Environmental DNA Detection Research Articles

The riverscape on a chip: High-throughput qPCR enables basin-wide fishery assessments

To achieve reliable detection of environmental DNA from multiple species, a method which combines the specificity of single-taxon assays with the broad taxonomic coverage of metabarcoding is needed. High-throughput quantitative PCR (HT-qPCR) has emerged as one such method that holds particular promise for ecological applications. Here, we present the development and validation of a HT-qPCR biochip (i.e., biochip) protocol for a suite of western North American fishes with high cultural, economic, and ecological significance. Coupling this biochip with systematically distributed eDNA samples collected by citizen scientists, we evaluated the distribution of multiple species throughout a large river basin in the U.S. Pacific Northwest. Patterns of detection using the biochip were comparable to those using single-taxon qPCR (ST-qPCR; mean concordance = 0.917), despite the biochip using 90% less DNA per taxon and reducing costs by an estimated 40%. Comparisons between biochip results and distributions derived from conventional methods demonstrate that this approach not only offers more reliable and high-resolution distribution data for these species but also achieves this more efficiently than traditional sampling methods.

Effects of storage conditions on the stability of qPCR reagents: implications for environmental DNA detection

ObjectiveEnvironmental DNA (eDNA) detection is a transformative tool for ecological surveys which in many cases offers greater accuracy and cost-effectiveness for tracking low-density, cryptic species compared to conventional methods. For the use of targeted quantitative PCR (qPCR)-based eDNA detection, protocols typically require freshly prepared reagents for each sample, necessitating systematic evaluation of reagent stability within the functional context of eDNA standard curve preparation and environmental sample evaluation. Herein, we assessed the effects of long-term storage and freeze–thaw cycles on qPCR reagents for eDNA analysis across six assays.ResultsResults demonstrate qPCR plates (containing pre-made PCR mix, primer-probe, and DNA template) remain stable at 4 °C for three days before thermocycling without fidelity loss irrespective of qPCR assay used. Primer-probe mixes remain stable for five months of − 20 °C storage with monthly freeze-thaw cycles also irrespective of qPCR assay used. Synthetic DNA stocks maintain consistency in standard curves and sensitivity for three months under the same conditions. These findings enhance our comprehension of qPCR reagent stability, facilitating streamlined eDNA workflows by minimizing repetitive reagent preparations.

Optimization and application of bacterial environmental DNA and RNA isolation for qualitative and quantitative studies

AbstractMolecular detection of environmental DNA (eDNA) and RNA (eRNA) allows highly sensitive qualitative (i.e., presence or absence) and quantitative (i.e., abundance) monitoring of aquatic bacteria. However, bacterial molecular diagnostics are limited by low positive predictive values. Protocols for bacterial eDNA and eRNA molecular monitoring have primarily focused on optimizing specimen collection, and the optimal method to purify bacterial nucleic material from postcollection aquatic specimens to maximize the analytical sensitivity of molecular diagnostics remains poorly defined. Accordingly, strategies to isolate bacterial eDNA and eRNA from fresh and saltwater were investigated. We evaluated two filtration and four nucleic acid purification systems as representative of current generation bacterial eDNA and eRNA isolation strategies for capacity to isolate bacterial eDNA and eRNA from prelysed (i.e., free‐nucleic acids) and viable (i.e., colony forming units, CFU) bacterial cells. We also compared the sensitivities of reverse transcription quantitative PCR (RT‐qPCR) and metagenomic shotgun microbiome sequencing. The optimal protocol used 0.7 μm borosilicate glass filters (Whatman plc) followed by extraction with the RNeasy PowerWater kit (Qiagen). The protocol had a very high analytical sensitivity (10−3–100 ng and 102–101 CFU detected in 500 mL) across multiple species of bacteria, when tested with either RTqPCR or metagenomic sequencing. Importantly, this study highlighted several limitations which are restrictive to both qualitative and quantitative bacterial eDNA and eRNA studies. First, a 12‐h time course between sampling and extraction revealed significant species‐specific changes in cell number and free‐nucleic acid concentrations can occur postspecimen collection. Second, we found Gram‐positive bacteria yielded less nucleic material compared to Gram‐negative bacteria suggesting bacterial eDNA and eRNA studies could be biased by microorganism genome stability and extraction efficiency. This study highlights the need to define the species‐specific diagnostic sensitivity of a protocol when monitoring aquatic bacterial eDNA and eRNA with molecular diagnostics.

