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All assays reported here were initially validated on a bench-top qPCR instrument, in both simplex and triplex formats, using either column-extracted DNA or gBlocks gene fragments. Assays run in both simplex and triplex formats yielded correct identification of all specimens/gBlocks tested and produced similar Ct values (Table 2; Supp File S3 [online only]). Although the Dendrolimus COI sub-assay typically produced a curve with a baseline drift, this phenomenon did not affect our ability to ascertain a positive amplification. In the case of the alternative triplex assay, where the DS sub-assay substitutes for the Eurasian Malacosoma sub-assay, we observed some interaction between the Dendrolimus COI and D. sibiricus ITS2 sub-assays, resulting in a reduction of the Fmax value for the latter sub-assay. This, however, had no impact on the conclusiveness of the assay.

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As a result of constraints on material availability, the assays described here were developed using moth legs as opposed to eggs, i.e., the life stage viewed as the primary target for molecular identification of intercepted lasiocampids in North America. While the accuracy of neither the bench-top nor the portable assay reported here was assessed using eggs as starting material, extensive testing of a similar assay targeting L. dispar, where eggs were employed for DNA extraction, showed that eggs provide good DNA yields, leading to efficient and accurate TaqMan species discrimination (Stewart et al. 2016, Stewart 2022). Based on these observations, the present assays are expected to work as well (if not better) with eggs as with moth legs.

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