A droplet digital PCR (ddPCR) assay to detect Helicoverpa armigera (Lepidoptera: Noctuidae) in bulk trap samples

Authors: ZINK, FRIDA - Colorado State University; TEMBROCK, LUKE - Colorado State University; TIMM, ALICIA - Colorado State University; FARRIS, ROXANNE - Animal And Plant Health Inspection Service (APHIS); Perera, Omaththage; GILLIGAN, TODD - Animal And Plant Health Inspection Service (APHIS)

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/18/2017
Publication Date: 5/31/2017
Publication URL: https://handle.nal.usda.gov/10113/5700682
Citation: Zink, F.A., Tembrock, L.R., Timm, A.E., Farris, R.E., Perera, O.P., Gilligan, T.M. 2017. A droplet digital PCR (ddPCR) assay to detect Helicoverpa armigera (Lepidoptera: Noctuidae) in bulk trap samples. PLoS One. doi:10.1371/journal.pone.0178704.

Interpretive Summary: Invasive old world bollworm, Helicoverpa armigera, and the native bollworm Helicoverpa zea are morphologically indistinguishable. Characteristics of male genitalia have been used to distinguish these two species, but the need to dissect every specimen for positive identification is especially problematic when conducting surveys for H. armigera adults and variation in the morphology of male genitalia may lead to misidentifications or inconclusive results. In this study, a digital droplet polymerase chain reaction (ddPCR) technique was used to extend the detection capability of a previously published method (Perera et. al. 2015, J. Insect Sci., DOI: 10.1093/jisesa/iev137) from one H. armigera in a pool of 25 insects to one H. armigera in a pool of 1,000 insects. This method will provide means for rapid detection of invasive H. armigera in North America.

Technical Abstract: Moths in the genus Helicoverpa are some of the most important agricultural pests in the world. Two species, H. armigera (Hübner) and H. zea (Boddie), cause the majority of damage to crops and millions of dollars are spent annually on control of these pests. The recent introduction of H. armigera into the New World has prompted extensive survey efforts for this species in the United States. Surveys are conducted using bucket traps baited with H. armigera pheromone, and, because the same pheromone compounds attract both species, these traps often capture large numbers of the native H. zea. Adult H. armigera and H. zea are very similar and can only be separated morphologically by minor differences in the genitalia. Thus, a time consuming genitalic dissection by a trained specialist is necessary to reliably identify either species, and every specimen must be dissected. Several molecular methods are available for differentiating and identifying H. armigera and H. zea, including two recently developed rapid protocols using real-time PCR. However, none of the published methods are capable of screening specimens in large batches. Here we detail a droplet digital PCR assay that is capable of detecting a single H. armigera in a background of up to 999 H. zea. The assay has been tested using bulk extractions of 1,000 legs from actual trap samples and is effective even when using poor quality samples. This study provides an efficient, rapid, reproducible, and scalable method for processing H. armigera survey trap samples in the U.S. and demonstrates the potential for applying ddPCR technology to screen and diagnose invasive species.