Use of environmental DNA for early detection of invasive Elodea species in interior Alaska

Abstract Invasive Canadian waterweed Elodea canadensis and western waterweed E. nuttallii are a threat to native salmon Onchorhynchus spp. in Alaska. Early detection is important to stop or mitigate spread. We evaluated detection of environmental DNA (eDNA) as a tool for early detection of these invasive species in Alaska. First, we evaluated four quantitative real-time PCR assays, one for each species, and two targeting either species, using samples taken at known infestations in Chena Lakes and Potter Marsh, Alaska. We also deployed E. nuttallii in screened containers at the Small Arms Complex Pond, Fort Wainwright, Alaska (Test Pond) in 2018 and 2019 to evaluate the detection of eDNA as a function of distance from the containers. At the known infestations, we detected the eDNA of both species in water samples. However, in our Test Pond, we only detected Elodea eDNA in 2 of 126 samples collected in 2019. Both detections were from samples collected within 10 cm of the containers. There were no detections in 60 samples collected in 2018 at the Test Pond. While there are potential uses for the eDNA markers we developed (e.g., species identification), we found no evidence to support their use as an early detection tool for Elodea in Alaska.

Evaluation of eDNA qPCR monitoring as an early detection tool for a non-native mysid in Great Lakes Waters

Early detection of aquatic invasive species (AIS) is vital to cost-effective prevention of their spread in the Great Lakes. Unfortunately, AIS surveillance has been generally too slow and geographically limited to support this purpose. Environmental DNA (eDNA) detection using quantitative polymerase chain reaction (qPCR) offers more rapid and affordable detection of likely AIS presence, but it does not directly discern live/dead status. Vital status verification using conventional surveys following positive eDNA qPCR detections could resolve this barrier, but only if the latter are adequately reliable and sensitive. Here we explore the reliability and sensitivity of eDNA qPCR monitoring for the bloody red shrimp (Hemimysis anomala), an AIS established in the southern Great Lakes but not yet widely distributed in Lake Superior, against conventional microscopy-based methods. We conducted this comparison using 1) harbor water from Muskegon Lake, MI where H. anomala is established, and 2) raw ballast water from ships transporting ballast from lower Lake Michigan to western Lake Superior. Our studies showed positive eDNA qPCR detections of H. anomala in all harbor and ballast samples for which conventional detection results were positive, and in some samples for which conventional results were negative. These results suggest that qPCR assays with adequate specificity could be an important tool in support of more effective and affordable early detection of target species in Great Lakes water, especially when combined with confirmatory conventional monitoring.

Systematic evaluation of the influence of Taq enzyme choice and amplification conditions on targeted environmental DNA assay performance and detection in field samples

AbstractTargeted environmental DNA (eDNA) studies mainly rely on quantitative real‐time polymerase chain reaction (qPCR) to amplify extremely low concentrations of DNA present in environmental samples. Understanding factors that influence targeted eDNA assay performance and detection in field samples, such as Taq DNA polymerase enzyme type and thermocycle protocol, is critical for the interpretation of qPCR results. We completed a systematic performance evaluation of five distinct targeted eDNA assays (eANFI6, eFISH1, eANBO5, eGLIN1, and eLICA3 targeting sablefish, general fish, Boreal toad, wood turtle, and the American bullfrog) by subjecting the same samples to analysis by five different Taq enzyme reagent mixes (Immolase, Environmental Master Mix (EMM), Amplitaq, QIAcuity, and QuantiNova) and the commonly used 2‐step (95°C, 60°C) and 3‐step (95°C, 64°C, 72°C) thermocycle protocols. We evaluated assay performance using a standardized dilution series of synthetic dsDNA target sequences and calculated limits of detection (LOD) and quantification (LOQ) and 95% confidence intervals for each combination of Taq enzyme and thermocycle protocol. All assays performed within acceptable performance criteria as defined by the Canadian national standard for targeted eDNA assays. Based on data generated by synthetic dsDNA fragments, the eDNA assays performed comparably regardless of the enzyme reagent mix and protocol used, except for eANFI6 and eGLIN1 using EMM and the 3‐step protocol, where no amplification was observed. On freshwater field samples, EMM and Immolase performed best. On marine field samples, Immolase, EMM, Qiacuity, and QuantiNova performed equally well, although EMM failed to amplify some samples. The work reveals that an enzyme reaction mix or thermocycle protocol can affect the result of an eDNA assay, but the appropriate choice also depends on the nature of the field sample. It is therefore imperative that these are considered when selecting appropriate reaction conditions and that they are clearly reported.

