A Multiplex Real-Time PCR Assay for Screening Gypsy Moths (Lepidoptera: Erebidae) in the United States for Evidence of an Asian Genotype

Abstract

European gypsy moth populations (Lymantria dispar L.) are well established and a proven destructive force in hardwood trees throughout the United States and Canada. Introduction of the exotic Asian gypsy moth into North America would be even more impactful, as Asian gypsy moth populations have wider host ranges, and are capable of naturally dispersing more rapidly due to female flight ability. To support early detection and exclusion of Asian gypsy moth, the U.S. Department of Agriculture (USDA) uses molecular techniques to screen moths trapped in North America for evidence of common Asian genotype. In order to strengthen U.S. domestic capacity to screen moths quickly and efficiently, we report a real-time PCR assay for this pest. A probe system using TaqMan 5' nuclease chemistry is reported for detection of an allele associated with common Asian gypsy moth genotypes. The targeted allele is located at the nuclear FS1 locus currently used by the USDA in conventional PCR tests to screen for evidence of Asian gypsy moth introductions or introgression. The diagnostic probe is successfully multiplexed with a conserved 18S probe system to detect reaction failure due to poor sample quality or quantity. The specificity, sensitivity, and repeatability of the FS1-18S multiplex real-time PCR assay were tested on laboratory-reared and field-collected moths to demonstrate diagnostic utility. Implications of the new assay as a screening tool for evidence of Asian gypsy moth introgression and introduction are discussed.

A Multi-Species TaqMan PCR Assay for the Identification of Asian Gypsy Moths (Lymantria spp.) and Other Invasive Lymantriines of Biosecurity Concern to North America

“…Other approaches that rely on the amplification and sequencing of mitochondrial markers [e.g., 6 ] or on the analysis of several microsatellite markers (e.g., [ 13 ]) have the potential of achieving levels of accuracy, resolution and scope similar to those of the present set of assays, but will require longer processing time. Conversely, the two AGM TaqMan assays developed earlier by other groups [ 14 , 24 ], although rapid, are limited in scope and subspecies resolution. With respect to cost, the suite of assays proposed here is relatively inexpensive to run (provided the necessary thermocycling equipment is available) and comparable in price to the sequencing of PCR products.…”

European gypsy moth (Lymantria dispar dispar L.) completes development and defoliates exotic radiata pine plantations in Spain

“…Their DNA was analysed using a Multiplex RealTime PCR Assay (Islam et al 2015) at the USDA-APHIS Center for Plant Health Science and Technology, Otis Laboratory, Buzzards Bay, MA, USA.…”

Barcoding the food chain: from Sanger to high-throughput sequencing

“…This will be critical as inspections and subsequent quarantine are reliant on the rapid identification of pests. One solution is the development of real-time PCR assays (see Text Box 1; Naaum et al 2012;Islam et al 2015), which are faster and often portable, making these suitable for scientists in the field or inspection officials surveying for a particular pest of interest. Here, species-specific primers must be developed, or we risk the cross-amplification of closely related species leading to potential false positives.…”