Forensic environmental DNA (eDNA) validation for protected species: An integrated framework for wildlife crime investigation

Environmental DNA (eDNA) analysis enables non-invasive detection of aquatic species, but no established framework integrates eDNA best practices with forensic validation standards. The critically endangered freshwater pearl mussel (Margaritifera margaritifera) exemplifies the need for forensically validated eDNA assays as traditional survey methods are invasive and time-consuming. This research provides a framework that bridges both ecological monitoring standards and forensic legal admissibility requirements in wildlife crime investigations. Here we describe the validation of a multiplex quantitative PCR (qPCR) assay targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene, using the highest forensic standards and eDNA best practice. In silico and in vitro testing confirmed species specificity, with alignment against reference sequences showing 100% identity. The multiplex assay LOD and LOQ were calculated at 2.5 and 3.14 copies/µL respectively, determined by the performance of the short fragment. The assay demonstrated robust repeatability (R², 0.986-1.000), precision (coefficient of variation, 6-28%), and accuracy (recovery, 62-137%), meeting forensic thresholds. Field validation detected a previously undetected Welsh population through intelligence-led sampling, while blind proficiency testing in the Lake District achieved 95.8-100% detection rates across juvenile release sites and successfully detected distribution patterns later confirmed by stakeholders. We establish the first forensic eDNA validation of a protected species, providing a transferable methodology for conservation, regulatory enforcement, and wildlife crime investigations, ready for deployment in an operational environment. We believe that this integrated approach can be transferred to similar forensic and conservation scenarios across a broad range of species and geographical contexts.