A new strategy based on a cascade amplification strategy biosensor for on-site eDNA detection and outbreak warning of crown-of-thorns starfish

Population outbreaks of the crown-of-thorns starfish (COTS) seriously threaten the sustainability of coral reef ecosystems. However, traditional ecological monitoring techniques cannot provide early warning before the outbreaks, thus preventing timely intervention. Therefore, there is an urgent need for a more accurate and faster technology to predict the outbreaks of COTS. In this work, we developed an electrochemical biosensor based on a programmed catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) cyclic amplification strategy for sensitive and selective detection of COTS environmental DNA (eDNA) in water bodies. This biosensor exhibited excellent electrochemical characteristics, including a low limit of detection (LOD = 18.4 fM), low limit of quantification (LOQ = 41.1 fM), and wide linear range (50 fM – 10 nM). The biosensing technology successfully allowed the detection of COTS eDNA in the aquarium environment, and the results also demonstrated a significant correlation between eDNA concentration and COTS number (r = 0.990; P < 0.001). The reliability and accuracy of the biosensor results have been further validated through comparison with digital droplet PCR (ddPCR). Moreover, the applicability and accuracy of the biosensor were reconfirmed in field tests at the COTS outbreak site in the South China Sea, which has shown potential application in dynamically monitoring the larvae before the COTS outbreak. Therefore, this efficient electrochemical biosensing technology offers a new solution for on-site monitoring and early warning of the COTS outbreak.

The Viability and Potential of Environmental DNA (eDNA) Detection of Freshwater Fish Based on Current Genetic Resources in Malaysia

Environmental DNA (eDNA) metabarcoding is a promising tool for regular biological monitoring, especially for freshwater fish, which are facing tremendous threats worldwide. The application of eDNA detection is a dramatic improvement on common methods of biomonitoring as it produces tangible results in a short time with low effort and little expense. However, the accuracy of the technique is largely dependent on the availability of genetic references for the target organisms. In this study, we investigated the availability of genetic resources for freshwater fish in Malaysia in three public depositories, National Center for Biotechnology Information (NCBI), Barcode of Life Data System (BOLD), and Mitochondrial Genome Database of Fish (MitoFish), focusing on seven targeted genes of mitochondrial DNA. We found that only 68.6% of freshwater fish found in Malaysia had information on at least one of the seven targeted genes, with data on Cytochrome C Oxidase Subunit I being most commonly available. Genetic information for threatened and endemic species were underrepresented (33.3%-41.7%), yet fish of commercial value and invasive species were well explored genetically. Although there is still room for improvement to achieve comprehensive and reliable genetic resource information for freshwater fish in Malaysia, the application of eDNA metabarcoding is still highly relevant. This is since the current decline in freshwater fish diversity in Malaysia is alarming and because the technique will assist in the ongoing effort to generate new genetic references for Malaysian freshwater fish.

Evaluating environmental DNA detection of a rare fish in turbid water using field and experimental approaches.

