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

Lewis, M ORCID: 0009-0006-2116-9253, Moir, C, Scott, K ORCID: 0000-0001-8660-3301, Mariani, S ORCID: 0000-0002-5329-0553, Hänfling, B and Dawnay, N (2026) Forensic environmental DNA (eDNA) validation for protected species: An integrated framework for wildlife crime investigation. Forensic Science International Genetics, 87. p. 103598. ISSN 1872-4973

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Abstract

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.

Item Type:	Article
Uncontrolled Keywords:	Environmental DNA; Forensic validation; Freshwater pearl mussel; Multiplex PCR; Wildlife crime; 31 Biological Sciences; 3105 Genetics; 15 Life on Land; 01 Mathematical Sciences; 06 Biological Sciences; 18 Law and Legal Studies; Legal & Forensic Medicine; 31 Biological sciences; 48 Law and legal studies; 49 Mathematical sciences
Subjects:	R Medicine > RA Public aspects of medicine > RA1001 Forensic Medicine. Medical jurisprudence. Legal medicine R Medicine > RS Pharmacy and materia medica
Divisions:	Biological and Environmental Sciences (from Sep 19) Pharmacy and Biomolecular Sciences
Publisher:	Elsevier
Date of acceptance:	28 July 2026
Date of first compliant Open Access:	18 August 2026
Date Deposited:	18 Aug 2026 14:23
Last Modified:	18 Aug 2026 14:23
DOI or ID number:	10.1016/j.fsigen.2026.103598
URI:	https://researchonline.ljmu.ac.uk/id/eprint/29158

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