A duplex real-time PCR with TaqMan probes for the distinction of Anoplophora chinensis (Forster) and Anoplophora glabripennis (Motschulsky) on different biological matrices

Anoplophora chinensis (Forster, 1771), the citrus longhorned beetle, and Anoplophora glabripennis (Motschulsky, 1853), the Asian longhorned beetle, are wood borers of great economic importance, infesting a wide range of ornamental trees and shrubs. Native to Asia, their presence has been detected in several countries on different continents. Both are included in the EPPO A2 list of pests recommended for regulation as quarantine pests. A duplex molecular test based on TaqMan probe technology for the identification of the two species has been set up, using specimens (adults and larvae) of the two species. To test the duplex real-time PCR on frass, an artificial frass (‘manufactured sawdust’) has been created using wood chips from healthy branches of Acer negundo, spiked with a known amount of DNA from larvae of the species. The analytical specificity, analytical sensitivity, repeatability and reproducibility of the duplex real-time PCR test were evaluated according to EPPO Standards. This new molecular tool allows distinction, with only one test, between these two Anoplophora species using DNA isolated from the adult, larvae and/or frass.