title: 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.
authors.affiliation: Department of Biology, University of Texas-Pan American, 1201 W University Drive, Edinburg, TX 78539.
authors.forename: M S
authors.initials: MS
authors.lastname: Islam
authors.affiliation: Center for Plant Health Science and Technology, Mission Laboratory, USDA-APHIS, Moore Air Base, Edinburg, TX 78541.
authors.forename: N B
authors.initials: NB
authors.lastname: Barr
authors.affiliation: Center for Plant Health Science and Technology, Mission Laboratory, USDA-APHIS, Moore Air Base, Edinburg, TX 78541.
authors.forename: W E
authors.initials: WE
authors.lastname: Braswell
authors.affiliation: Department of Biology, University of Texas-Pan American, 1201 W University Drive, Edinburg, TX 78539.
authors.forename: M
authors.initials: M
authors.lastname: Martinez
authors.affiliation: Center for Plant Health Science and Technology, Mission Laboratory, USDA-APHIS, Moore Air Base, Edinburg, TX 78541.
authors.forename: L A
authors.initials: LA
authors.lastname: Ledezma
authors.affiliation: Center for Plant Health Science and Technology, Otis Laboratory, USDA-APHIS, 1398 W. Truck Rd., Joint Base Cape Cod, Buzzards Bay, MA 02542.
authors.forename: J
authors.initials: J
authors.lastname: Molongoski
authors.affiliation: Center for Plant Health Science and Technology, Otis Laboratory, USDA-APHIS, 1398 W. Truck Rd., Joint Base Cape Cod, Buzzards Bay, MA 02542.
authors.forename: V
authors.initials: V
authors.lastname: Mastro
authors.affiliation: Department of Biology, University of Texas-Pan American, 1201 W University Drive, Edinburg, TX 78539. Department of Biology, University of Texas Rio Grande Valley, 1201 W University Drive, Edinburg, TX 78539. erin.schuenzel@utrgv.edu.
authors.forename: E L
authors.initials: EL
authors.lastname: Schuenzel
mesh_terms: D000818:Animals; D001208:Asia; D005838:Genotype; D019476:Insect Proteins / Q000235:genetics; D009036:Moths / Q000145:classification* / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014481:United States
chemical_list: D019476:Insect Proteins; D012337:RNA, Ribosomal, 18S
keywords: Asian gypsy moth haplotype; Erebidae; gypsy moth; real-time PCR assay