Molecular Identification of <scp><i>Agrilus anxius</i></scp> (Coleoptera: Buprestidae) Using a <scp>qPCR</scp> Assay With Locked Nucleic Acid (<scp>LNA</scp>) Probe

ABSTRACT In the present study, a biomolecular diagnostic assay based on qPCR‐LNA (Locked Nucleic Acid) probes was developed for the identification of Agrilus anxius (Coleoptera: Buprestidae) from adult insects. The performance of the new protocol was also evaluated for indirect diagnosis of the insect's presence on artificially contaminated frass. The designed primer and probe were able to distinguish in silico all A. anxius samples from nontarget species, with a 100% match with homologous sequences found in GenBank databases. The molecular assay was sensitive, specific and repeatable. The analytical sensitivity (limit of detection—LoD) for A. anxius adults and artificially contaminated frass was 6.4 fg/μL and 0.08 pg/μL, respectively. This assay, by analysing eDNA samples, will allow the insect's early detection in an area before it has caused major impact. eDNA analysis is becoming an increasingly used tool in the spatial survey programs of phytosanitary services and could play a decisive role in pest surveillance.