Detection sensitivity of aquatic species using environmental DNA (eDNA) generally decreases in turbid water but is poorly characterized. In this study, eDNA detection targeted delta smelt (Hypomesus transpacificus), a critically endangered estuarine fish associated with turbid water. eDNA sampling in the field was first paired with a trawl survey. Species-specific detection using a Taqman qPCR assay showed concordance between the methods, but a weak eDNA signal. Informed by the results of field sampling, an experiment was designed to assess how turbidity and filtration methods influence detection of a rare target. Water from non-turbid (5 NTU) and turbid (50 NTU) estuarine sites was spiked with small volumes (0.5 and 1 mL) of water from a delta smelt tank to generate low eDNA concentrations. Samples were filtered using four filter types: cartridge filters (pore size 0.45 μm) and 47 mm filters (glass fiber, pore size 1.6 μm and polycarbonate, pore sizes 5 and 10 μm). Prefiltration was also tested as an addition to the filtration protocol for turbid water samples. eDNA copy numbers were analyzed using a censored data method for qPCR data. The assay limits and lack of PCR inhibition indicated an optimized assay. Glass fiber filters yielded the highest detection rates and eDNA copies in non-turbid and turbid water. Prefiltration improved detection in turbid water only when used with cartridge and polycarbonate filters. Statistical analysis identified turbidity as a significant effect on detection probability and eDNA copies detected; filter type and an interaction between filter type and prefilter were significant effects on eDNA copies detected, suggesting that particulate-filter interactions can affect detection sensitivity. Pilot experiments and transparent criteria for positive detection could improve eDNA surveys of rare species in turbid environments.

Detection and transport of environmental DNA from two federally endangered mussels.

Environmental DNA (eDNA) offers a novel approach to supplement traditional surveys and provide increased spatial and temporal information on species detection, and it can be especially beneficial for detecting at risk or threatened species with minimal impact on the target species. The transport of eDNA in lotic environments is an important component in providing more informed descriptions of where and when a species is present, but eDNA transport phenomena are not well understood. In this study, we used species-specific assays to detect eDNA from two federally endangered mussels in two geographically distinct rivers. Using the eDNA concentrations measured from field samples, we developed a one-dimensional (1D) hydrodynamic transport model to predict the downstream fate and transport of eDNA. We detected eDNA from both federally endangered mussels across several seasons and flow rates and up to 3.5 km downstream from the source populations, but the detection rates and eDNA concentrations were highly variable across and within rivers and study reaches. Our 1D transport models successfully integrated the variability of the eDNA field samples into the model predictions and overall model results were generally within ±1 standard error of the eDNA field concentration values. Overall, the results of this study demonstrate the importance of optimizing the spatial locations from where eDNA is collected downstream from a source population, and it highlights the need to improve understanding on the shedding mechanisms and magnitude of eDNA from source populations and biogeomorphic processes that influence eDNA transport.

Environmental DNA detection of spot-tailed quoll from soil is unlikely to be useful for routine monitoring

Context Accurate monitoring data on species presence and distribution are crucial for effective conservation management. Environmental DNA (eDNA)-based techniques, in which species are detected from trace amounts of DNA found throughout the environment, are promising tools that may complement traditional monitoring methods and improve detection. However, imperfect detection is a feature of all survey methods that should be properly assessed so that the probability of detecting a target species’ DNA at a site where it is present (i.e. the sensitivity of the method) can be determined. The spot-tailed quoll (Dasyurus maculatus), a carnivorous marsupial found in eastern Australia, is a difficult species to detect as it is rare and has large home ranges, often in remote and difficult to access habitat. Aims In this study, we aimed to evaluate the feasibility of using eDNA soil analysis as a viable alternative or complement to traditional monitoring techniques for detecting spot-tailed quoll. Methods We developed a species-specific assay and validated it using synthetic oligos, tissue samples and soil collected from a captive quoll enclosure. We then assessed the assay on natural environment soil samples taken from the Snowy River region from communal quoll defecation sites (latrines) and from broader quoll habitat. We used amplification success data to model the concentration of quoll DNA in soil from different site types and calculate the sensitivity of our assay. Key results Sensitivity was highest at latrine sites, but decreased sharply when sampling just 1 m away. In non-latrine habitat, the positive amplification rate was too low to allow for meaningful statistical analyses, suggesting that a prohibitively large number of samples would need to be analysed for detection probabilities to be adequate for routine monitoring programs. Conclusions Overall, we found that low sensitivity was driven by the low concentration of spot-tailed quoll DNA at many of the surveyed sites. Implications Given that quoll latrines can usually be identified from the accumulation of scats, and scats themselves can be sampled for DNA, we suggest that eDNA analysis of soil is unlikely to offer improvements over current spot-tailed quoll monitoring methods.

