Development of novel LAMP and qPCR assays for rapid and specific identification of Bronze birch borer (Agrilus anxius)

Abstract

Buprestids are an emerging threat to broadleaf forests across the world. Bronze birch borer (Agrilus anxius, BBB) poses a serious threat to European birch species if the insect were to be introduced. Due to their cryptic lifestyle feeding on the vascular tissue of their host plants, buprestids and other wood borers can be difficult to observe or detect. Early detection tools are vital to swiftly implement eradication measures and prevent the establishment of introduced species. In this study, we developed novel qPCR and LAMP assays for BBB and investigated the specificity and sensitivity for their use as early detection tools in European forests. Plant chemicals may limit these assays, so we conducted sensitivity testing with extracted foliage and plant vascular tissues to determine potential inhibition effects on DNA amplification. Both assays were specific to the target species when tested against the DNA of 17 other European Agrilus/buprestid species, two Scolytinae, and five Cerambycids (N = 24). Both assays varied in sensitivity with the qPCR assay amplifying at a concentration as low as 20 fg/μL, whereas the LAMP assay amplified as low as 3.2 pg/μL. Plant chemicals in DNA extracts from leaves did not impact the sensitivity of either assay, reaching similar detection levels. In contrast, vascular tissue reduced the sensitivity of the LAMP assay, amplifying as low as 0.04 ng/μL compared with 0.008 ng/μL in the control. These results demonstrate that both assays are highly specific and sensitive tools that can be used to detect frass and identify larvae as well as monitor the spread of A. anxius. qPCR resulted in more sensitive than LAMP overall. Thus, if results are needed quickly to make fast management decisions or as an initial screening of samples, the LAMP method is optimal. However, if fine detection is critical, then qPCR is preferential.