{Reference Type}: Journal Article
{Title}: Drop it all: extraction-free detection of targeted marine species through optimized direct droplet digital PCR.
{Author}: Scriver M;von Ammon U;Youngbull C;Pochon X;Stanton JL;Gemmell NJ;Zaiko A;
{Journal}: PeerJ
{Volume}: 12
{Issue}: 0
{Year}: 2024
{Factor}: 3.061
{DOI}: 10.7717/peerj.16969
{Abstract}: Molecular biomonitoring programs increasingly use environmental DNA (eDNA) for detecting targeted species such as marine non-indigenous species (NIS) or endangered species. However, the current molecular detection workflow is cumbersome and time-demanding, and thereby can hinder management efforts and restrict the "opportunity window" for rapid management responses. Here, we describe a direct droplet digital PCR (direct-ddPCR) approach to detect species-specific free-floating extra-cellular eDNA (free-eDNA) signals, i.e., detection of species-specific eDNA without the need for filtration or DNA extraction, with seawater samples. This first proof-of-concept aquarium study was conducted with three distinct marine species: the Mediterranean fanworm Sabella spallanzanii, the ascidian clubbed tunicate Styela clava, and the brown bryozoan Bugula neritina to evaluate the detectability of free-eDNA in seawater. The detectability of targeted free-eDNA was assessed by directly analysing aquarium marine water samples using an optimized species-specific ddPCR assay. The results demonstrated the consistent detection of S. spallanzanii and B. neritina free-eDNA when these organisms were present in high abundance. Once organisms were removed, the free-eDNA signal exponentially declined, noting that free-eDNA persisted between 24-72 h. Results indicate that organism biomass, specimen characteristics (e.g., stress and viability), and species-specific biological differences may influence free-eDNA detectability. This study represents the first step in assessing the feasibility of direct-ddPCR technology for the detection of marine species. Our results provide information that could aid in the development of new technology, such as a field development of ddPCR systems, which could allow for automated continuous monitoring of targeted marine species, enabling point-of-need detection and rapid management responses.