scholarly journals Development of an eDNA metabarcoding tool for surveying the world’s largest amphibian

2021 ◽  
Author(s):  
Jie Wang ◽  
Ping Liu ◽  
Jiang Chang ◽  
Cheng Li ◽  
Feng Xie ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Survey Methods ◽  
Environmental Dna ◽  
Cost Effective ◽  
Isolated Populations ◽  
Specificity And Sensitivity ◽  
Chinese Giant Salamander ◽  
Elusive Species ◽  
Primer Sets ◽  
Species Specific

Abstract Due to the overexploitation of farming, as well as habitat loss or degradation, the wild population of Chinese giant salamander Andrias davidianus (CGS), a species with seven genetically distinct lineages, has decreased by over 80% in the past 70 years. Traditional survey methods have proven to be unsuitable for finding this rare and elusive species. We evaluated the efficacy of environmental DNA (eDNA) sampling to detect CGS indirectly from its aquatic environment. We developed several species-specific primer sets; validated their specificity and sensitivity; and assessed their utility in silico, in the laboratory, and in two field sites having released farm-bred CGSs. We detected the presence of CGS DNA by using polymerase chain reaction (PCR) and Sanger sequencing. We also sequenced an amplicon mixture of seven haplotype-represented samples using high-throughput sequencing. Our eDNA methods could detect the presence of CGS at moderate densities reported across its range, proving them as a cost-effective way to establish broad-scale patterns of occupancy for CGS. In addition, our primers enabled the detection of a mitochondrial lineage mixture or introduced individuals from geographically isolated populations of CGS.

An eDNA approach to detect eastern hellbenders (Cryptobranchus a. alleganiensis) using samples of water

2012 ◽  
Vol 39 (7) ◽  
pp. 629 ◽  
Author(s):  
Zachary H. Olson ◽  
Jeffrey T. Briggler ◽  
Rod N. Williams
Keyword(s):  
Water Samples ◽  
Effective Means ◽  
Environmental Dna ◽  
Cost Effective ◽  
Negative Control ◽  
Specificity And Sensitivity ◽  
Genetic Sampling ◽  
Conservation Concern ◽  
Species Specific ◽  
Eastern Hellbender

Context Environmental DNA, or eDNA, methods are a novel application of non-invasive genetic sampling in which DNA from organisms is detected via sampling of water or soil, typically for the purposes of determining the presence or absence of an organism. eDNA methods have the potential to revolutionise the study of rare or endangered taxa. Aims We evaluated the efficacy of eDNA sampling to detect populations of an amphibian of conservation concern, the eastern hellbender (Cryptobranchus a. alleganiensis), indirectly from their aquatic environments. Methods We developed species-specific primers, validated their specificity and sensitivity, and assessed the utility of our methods in silico and in laboratory trials. In the field, we collected water samples from three sites with known densities of hellbenders, and from one site where hellbenders do not occur. We filtered water samples, extracted DNA from filters, and assayed the extraction products for hellbender DNA by using polymerase chain reaction (PCR) and gel electrophoresis. Key results Our methods detected hellbenders at densities approaching the lowest of reported natural densities. The low-density site (0.16 hellbenders per 100 m2) yielded two positive amplifications, the medium-density site (0.38 hellbenders per 100 m2) yielded eight positive amplifications, and the high-density site (0.88 hellbenders per 100 m2) yielded 10 positive amplifications. The apparent relationship between density and detection was obfuscated when river discharge was considered. There was no amplification in any negative control. Conclusion eDNA methods may represent a cost-effective means by which to establish broad-scale patterns of occupancy for hellbenders. Implications eDNA can be considered a valuable tool for detecting many species that are otherwise difficult to study.

scholarly journals Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

2017 ◽  
Author(s):  
Lynsey R. Harper ◽  
Lori Lawson Handley ◽  
Christoph Hahn ◽  
Neil Boonham ◽  
Helen C. Rees ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Detection Threshold ◽  
Environmental Dna ◽  
Cost Effective ◽  
Triturus Cristatus ◽  
Species Detection ◽  
Survey Tool ◽  
Crested Newt ◽  
Species Specific ◽  
Freshwater Monitoring

SummaryEnvironmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and ‘metabarcoding’ have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real-time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using High-Throughput Sequencing technology. With qPCR and a detection threshold of 1/12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4/12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species-specific surveys.

scholarly journals DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

2015 ◽  
Vol 8 ◽  
pp. MBI.S29736 ◽  
Author(s):  
Kenjiro Nagamine ◽  
Guo-Chiuan Hung ◽  
Bingjie Li ◽  
Shyh-Ching Lo
Keyword(s):  
Safety Concern ◽  
Rapid Development ◽  
High Sensitivity ◽  
High Specificity ◽  
Clostridium Tetani ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Pcr Assays ◽  
Primer Sets ◽  
Species Specific

Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

scholarly journals Detection of four imperiled western North American freshwater mussel species from environmental DNA with multiplex qPCR assays

2020 ◽  
Author(s):  
Torrey W. Rodgers ◽  
Joseph C. Dysthe ◽  
Cynthia Tait ◽  
Thomas W. Franklin ◽  
Michael K. Schwartz ◽  
...  
Keyword(s):  
Freshwater Mussel ◽  
Environmental Dna ◽  
Vulnerable Species ◽  
Western North America ◽  
Mussel Species ◽  
Multiplex Assays ◽  
Specificity And Sensitivity ◽  
Pcr Assays ◽  
Species Specific ◽  
Management And Conservation

AbstractWe developed multiplexed, species-specific, quantitative PCR assays for the detection of four freshwater mussel species native to western North America, Gonidea angulata, Margaritifera falcata, Anodonta nuttalliana and Anodonta oregonensis, from environmental DNA (eDNA). These species have experienced dramatic declines over the last century and are currently threatened in many portions of their ranges. Therefore, improved tools for detecting and monitoring these species are needed. Species-specificity and sensitivity of assays were empirically tested in the lab, and multiplex assays were also validated with field collected eDNA samples. All assays were species-specific, sensitive, and effective for detection from eDNA samples collected from streams and rivers. These assays will aid in the detection, monitoring, management, and conservation of these vulnerable species.

scholarly journals Novel Universal Primers for Metabarcoding eDNA Surveys of Marine Mammals and Other Marine Vertebrates

2019 ◽  
Author(s):  
Elena Valsecchi ◽  
Jonas Bylemans ◽  
Simon J. Goodman ◽  
Roberto Lombardi ◽  
Ian Carr ◽  
...  
Keyword(s):  
Marine Mammals ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
Universal Primers ◽  
Marine Biodiversity ◽  
Primer Sets ◽  
Marine Megafauna

ABSTRACTMetabarcoding studies using environmental DNA (eDNA) and high throughput sequencing (HTS) are rapidly becoming an important tool for assessing and monitoring marine biodiversity, detecting invasive species, and supporting basic ecological research. Several barcode loci targeting teleost fish and elasmobranchs have previously been developed, but to date primer sets focusing on other marine megafauna, such as marine mammals have received less attention. Similarly, there have been few attempts to identify potentially ‘universal’ barcode loci which may be informative across multiple marine vertebrate Orders. Here we describe the design and validation of four new sets of primers targeting hypervariable regions of the vertebrate mitochondrial 12S and 16S rRNA genes, which have conserved priming sites across virtually all cetaceans, pinnipeds, elasmobranchs, boney fish, sea turtles and birds, and amplify fragments with consistently high levels of taxonomically diagnostic sequence variation. ‘In silico’ validation using the OBITOOLS software showed our new barcode loci outperformed most existing vertebrate barcode loci for taxon detection and resolution. We also evaluated sequence diversity and taxonomic resolution of the new barcode loci in 680 complete marine mammal mitochondrial genomes demonstrating that they are effective at resolving amplicons for most taxa to the species level. Finally, we evaluated the performance of the primer sets with eDNA samples from aquarium communities with known species composition. These new primers will potentially allow surveys of complete marine vertebrate communities in single HTS metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators.

