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

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.

Field-based recombinase polymerase amplification and lab-based qPCR assays for detection of Helicoverpa armigera

“…The RPA assay had lower detection sensitivity than the qPCR assay developed in this study, but it was able to detect a smaller quantity of purified H. armigera DNA (10 pg vs. 50 pg) than the LAMP assay (Amano and Nomura 2020). When the crude DNA from whole moths was used, the RPA assay could detect one H. armigera in 999 H. zea , the same ratio as reported by ddPCR assay using the crude DNA of moth legs (Zink et al 2017) and lower than the ratio in the qPCR assays (Perera et al 2015, Oliveira et al 2021). The LAMP assay developed by Amano and Nomura (2020) was not tested with crude DNA from a mixture of H. armigera and H. zea , thus no direct comparison can be made for the crude DNA samples.…”

“…However, with the new crude DNA extraction protocol developed for whole moths, the qPCR assay in this study was able to detect one H. armigera from within a pool of 999 H. zea , as well as from crude DNA of H. armigera spiked with up to 99999 H. zea extract volume equivalents. This represents a lower H. armigera : H. zea ratio than those reported in the qPCR assays using crude DNA from moth legs (down to 1:24 and 1:500) (Perera et al 2015, Oliveira et al 2021). The high detection sensitivity with the crude DNA in this study was likely attributed to the higher amount of DNA extracted from the whole moths.…”

“…The high resolution melt qPCR assay developed by Perera et al (2015) can detect the DNA of one H. armigera mixed with up to 24 H. zea legs. The probe-based real-time PCR assay developed by Oliveira et al (2021) can detect the DNA of one H. armigera mixed with up to 500 H. zea legs. The droplet digital PCR (ddPCR) method developed by Zink et al (2017) had the highest detection sensitivity and was capable of detecting the DNA of a single H. armigera leg in the background of 999 H. zea legs; however, its associated equipment ($100,000 vs. $30,000) and reaction costs ($2 vs. $0.5 per reaction) are much more expensive than qPCR.…”

Development of LAMP and droplet digital PCR methods for differentiation between Cydia pomonella (L.) and Grapholita molesta (Busck)

“…Thus, the eDNA from the frass or other debris may provide a clue about the target species 35,36,37,38 . For the detection of eDNA, the ddPCR has higher sensitivity over the real-time quantitative PCR, LAMP, and conventional PCR 29,30,39,40 . In this study, the ddPCR method can detect the DNA of C. pomonella at a minimum concentration of 1.43×10 − 2 ng/uL, which provides a potential tool to detect the eDNA of C. pomonella in the eld.…”