An Improved Bulk DNA Extraction Method for Detection of Helicoverpa armigera (Lepidoptera: Noctuidae) Using Real-Time PCR.

Simple Summary: Old World bollworm is a moth species that causes significant damage to a wide range of agricultural crops. This species was once confined to the eastern hemisphere but has recently spread throughout South America and the Caribbean and has the potential to establish in North America. Molecular detection methods for Old World bollworm have been developed to track its spread and rapidly differentiate it from the native sibling species, corn earworm. Currently, droplet digital PCR (ddPCR) is a preferred method for bulk screening as it is highly accurate and tolerant of PCR inhibitors; however, real-time PCR is less expensive and more widely available in molecular labs. Improvements to DNA extraction yield and purity are crucial for real-time PCR assay optimization, but these improvements must be time- and cost-efficient. In this study, we improve upon previously published bulk DNA extraction methods by reducing bench time and costly materials. Our results indicate that the addition of caffeine and RNase A improves DNA extraction, resulting in higher amounts of target DNA in real-time PCR. Such improvements will enable the use of high throughput screening methods across multiple platforms to improve the probability of detection of Old World bollworm. Helicoverpa armigera is among the most problematic agricultural pests worldwide due to its polyphagy and ability to evolve pesticide resistance. Molecular detection methods for H. armigera have been developed to track its spread, as such methods allow for rapid and accurate differentiation from the native sibling species H. zea. Droplet digital PCR (ddPCR) is a preferred method for bulk screening due to its accuracy and tolerance to PCR inhibitors; however, real-time PCR is less expensive and more widely available in molecular labs. Improvements to DNA extraction yield, purity, and throughput are crucial for real-time PCR assay optimization. Bulk DNA extractions have recently been improved to where real-time PCR sensitivity can equal that of ddPCR, but these new methods require significant time and specialized equipment. In this study, we improve upon previously published bulk DNA extraction methods by reducing bench time and materials. Our results indicate that the addition of caffeine and RNase A improves DNA extraction, resulting in lower Cq values during real-time PCR while reducing the processing time and cost per specimen. Such improvements will enable the use of high throughput screening methods across multiple platforms to improve the probability of detection of H. armigera. [ABSTRACT FROM AUTHOR]