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

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 (ddPCR) 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.

A needle in a haystack: a multigene TaqMan assay for the detection of Asian gypsy moths in bulk pheromone trap samples

“…Although ddPCR clearly provided excellent performance for these two assays, the level of sensitivity we achieved in the present TaqMan qPCR assay is at least as good as that reported by this group (1:1000, Zink et al 2017;1:200, Zink et al 2018). The qPCR TaqMan assay also has the advantage of requiring a type of thermocycler that is less expensive and more ubiquitous than the ddPCR equipment employed by Zink et al (2017Zink et al ( , 2018. In addition, the work involved in data analysis and interpretation for the present assay is simpler and more easily automated than it is for the ddPCR technology.…”

“…For these two assays, the authors opted for the use of a different, more recent PCR technology known as droplet digital PCR (ddPCR), largely because of its high reported sensitivity (Milbury et al 2014). Although ddPCR clearly provided excellent performance for these two assays, the level of sensitivity we achieved in the present TaqMan qPCR assay is at least as good as that reported by this group (1:1000, Zink et al 2017;1:200, Zink et al 2018). The qPCR TaqMan assay also has the advantage of requiring a type of thermocycler that is less expensive and more ubiquitous than the ddPCR equipment employed by Zink et al (2017Zink et al ( , 2018.…”

“…For example, networks of pheromone traps deployed for the detection of AGM can easily produce situations where an AGM individual, if caught, is greatly outnumbered by local EGMs. Unlike other molecular assays designed by others to detect a single species in a large background of another species (e.g., Zink et al 2017Zink et al , 2018, the assay reported here had to overcome the additional hurdle of distinguishing taxa within the AGM complex. To this end, we developed independent sets of qPCR primers and TaqMan probes for AGM species/subspecies, using SNPs identified b Fig.…”

“…Examples of other assays designed for the detection of invasive species in bulk pheromone trap samples include the one developed for the detection of the invasive noctuid Helicoverpa armigera among large numbers of the native congener H. zea (Zink et al 2017), and another one that targets the silver Y moth, Autographa gamma, in a background of several closely related species (Zink et al 2018). For these two assays, the authors opted for the use of a different, more recent PCR technology known as droplet digital PCR (ddPCR), largely because of its high reported sensitivity (Milbury et al 2014).…”

Optimizing Droplet Digital PCR for Environmental Samples

“…There is no uniform cause, nor approach for dealing with 'rain' as it may have different causes [14]. For example, droplet coagulation [13] or variation in droplet size could affect fluorescence [15].Variation in target amplification efficiency may also arise through physical problems of template DNA [16] including fragmentation of DNA incurred during extraction or storage [17].…”