LAMPSEQ: Colorimetric LAMP and nanopore sequencing for rapid species identification in the illegal wildlife trade

The illegal ivory trade in animal parts such as ivory, horn, and bone, threatens the survival of many protected species including elephants, rhinoceros, hippopotamus, and walrus. Accurate and reliable species identification is critical for enforcement under CITES, yet traditional DNA-based methods are time-consuming, resource-intensive, and are often inaccessible in regions most affected by poaching. This proof-of-concept study presents a rapid, portable, two-step workflow combining colorimetric loop-mediated isothermal amplification (LAMP) with nanopore sequencing for the authentication of elephant ivory and its legal and illegal substitutes. Novel LAMP assays were developed for elephants, mammoths, rhinoceros, hippopotamus, walrus, water buffalo, and common domestic species, targeting conserved mitochondrial DNA regions to enable both presumptive detection and sequencing-based confirmation. Using synthetic DNA, all assays demonstrated high sensitivity, with limits of detection down to 1-100 fg, with good consistency between replicates. A multiplexing strategy was designed to group closely related species into a single assay. Separate assays were designed for all elephant species, all mammoths, and the two closely related rhinoceros species (R. sondaicus and R. unicornis), enabling rapid presumptive screening followed by species-level confirmation via sequencing. To address challenges associated with the tandem repeats present in LAMP products, LAMPSEQ, a custom bioinformatics pipeline, was developed to extract, align, and accurately identify species from LAMP sequencing data. This sensitive, scalable, and field-deployable workflow expands the molecular toolkit, delivering innovative solutions to wildlife crime investigations and supports the protection of the world's most vulnerable and treasured species from extinction.