A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World

The Old World bollworm, Helicoverpa armigera (Hübner), and the corn earworm, H. zea (Boddie), are two of the most important agricultural pests in the world. Diagnosing these two species is difficult—adults can only be separated with a complex dissection, and larvae cannot be identified to species using morphology, necessitating the use of geographic origin for identification in most instances. With the discovery of H. armigera in the New World, identification of immature Helicoverpa based on origin is no longer possible because H. zea also occurs in all of the geographic regions where H. armigera has been discovered. DNA barcoding and restriction fragment length polymorphism (RFLP) analyses have been reported in publications to distinguish these species, but these methods both require post-PCR processing (i.e., DNA sequencing or restriction digestion) to complete. We report the first real-time PCR assay to distinguish these pests based on two hydrolysis probes that bind to a segment of the internal transcribed spacer region 2 (ITS2) amplified using a single primer pair. One probe targets H. armigera, the second probe targets H. zea, and a third probe that targets a conserved segment of 18S rDNA is used as a control of DNA quality. The assay can be completed in 50 minutes when using isolated DNA and is successfully tested on larvae intercepted at ports of entry and adults captured during domestic surveys. We demonstrate that the assay can be run in triplex with no negative effects on sensitivity, can be run using alternative real-time PCR reagents and instruments, and does not cross react with other New World Heliothinae.