Drop it all: extraction-free detection of targeted marine species through optimized direct droplet digital PCR.

Autor:	Scriver M; Biosecurity Group, Cawthron Institute, Nelson, New Zealand.; Institute of Marine Science, University of Auckland, Auckland, New Zealand., von Ammon U; Biosecurity Group, Cawthron Institute, Nelson, New Zealand., Youngbull C; Nucleic Sensing Systems, LCC, Saint Paul, Minnesota, United States., Pochon X; Biosecurity Group, Cawthron Institute, Nelson, New Zealand.; Institute of Marine Science, University of Auckland, Auckland, New Zealand., Stanton JL; Department of Anatomy, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand., Gemmell NJ; Department of Anatomy, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand., Zaiko A; Biosecurity Group, Cawthron Institute, Nelson, New Zealand.; Sequench Ltd, Nelson, New Zealand.
Jazyk:	angličtina
Zdroj:	PeerJ [PeerJ] 2024 Feb 23; Vol. 12, pp. e16969. Date of Electronic Publication: 2024 Feb 23 (Print Publication: 2024).
DOI:	10.7717/peerj.16969
Abstrakt:	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. Competing Interests: Xavier Pochon and Anastasija Zaiko are Academic Editors for PeerJ. Anastasija Zaiko is employed by Sequench Limited and Cody Youngbull is employed by Nucleic Sensing Systems, LLC. (© 2024 Scriver et al.)
Databáze:	MEDLINE
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