommon and expensive. Here, we optimized a newly designed assay for accurate and repeatable detection of H. armigera in bulk samples across both ddPCR and less costly, and more common, real-time PCR methods. Improvements over previously designed assays were sought through multiple means. Our results suggest bulk real-time PCR assays can be improved through changes in DNA extraction and purification, so that real-time PCR can be substituted for ddPCR in screening projects. While ddPCR remains a more sensitive method for detection of H. armigera in bulk samples, the improvements in assay design, DNA extraction, and purification presented here also enhance assay performance over previous protocols.