Establishment of a Microfluidic Chip Based Rapid Detection Method for <i>Wasmannia auropunctata</i>

Abstract

Invasive alien ants are one of the most aggressive, competitive, and widespread invasive alien species around the world. <i>Wasmannia auropunctata, </i>native to the neotropical zoogeographic area, has been inadvertently introduced all over the world. The first disclosed record of field establishment of <i>W. auropunctata</i> in Chinese mainland was reported in 2022. In this paper, we aim to establish a rapid detection method targeting the <i>W. auropunctata </i>polymorphic microsatellite locus Waur-275, based on the microfluidic chip technology, to contribute to an active response to the crisis caused by this invasive pest. Eight primer sets were generated using the PrimerExplorer v5 online service, and the fifth primer set selected had the lowest Ct in the volume ratio of 8 (FIP/BIP:F3/B3). Method validity with six <i>W. auropunctata </i>samples confirmed the efficiency of this assay. High specificity was demonstrated by the positive result for <i>W. auropunctata</i> out of 6 related insect samples tested. Sensitivity analysis showed that this microfluidic chip method could achieve the detection limit of 1.00×10<sup>1</sup> copies/μL. A rapid screening method is essential for the whole process of interception and control of <i>W. auropunctata </i>in China. It can also help determine the origin of invasion, clarify the path of introduction to ensure biosecurity, and also uncover other possible undetected establishments in the wild.