CRISPR‐based environmental DNA detection for a rare endangered estuarine species

AbstractEnvironmental DNA (eDNA) methods complement traditional aquatic monitoring surveys and are especially advantageous for rare and listed species to detect spatial and temporal distribution patterns. However, improvements in ease of use and portability could increase the utility of eDNA methods, leading to more widespread application, including expanding its role in management decision‐making processes. We describe the development of an eDNA detection assay for delta smelt (Hypomesus transpacificus), an endangered fish in the San Francisco Estuary, using SHERLOCK (Specific High‐Sensitivity Enzymatic Reporter Unlocking). SHERLOCK is a clustered regularly interspaced short palindromic repeats (CRISPR)‐based diagnostic tool with the ability to detect species‐specific genetic variants, making it ideal for genetic‐based taxonomic identification of any organism. Because of its high sensitivity and specificity, SHERLOCK is adaptable to eDNA detection in water samples. Here, we describe adaptation of a delta smelt SHERLOCK assay for use with estuarine water eDNA samples. This version of the assay exhibits increased sensitivity compared to the original delta smelt SHERLOCK protocol (new limit of detection approximately three copies per reaction compared to ~300 in original assay) and successfully detected delta smelt eDNA in both experimental and natural contexts. Overall, our results demonstrate that SHERLOCK eDNA detection offers managers an alternative, isothermal methodology, and highlights some challenges for detection of rare, endangered species at low abundance.

PCR and LAMP detection of environmental DNA of the invasive clam &lt;i&gt;Corbicula fluminea&lt;/i&gt;

PCR and LAMP detection of environmental DNA of the invasive clam &lt;i&gt;Corbicula fluminea&lt;/i&gt;

New insights into the on-site monitoring of probiotics eDNA using biosensing technology for heat-stress relieving in coral reefs

Coral probiotics can improve the tolerance of corals to heat stress, thus mitigating the process of coral thermal bleaching. Sensitive and specific detection of coral probiotics at low abundances is highly desirable but remains challenging, especially for rapid and on-site detection of coral probiotics. Since the electrochemical biosensor has been recently used in the field of environmental DNA (eDNA) detection, herein, an efficient electrochemical biosensor was developed based on CoS2/CoSe2-NC HNCs electrode material with a specific DNA probe for the C. marina detection. After optimization, the lower limit of detection (LOD) values of such biosensors for the target DNA and genomic DNA were 1.58 fM and 6.5 pM, respectively. On this basis, a portable device was constructed for the practical detection of C. marina eDNA, and its reliability and accuracy were verified by comparison with the ddPCR method (P > 0.05). For each analysis, the average cost was only ∼ $1.08 and could be completed within 100 min with reliable sensitivity and specificity. Overall, the biosensor could reflect the protective effect of probiotics on coral heat stress, and the proposed technique will put new insights into the rapid and on-site detection of coral probiotics to assist corals against global warming.