Novel Primer Sets for Rapid Detection of Zymoseptoria tritici in Wheat

Plant Disease ◽  
2020 ◽  
pp. PDIS-02-20-0318
Author(s):  
Adam Kuzdraliński ◽  
Justyna Leśniowska-Nowak ◽  
Michał Nowak ◽  
Magdalena Kawęcka ◽  
Anna Kot ◽  
...  
Keyword(s):  
High Specificity ◽  
Septoria Tritici Blotch ◽  
Septoria Tritici ◽  
In Vitro Tests ◽  
Zymoseptoria Tritici ◽  
Specificity And Sensitivity ◽  
Wheat Leaf ◽  
Primer Sets ◽  
Species Specific

Zymoseptoria tritici is a fungal pathogen causing losses in wheat yields. Here, we present new primer sets for species-specific identification of this microorganism in wheat leaf samples using conventional PCR. Primer sets were validated in silico using tools available in genetic databases. Furthermore, in vitro tests were also carried out on 190 common wheat samples with visual symptoms of Septoria tritici blotch (STB) collected in Poland in three growing seasons (2015, 2016, 2017). The designed primer sets showed full hybridization to the available genetic resources deposited in the NCBI GenBank database, and their high specificity and sensitivity were demonstrated on wheat leaf samples and selected fungal strains.

scholarly journals Scanning ferry routes: looking for eDNA traces of marine mammals and their preys

2021 ◽  
Vol 4 ◽  
Author(s):  
Elena Valsecchi
Keyword(s):  
Marine Mammals ◽  
High Throughput Sequencing ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Trophic Levels ◽  
Systematic Sampling ◽  
Marine Biodiversity ◽  
Tyrrhenian Sea ◽  
Night Time ◽  
Species Specific

Marine environmental DNA (eDNA) surveys are becoming a promising approach to monitor biodiversity status and its variation over time. However, monitoring offshore areas could be extremely costly when using dedicated vessels, beside the impossibility to sample simultaneously geographically distant (even if adjacent) areas. The unexplored possibility of availing on operating ferries as an opportunistic platform for eDNA sampling offers several advantages besides opening limitless opportunities for systematic surveys on marine biodiversity.We present the results of both metabarcoding and barcoding approaches obtained from the analysis of water samples collected on board of a ferry boat along a pilot Mediterranean route crossing the Pelagos Sanctuary for Mediterranean Marine Mammals. The recently described MarVer primer sets (12SrDNA and 16SrDNA regions), specifically designed for the simultaneous detection of marine mammals and other marine vertebrates, were employed. The High Throughput Sequencing (HTS) outcome showed that the markers successfully detected most trophic levels of vertebrate marine communities, and classes, including bony fish, rays, cetaceans and birds. Ferry-based sampling allow to collect sample at any time of the day, and we indeed found diel differences in both quantitative and qualitative distribution of read counts. For instances, we observed an increased abundance of lantern fish amplicons in night-time collect samples (50%), reflecting nocturnal migration through the water column. In general, the number of read counts was significantly higher in nocturnal samples. Such diel differences within our sample indirectly provides evidence of the efficiency of the eDNA approach to detect contemporary signals in the sampled environment. Similarly, cetaceans were detected in correspondence of visual sightings (when these occurred, supplementary samples were collected). Rare species, such as the monk seal, are difficult to be detected in metabarcoding surveys, thus we opted to side the screening of the ferry-samples with a panel of species-specific qPCR assays, which were able to detect DNA traces of the endangered pinniped in the Tuscany archipelago (Tyrrhenian Sea) long before visual observations witnessed its presence in the same area. The study demonstrates the feasibility of using commercial shipping as a platform for eDNA marine sampling without dedicated survey cruises. Commercial shipping routes have potential to act as regular systematic sampling transects which can contribute to evaluating and monitoring marine biodiversity.

scholarly journals COI metabarcoding primer choice affects richness and recovery of indicator taxa in freshwater systems

2019 ◽  
Author(s):  
Mehrdad Hajibabaei ◽  
Teresita M. Porter ◽  
Michael Wright ◽  
Josip Rudar
Keyword(s):  
Beta Diversity ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
Sequencing Technology ◽  
Indicator Taxa ◽  
Combined Use ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
Major Attention ◽  
Biodiversity Analysis

