Rapid and Cost-Effective Platypus eDNA Detection in Waterways Using Loop-Mediated Isothermal Amplification Assay: Advancing Conservation Efforts

Abstract

Freshwater ecosystems, home to a remarkable diversity of species, are facing severe threats from human activities such as climate change, habitat degradation, over-extraction of water for irrigation, and pollution. The platypus, an iconic species in freshwater ecosystems around Australia, is threatened by all these activities, both singly and in combination. The scale and complexity of these intersecting and reinforcing threats makes cost-effective monitoring tools essential to better understand how platypus populations are responding. In this study, we optimized a loop-mediated isothermal amplification (LAMP) assay for the rapid and cost-effective detection of platypus DNA in environmental water samples, offering an attractive alternative to quantitative polymerase chain reaction (qPCR). We improved a water filtration protocol for in-field use, employing suitable filter membranes for processing large volumes of water, thereby maximizing DNA recovery from dilute samples. The limit of detection for the platypus LAMP (Plat-LAMP) assay was determined to be 12.4 copies/μL using a standard plasmid positive reference and 7 × 10⁻⁶ ng/μL when applied to DNA extracted from platypus tissue, with improved sensitivity achieved through the incorporation of locked nucleic acid primers. In comparative testing against qPCR, the Plat-LAMP assay exhibited greater sensitivity in detecting platypus DNA in known positive water samples collected from platypus habitat at Healesville Sanctuary. Furthermore, the Plat-LAMP assay demonstrated 100% specificity when performed on water samples collected from non-platypus habitats. In field testing across waterways in Victoria and New South Wales, the Plat-LAMP assay detected platypus in 36.96% of samples, compared to 54.35% of samples using qPCR. These findings underscore the Plat-LAMP assay's potential as a faster and more cost-effective complementary method to qPCR, rendering it suitable for point-of-application water testing. The ability to conduct eDNA surveys without the need for cold-chain logistics would significantly assist conservation organizations and water managers map platypus distributions and facilitate conservation efforts around Australia.