Environmental DNA detection range for Hydrilla verticillata, a prolific invasive plant

Environmental DNA detection range for <i>Hydrilla verticillata</i>, a prolific invasive plant

A preliminary study into the detection of fish environmental DNA in selected South African estuaries

Environmental DNA (eDNA) is a powerful technique for the non-invasive and comprehensive monitoring of entire ecosystems. Despite its many advantages, the use of eDNA in South Africa as an assessment tool for fish biodiversity is largely underutilised. This study was performed as an initial screening to assess the efficiency of the eDNA method for characterising fish communities in South African waters using the metabarcoding approach. Water samples were collected from eight estuaries and vacuum filtered using 0.45 μm nylon membrane filters. DNA was extracted from the filters, and sample extracts underwent PCR assaying using MiFish primers for the 12S rRNA gene, prior to being sequenced on the Illumina MiSeq platform. The resulting sequence data produced 1 574 092 raw reads, which resulted in 119 operational taxonomic units (OTUs) after filtering and clustering. Taxonomic assignment of the OTUs produced four different taxonomic groups and one group representing unidentified sequences. The group with the highest number of OTUs (54%) was assigned to fish taxa consisting of 12 families and 22 genera identified across all estuaries, with only 16 taxa identified to species level. As this was a preliminary study, few samples were collected, and therefore several recommendations for a more comprehensive study design are provided. Overall, these results support the hypothesis that the eDNA metabarcoding approach can efficiently detect fish species in South African estuarine environments.

Distribution and habitat preference of polar cod (Boreogadus saida) in the Bering and Chukchi Seas inferred from species-specific detection of environmental DNA

Ongoing warming and sea-ice reductions in the Arctic can seriously impact cold-water species, such as polar cod (Boreogadus saida), necessitating biomonitoring to reveal the ecological consequences. Recent methodological advancements in environmental DNA (eDNA) techniques have increased our ability to conduct ecological monitoring at various locations, including the Arctic. This study aimed to provide an overview of the distribution of polar cod across the Bering and Chukchi Seas by employing species-specific detection of eDNA. First, we successfully developed novel species-specific qPCR assay targeting the mitochondrial D-loop region, which exclusively amplifies eDNA derived from polar cod. Subsequently, polar cod eDNA was detected using the assay from the samples that we collected latitudinally across the study area during the open-water season. Polar cod eDNA was primarily detected in the surface water from the central Chukchi Sea shelf and the northernmost observation line (75°N), which was located on the shelf slope, off the Point Barrow, and in the marginal ice zone. In contrast, only trace amounts of eDNA were detected in the Bering Sea. This pattern corresponded well with the distribution of water masses classified based on environmental conditions. The detection of eDNA in surface water was clearly limited to cold (-1 to 5°C) and low salinity (25–32) water, whereas it was detected in a higher salinity range (32–34) in the middle and bottom layers. These findings are consistent with current knowledge about the distribution and habitat of the polar cod, suggesting that eDNA can be regarded as a reliable tool to replace or supplement conventional methods. Incorporating eDNA techniques into large-scale oceanographic surveys can improve the spatial and temporal resolution of fish species detection with a reasonable sampling effort and will facilitate the continuous monitoring of Arctic ecosystems.

Building a Teleost Fish Traceability Program Based on Genetic Data from Pacific Panama Fish Markets.

Fish tissue samples from 203 adult individuals were collected in the main ports and markets of the Pacific coast of Panama. Molecular identification based on a cytochrome oxidase I gene segment of all species was verified by GENBANK reference sequences. A total of 34 species from 14 families (Ariidae, Caranjidae, Centropomidae, Gerreidae, Haemulidae, Lobotidae, Lutjanidae, Malacanthidae, Mugilidae, Scianidae, Scombridae, Serranidae, Sphyraenidae, Stromateidae) were identified at the species level from 164 sequences. Additionally, three Caribbean species were molecularly identified among the analyzed samples (Mycteroperca xenarcha, Paralonchurus brasilensis and Lobotes surinamensis). Species diversity was slightly higher in the Gulf of Panama than in the Gulf of Chiriquí. For species with five or more individual sequences, genetic diversity and genetic connectivity parameters such as total number of haplotypes (H), haplotype diversity (Hd), and nucleotide diversity (π) were calculated. Overall, pelagic-migratory species showed higher values of genetic diversity than coastal and estuarine species with some exceptions. Connectivity between Gulf areas was compared using values of genetic distances and genetic differentiation (Fst). The high level of connectivity observed between the Gulf of Chiriqui and the Gulf of Montijo indicates the existence of a single stock in that area for the following species: Scomberomorus sierra, Caranx caninus and Lutjanus guttatus. The demographic history of the most common species was examined using Tajima's D values, suggesting population expansion for two snapper species, L. peru and L. argentiventris, having significant and higher values. Another important contribution from this research was the production of primers and dual-labeled probes for environmental DNA detection using qPCR for the five most abundant species (spotted rose snapper, yellow snapper, green jack, Pacific crevalle jack and the Pacific sierra fish). These markers represent a new set of tools for environmental DNA (eDNA) detection and molecular traceability of three commercially important fish species along the supply chain including landing sites and markets of the main fishery areas.

