Optimization of a bulk DNA extraction method for the detection of Helicoverpa armigera (Lepidoptera: Noctuidae) using real-time PCR

Due to the threat of Helicoverpa armigera (Hübner) invading and establishing populations in the United States, molecular methods for screening bulk samples are needed. In order to screen large numbers of individuals in a short period of time, these methods should be deployable across many different labs. Currently, ddPCR is used for bulk screening as it is highly accurate and tolerant of PCR inhibitors, however it is costly and not widely available. Real-time PCR machines present a lower barrier to entry but are currently only used for screening individual specimens. In a recent study, an optimized real-time PCR assay was developed that that matched the efficiency of ddPCR for detecting H. armigera in bulk samples. Further optimization of this assay is needed to simplify the workflow. The utility of real-time PCR in accurate detection of a target species in complex DNA samples depends on developing a rapid but efficient DNA extraction method. This study aims to improve upon previously published bulk DNA extraction methods by reducing bench time, decreasing PCR inhibitors, and maximizing extraction efficiency. Results indicate that the addition of both caffeine and the enzyme RNase A can improve DNA extraction, resulting in a lower Cq value during real-time PCR analysis, while maintaining minimal bench time. A more efficient DNA extraction method will help enable widespread use of real-time PCR for the detection of H. armigera in bulk samples across many different labs.