A triplex real-time polymerase chain reaction assay to diagnose Asian variant of gypsy moth (Lymantria dispar) (Lepidoptera: Lymantriidae)

Introduction of the exotic Asian gypsy moth (AGM) (Lymantria dispar ssp. L.) into North America would have a serious impact on the hardwood and softwood trees in forests and orchards. Early detection programs in support of pest exclusion require diagnostic tools that can reliably distinguish AGM from European gypsy moth (EGM) (Lymantria dispar dispar. L.) populations present in the United States. The USDA currently uses two molecular methods to identify trapped gypsy moths to a subspecies: 1) a conventional PCR of a Sequence Characterized Amplified Region (SCAR) called FS1 and 2) PCR- Restriction Fragment Length Polymorphism (RFLP) of a fragment of the mitochondrial cytochrome oxidase I (COI) gene. In order to enhance AGM detection and identification methods, we have developed these genetic loci into a real-time PCR assay. The assay multiplexes three TaqMan® real-time PCR probe systems (triplex) to target DNA segments of the nuclear FS1 locus, mitochondrial COI gene, and nuclear 18S rRNA gene. The 18S probe recognizes a DNA sequence conserved in all variants of gypsy moths and serves as a control. The other probes recognize segments of the FS1 and COI loci that are present in the AGM but not found in the EGM.