International timber trade has accelerated the global spread of the invasive red-haired pine bark beetle H. ligniperda, posing persistent challenges to phytosanitary inspection and border biosecurity. Rapid isothermal amplification assays are increasingly deployed in frontline quarantine settings to support timely regulatory decisions. However, their performance under the heterogeneous biological backgrounds typical of traded timber remains insufficiently evaluated, particularly with respect to the practical implications of low-level false-positive signals. We re-evaluated a previously reported isothermal assay for H. ligniperda using conditions that simulate timber transport and routine customs workflows. Fifty non-target arthropod species (predominantly insects), selected from quarantine interception records, were included to represent taxa likely to co-occur in operational contexts. Material from Lema decempunctata consistently generated weak but reproducible amplification signals across replicates. Sanger sequencing excluded contamination, confirming low-level non-target amplification in complex biological matrices. Although the signals were faint, ambiguous results in quarantine settings may trigger shipment detention, confirmatory laboratory testing, or temporary trade restrictions, thereby increasing inspection workload, delaying clearance, and generating avoidable compliance costs. These findings indicate that trade-mediated species assemblages can compromise assay performance beyond expectations derived from conventional taxonomy-based specificity testing. To reduce interpretive uncertainty and associated regulatory burden, we propose a tiered diagnostic workflow combining rapid on-site isothermal screening with specificity-oriented SYBR Green qPCR confirmation. This strategy enhances diagnostic reliability while preserving operational efficiency in applied biosecurity surveillance.
Han et al. (Thu,) studied this question.