Development of a rapid and sensitive real-time diagnostic assay to detect and quantify Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome.

The oomycete Aphanomyces invadans causes epizootic ulcerative syndrome (EUS), a World Organization for Animal Health (WOAH)-listed disease that has seriously impacted a wide range of fish worldwide. Currently, only three conventional polymerase chain reaction (PCR) assays are recommended for the detection of A. invadans. The robust quantitative PCR (qPCR) assay has recently become more important due to its highly accurate nature and the applicability of qPCR-based environmental DNA (eDNA) detection in the monitoring of pathogens in aquatic environments. Therefore, in this study, we developed a novel TaqMan probe-based qPCR method to sensitively and quantitatively detect A. invadans. The assay limit of detection was determined using 10-fold serial dilutions of linearized A. invadans plasmid. Assay sensitivity was assessed in the presence of interfering substances and compared to three WOAH-listed primers using the mycelia and zoospores of A. invadans with and without fish muscle tissue. The assay specificity was also theoretically and experimentally assessed against other oomycetes, fish muscle tissue, and water samples. The assay's repeatability and reproducibility were determined. In this study, the limit of detection of the developed assay was 7.24 copies of A. invadans genomic DNA per reaction (95% confidence interval (CI): 2.75 to 19.05 copies/reaction). The assay showed the same sensitivity in the presence of other substances. Compared to the WOAH-recommended PCR assays, this assay had 10-times higher sensitivity for all tested samples. There were no cross-reactions with other closely related oomycetes, fish muscle, or water samples, indicating that the assay was highly specific for A. invadans. The repeatability and reproducibility tests showed little variation, ranging from 0.1-0.9% and 0.04-1.1%, respectively, indicating the high consistency, repeatability, and reliability of the developed assay. This highly rapid, sensitive, specific, and consistent EUS qPCR assay would be of importance in transboundary disease management and the monitoring of pathogens in aquatic environments.

Influence of distance from source population and seasonality in eDNA detection of white‐clawed crayfish, through qPCR and ddPCR assays

AbstractThe white‐clawed crayfish (Austropotamobius pallipes) is an emblematic taxon of European rivers, found mainly in oxygenated streams, known to be an excellent indicator of river quality. Since several decades, the population of A. pallipes declined in relation to anthropogenic pressure, habitat loss, and competition with pests (invasive crayfish, crayfish plague). This endangered species is now submitted to conservation strategies by freshwater managers in order to survey and protect the remaining populations. In France, traditional surveys in freshwater environments were performed by electric fishing, kick‐net fishing, or trapping, particularly disruptive for the environment and very time‐consuming. However, with the rise of molecular genetic technology, new methods based on the detection of environmental DNA (eDNA) have emerged. We present here the results of an optimized study for the detection of the endangered crayfish Austropotamobius pallipes in France, considering certain environmental co‐factors and comparing two PCR methods (qPCR and ddPCR). After improving laboratory procedures, we were able to detect the presence of the crayfish up to 2 km downstream from a known point of presence and unfortunately highlight the disappearance of a historical population, after sampling two consecutive years. Such a level of precision is interesting because it makes it possible to precise the presence of specimens in a relatively restricted area and to orient traditional prospecting, necessary for certain additional studies. During our study, we observed better probabilities of detection during the summer period, but in a growing context of climate change, we advise to adapt the sampling year by year. That said, this methodology is a very useful tool for the detection of rare and/or endemic species and we did not observe any difference between the two PCR methods used.