Wanted Dead or Alive: Enhancing Spatiotemporal Resolution of Environmental Nucleic Acid Techniques in Macro-Organism Biosecurity

Highly sensitive and non-invasive detection of macroorganisms using environmental nucleic acids (eNA) has transformed biosecurity surveillance. However, the persistence of legacy DNA compromises the spatiotemporal accuracy of environmental DNA (eDNA)-based detection, leading to false indications of contemporary species presence. This review critically evaluates emerging molecular approaches aimed at improving the temporal resolution of eNA signals and distinguishing living organisms from historical residues. We examine environmental RNA (eRNA), long-fragment eDNA (LFeDNA), propidium monoazide (PMA) treatment, and organelle-to-nuclear DNA ratios as indicators of eDNA age, assessing their principles, technical challenges, and practical potential. While eRNA offers the strongest theoretical link to organism viability, its application is constrained by rapid decay and stringent handling requirements. LFeDNA presents a more practical alternative but requires careful assay design. PMA treatment has shown limited effectiveness in excluding legacy DNA, whereas organelle-to-nuclear DNA ratios remain promising but underexplored. We identify key research priorities needed to transition these approaches from experimental studies to operational biosecurity tools. Addressing these gaps will improve interpretation of eNA signals, enabling more accurate detection of living invasive organisms and enhancing the reliability of biosecurity surveillance.