Development of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Molecular Identification of the Bark...

Ips hauseri Reitter is the most important wood-boring pest of natural Tianshan spruce (Picea schrenkiana) forests and is listed as an A2 quarantine pest by the European and Mediterranean Plant Protection Organization (EPPO). Because the beetle is small and cryptic and its immature stages cannot be reliably identified by morphology, a rapid molecular identification method is urgently needed. Here, we targeted the mitochondrial cytochrome c oxidase subunit I (COI) gene, designed a set of I. hauseri-specific LAMP primers, and established a visually interpretable LAMP assay using a WarmStart colorimetric system. The optimal reaction conditions were 63.8 °C for 35 min. The assay amplified only I. hauseri, with no cross-reaction with the related species Pityogenes spessivtsevi, Ips typographus, Ips subelongatus, and Hylurgus ligniperda; its limit of detection was 100 pg of genomic DNA; and it amplified efficiently from adults, larvae, pupae, and a single egg. Positive results could be scored directly by eye from the red-to-yellow colour change in the reaction, requiring no precision instruments at any step. Using a non-destructive alkaline crude-DNA extraction (HotSHOT), the assay also amplified I. hauseri directly from single adults and larvae, confirming its compatibility with a rapid, field-simple sample preparation. The LAMP assay was rapid, specific within the tested panel, and simple. However, broader validation with additional sympatric non-target species and geographically distinct populations of I. hauseri is needed before routine field or quarantine application.