AbstractDNA-based biodiversity analysis has gained major attention due to the use of high throughput sequencing technology in approaches such as mixed community or environmental DNA metabarcoding. Many cytochrome c oxidase subunit I (COI) primer sets are now available for such work. The purpose of this study is to look at how COI primer choice affects the recovery of arthropod richness, beta diversity, and recovery of site indicator taxa in benthos kick-net samples typically used in freshwater biomonitoring. We examine 6 commonly used COI primer sets, on samples collected from 6 freshwater sites. Richness is sensitive to primer choice and the combined use of additional multiple COI amplicons recovers higher richness. Thus, to recover maximum richness, multiple primer sets should be used with COI metabarcoding. Samples consistently cluster by site regardless of amplicon choice or PCR replicate. Thus, for broadscale community analyses, overall beta diversity patterns are robust to COI marker choice. Additionally, the recovery of traditional freshwater bioindicator assemblages such as Ephemeroptera, Trichoptera, Plectoptera, and Diptera may not fully capture the diversity of broadscale arthropod site indicators that can be recovered from COI metabarcoding. Based on these results, studies that use different COI amplicons may not be directly comparable. This work will help future biodiversity and biomonitoring studies develop not just standardized, but optimized workflows that maximize taxon-detection or order taxa along gradients.

scholarly journals Development of a 16S metabarcoding assay for the environmental DNA (eDNA) detection of aquatic reptiles across northern Australia

2020 ◽  
Author(s):  
Katrina West ◽  
Matthew Heydenrych ◽  
Rose Lines ◽  
Tony Tucker ◽  
Sabrina Fossette ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Environmental Dna ◽  
Cost Effective ◽  
Distribution Data ◽  
Freshwater Turtles ◽  
Northern Australia ◽  
Non Invasive ◽  
Sea Snakes ◽  
Reptile Species ◽  
Species Specific

AbstractA severe lack of distribution data for aquatic reptiles in northern Australia leaves many taxa vulnerable to extirpation and extinction. Environmental DNA (eDNA) technologies offer sensitive and non-invasive genetic alternatives to trapping and visual surveys and are increasingly employed for the detection of aquatic and semi-aquatic reptiles. However, at present, these studies have largely applied species-specific primers which do not provide a cost-effective avenue for the simultaneous detection of multiple reptilian taxa. Here, we present a 16S rRNA metabarcoding assay for the broad detection of aquatic and semi-aquatic reptile species. This assay is tested on water samples collected at multiple sampling sites at two tropical locations: 12 marine/estuarine sites in Roebuck Bay, Western Australia, and 4 estuarine sites in Cooktown, Queensland, Australia. A total of nine reptile taxa were detected from 10 of the 16 sampled sites, including marine and freshwater turtles, aquatic and semi-aquatic/terrestrial snakes, and terrestrial skinks. However, inconsistencies in the detection of previously observed aquatic reptiles at our sampled sites, such as saltwater crocodile and sea snakes, indicates that further research is required to assess the reliability, strengths and limitations of eDNA methods for aquatic reptile detection before it can be integrated as a broad-scale bioassessment tool.

scholarly journals Environmental DNA and Specific Primers for Detecting the Invasive Species Ectopleura crocea (Hydrozoa: Anthoathecata) in Seawater Samples

2020 ◽  
Vol 12 (6) ◽  
pp. 2360 ◽  
Author(s):  
Philjae Kim ◽  
Tae Joong Yoon ◽  
Sook Shin
Keyword(s):  
Invasive Species ◽  
Survey Methods ◽  
Environmental Dna ◽  
Visual Assessment ◽  
Molecular Techniques ◽  
Mitochondrial Cytochrome ◽  
Specific Primer ◽  
Specific Primers ◽  
Seawater Samples ◽  
Species Specific

In marine environments, environmental DNA (eDNA) can be effectively detected and possibly quantified when combined with molecular techniques, as demonstrated by several recent studies. In this study, we developed a species-specific primer set and a probe to detect the distribution and biomass of an invasive hydrozoan in South Korea, Ectopleura crocea. These molecular markers were designed to amplify a 187 bp region based on mitochondrial cytochrome c oxidase subunit I (COI) of E. crocea and were tested on seawater samples from 35 Korean harbors in 2017. Of the 35 sites we investigated, only nine harbors returned positive detections when using traditional survey methods, while surveys based on the use of eDNA techniques detected E. crocea DNA in all seawater samples. These results suggest that eDNA surveys based on molecular techniques are more effective at identifying species distribution and estimating biomass than traditional surveys based on visual assessment of morphology.

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