The stakes are considerable. Globally, dugong numbers have fallen by roughly 20 percent over the past ninety years, and some regional populations face outright extirpation. The species is listed as Vulnerable on the IUCN Red List, with certain subpopulations, such as those around the Nansei Islands of Japan and along the East African coast, classified as Critically Endangered, and the New Caledonia subpopulation listed as Endangered. Habitat loss, marine pollution, and bycatch mortality all contribute to the decline, and the animal’s naturally low fecundity makes recovery slow even when pressures ease.

Traditional monitoring methods struggle with an animal like the dugong. In the Red Sea, which hosts the third largest dugong population in the world, most knowledge has come from aerial and surface observations, photo identification, analysis of grazing trails left in seagrass meadows, and interviews with fishermen. Because dugongs are timid and spend limited time near the surface, visual surveys are inefficient and prone to non-detection error, meaning animals can be present but simply missed. Environmental DNA techniques promise to sidestep these problems by detecting the genetic material organisms leave behind in water, mucus, feces, and sloughed cells.

The research team, drawn from King Abdullah University of Science and Technology in Saudi Arabia, Curtin University in Australia, eDNA Frontiers, Red Sea Global, the University of Milano-Bicocca, and the MaRHE Center in the Maldives, began by screening three candidate mitochondrial regions: the control region, the 12S rRNA gene, and the 16S rRNA gene. Eight candidate primer sets were designed in silico, four from the 12S region, three from the 16S region, and one from the control region. The designers required at least five variant positions within the primer annealing regions compared with 103 marine mammal mitogenomes, including manatees, seals, and dozens of whale and dolphin species, with stringent selection for at least one discriminatory mismatch at the 3-prime terminus of the primer.

That terminal mismatch matters because the final bases of a primer largely determine whether DNA polymerase will extend the reaction. A mismatch at the 3-prime end effectively blocks amplification of non-target DNA, giving the assay its specificity. The team also kept amplicons short, between 87 and 232 base pairs, because eDNA in aquatic systems is typically fragmented and degraded, and only small templates amplify reliably. After in vitro testing of the five most promising primer sets, one assay, designated Ddu_16S_07, emerged as the winner. It amplifies an 87 base pair fragment of the mitochondrial 16S rRNA gene using a dugong-specific reverse primer paired with a universal marine-vertebrate forward primer, and it relies on inexpensive SYBR Green chemistry rather than costly fluorescent probes.

The performance figures are impressive for a non-probe assay. Using synthetic DNA standards, the researchers modeled a limit of detection of 2.88 copies per reaction, meaning the assay can flag dugong DNA at extraordinarily low concentrations, and a limit of quantification of 66 copies per reaction, the threshold at which copy numbers can be measured reliably. The standard curve showed strong linearity, with an R-squared of 0.985 and an amplification slope of -3.28, corresponding to an efficiency of 101.78 percent, comfortably within the accepted 90 to 110 percent range for qPCR. In silico testing against hundreds of reference sequences found no binding to non-target species with four or fewer mismatches; the only predicted cross-binding at five mismatches was to Steller’s sea cow, which has been extinct since the eighteenth century.

Empirical validation backed up the computational predictions. The assay amplified dugong DNA from tissue samples and from water collected in the dugong exhibit at SeaWorld Abu Dhabi, while showing no amplification for six sympatric cetacean species tested in the laboratory, including Risso’s dolphin, spinner dolphin, Indian Ocean humpback dolphin, pantropical spotted dolphin, Indo-Pacific bottlenose dolphin, and humpback whale. Positive amplicons were confirmed by melt curve analysis, fragment sizing, and Sanger sequencing. The team also ran inhibition tests, spiking environmental water and seagrass samples with a known quantity of synthetic DNA. Neat samples showed a cycle delay of three or more cycles and incomplete amplification, but when samples were diluted tenfold, all replicates amplified, indicating that inhibitors are present in Red Sea eDNA matrices and that running samples both neat and diluted is best practice.

The real-world test involved 40 environmental samples: 28 seawater samples and 11 seagrass roller samples collected along the Saudi Arabian Red Sea coast, plus the aquarium water sample. Water was collected with Niskin bottles at one meter depth and filtered on site through 1.2 micrometer cellulose acetate cartridges. The roller samples were an experimental twist: because dugongs graze on seagrass, the team used sterile miniature paint rollers, wielded by snorkelers or an extendable handle from the boat, to swab seagrass surfaces in the hope that grazing activity would leave detectable DNA on the plants. Two field water samples produced positive amplification, each in two of three technical replicates, with melt temperatures consistent with the expected product. The aquarium sample amplified in all neat replicates. Notably, none of the seagrass roller samples amplified, despite being paired with two of the positive water sites.

The roller result is a cautionary tale about eDNA substrates. The authors suggest that substrate-specific differences in how DNA accumulates, persists, and is recovered likely explain the discrepancy: the dugong signal may simply not have been present at the exact swabbed spot, or plant material and surface debris may have diluted it. They propose that future roller sampling could be improved by increasing replicate numbers, covering broader areas of seagrass beds, or targeting sites showing fresh feeding trails. Similarly, the limited field detection rate probably reflects low and spatially patchy eDNA concentrations, and the authors note that filtering larger water volumes and sampling at greater depths, where dugongs are more active, could raise detection probabilities for this rare species.

Under the standardized validation framework proposed by Thalinger and colleagues, which grades eDNA assays from Level 1 (Incomplete) to Level 5 (Operational), the dugong assay meets the criteria for Level 4 (Substantial), reflecting demonstrated sensitivity plus expanded in vitro and field testing. Reaching Level 5 would require statistical estimation of detection probabilities and exhaustive assessment of environmental variables affecting performance. Even so, the assay fills a genuine gap: no species-specific qPCR assay for Dugong dugon had previously been published, although comparable assays exist for manatees, the dugong’s closest living relatives. The authors report that their assay performs comparably to a validated probe-based manatee assay, and its modeled detection limit is actually lower, though the two studies used different estimation approaches. For conservationists working across the Indo-Pacific, where dugong distribution data are often decades old or nonexistent, a cheap, scalable, probe-free test that can confirm the presence of dugongs from a few liters of seawater could transform monitoring, enabling managers to detect population change, target protection measures, and evaluate whether interventions are actually working before it is too late for the species’ most imperiled populations.

Subject of Research: Development of a species-specific environmental DNA qPCR assay for detecting the threatened dugong

Article Title: Development of a species-specific qPCR assay for the detection of the threatened dugong (Dugong dugon)

Article References: Development of a species-specific qPCR assay for the detection of the threatened dugong (Dugong dugon). (n.d.). https://doi.org/10.3389/fmamm.2026.1915420

Image Credits: AI Generated

DOI: 10.3389/fmamm.2026.1915420

Keywords: dugong, environmental DNA, qPCR, eDNA biomonitoring, Red Sea, marine mammal conservation, mitochondrial 16S rRNA, species-specific assay, seagrass, IUCN Vulnerable species, non-invasive monitoring, primer design