Drop it all: Extraction-free detection of non-indigenous marine species through optimized direct-droplet digital PCR

Molecular biosecurity surveillance programs increasingly use environmental
DNA (eDNA) for detecting marine non-indigenous species (NIS). However, the
current molecular detection workflow is cumbersome, prone to errors and
delays, and thereby can hinder management efforts and restrict the
“opportunity window” for a rapid response to new marine NIS incursions.
Here, we describe the first method to use direct digital droplet PCR
(direct-ddPCR) 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 sample. This
first proof-of-concept aquarium study was conducted with three distinct
marine NIS: 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 Sabella spallanzanii and Bugula 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 hours. 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 for marine biosurveillance, enabling point-of-need
detection and rapid management response to biosecurity threats.