Development of a Quantitative PCR Assay for Four Salmon Species Inhabiting the Yangyangnamdae River Using Environmental DNA

A species-specific quantitative PCR (qPCR) assay using environmental DNA (eDNA) is a promising tool for both qualitative and quantitative analyses of target species directly from water samples. Despite its reliability, an eDNA-based qPCR assay pipeline has not yet developed to monitor salmon species inhabiting Korean waters, which have been rapidly decreasing. We designed species-specific primers for four Oncorhynchus species inhabiting the eastern coastal waters along the Korean Peninsula. These include primers for two native species (Oncorhynchus keta and O. masou) and two that were introduced (O. mykiss and O. kisutch). The limit of detection and limit of quantification for the four qPCR assays ranged from 4.11 to 10.38 copies and from 30 to 81 copies, respectively, indicating a high sensitivity and specificity across all four species. Following optimization, the qPCR assays were used for the quantitative analyses of the four Oncorhynchus species in the Yangyangnamdae River during the spawning and non-spawning seasons in the year 2019–2020, one of the main rivers where salmon migrate during the spawning season in Korea. The raw copy numbers in all of the examined samples were normalized by PCR inhibition rates to standardize and compare with other studies. Among the four Oncorhynchus species examined, the eDNA concentration of O. keta increased significantly (63.60-fold, p < 0.0001) during the spawning season (November) compared with that in the non-spawning season (March), suggesting that O. keta is the main salmon species migrating through the Yangyangnamdae River. In contrast, we did not detect any differences in eDNA concentration for the other three Oncorhynchus species between the spawning and non-spawning seasons, indicating that their presence does not alter during the year. Their eDNA concentration is also relatively low compared to O. keta, which suggests that small numbers of these three species are present in the river. Overall, these newly developed qPCR assays represent useful monitoring tools for the management of four salmon species in Korean waters.

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Environmental DNA Based Surveillance for the Highly Invasive Carpet Sea Squirt Didemnum vexillum: A Targeted Single-Species Approach

Frontiers in Marine Science ◽

10.3389/fmars.2021.728456 ◽

2021 ◽

Vol 8 ◽

Author(s):

Iveta Matejusova ◽

Jennifer Graham ◽

Fiona Bland ◽

Jean-Pierre Lacaze ◽

Guillaume Herman ◽

...

Keyword(s):

Water Samples ◽

Native Species ◽

Limit Of Detection ◽

Sampling Site ◽

Monitoring Program ◽

Environmental Dna ◽

Qpcr Assay ◽

Didemnum Vexillum ◽

Sampling Points ◽

The Impact

The presence and diversity of marine non-native species, the number of new invasions, and the impact on native communities and habitats are important metrics used to assess the health of marine ecosystems. Monitoring for marine non-native species, using traditional approaches such as rapid assessment surveys (RASs), requires taxonomic expertise and may still fail to detect rare or inconspicuous species. This study reports a validation process for a quantitative PCR (qPCR) assay based on the cytochrome oxidase 1 gene, designed to detect highly invasive tunicate Didemnum vexillum by targeting environmental DNA (eDNA) present in water samples. The D. vexillum qPCR assay showed high sensitivity, with the threshold limit of detection (LOD) and modeled LOD3 (based on triplicate qPCR reactions) estimated as 9.187 and 1.117 copies reaction–1, respectively and the limit of quantification (LOQ) was calculated as 18 copies reaction–1. Analyses of water samples collected from selected Pacific oyster farms and recreational marinas in Scotland showed 100% concordance between the historical data on presence of D. vexillum from RASs and detection of D. vexillum eDNA. Consistency of detection of D. vexillum eDNA among different sampling points within each infected sampling site varied, ranging between 100% positive throughout the site to some sampling points testing “negative” or only as “suspected” for D. vexillum. Sites with lower within-site detection consistency included sites with a low density of D. vexillum as reported by RASs or were sites undergoing D. vexillum management. The present pilot monitoring program demonstrates the potential to generate important data on presence of D. vexillum. This program will be scaled up across large geographic regions and used in the first instance to focus and target the traditional RASs to D. vexillum eDNA-positive sites in a cost-effective way, with an aim to verify the species presence by visual observation and direct Sanger sequencing of positive qPCR products.

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Development of a quantitative PCR assay for detection of redside shiner (Richardsonius balteatus) from environmental DNA

BMC Research Notes ◽

10.1186/s13104-019-4819-6 ◽

2019 ◽

Vol 12 (1) ◽

Author(s):

Marshal S. Hoy ◽

Carl O. Ostberg

Keyword(s):

Quantitative Pcr ◽

Water Samples ◽

Effective Means ◽

Environmental Dna ◽

Sympatric Species ◽

Negative Control ◽

Control Site ◽

Qpcr Assay ◽

Redside Shiner ◽

Richardsonius Balteatus

Abstract Objective A quantitative PCR (qPCR) assay for the detection of redside shiner (Richardsonius balteatus) environmental DNA (eDNA) was designed as a side product of a larger project aimed at using eDNA to determine the presence and geographic extent of native and non-native fishes in the reservoirs and associated tributaries above the three mainstem dams (Ross, Diablo, Gorge) on the Skagit River, Washington, USA. The eDNA survey results can be used to help guide additional sampling efforts that include traditional sampling methods, such as electrofishing and netting. Results The redside shiner qPCR assay (RSSCOI_540-601) was validated by testing for sensitivity using redside shiner genomic DNA from three different populations and by testing for specificity against 30 potentially sympatric species. No non-target amplification was observed in our validation tests. We then evaluated the assay on field-collected water samples where there are known populations of redside shiner and a negative control site where the target species is known to be absent. The field-collected water samples tested positive at the redside shiner sites and tested negative at the negative control site. The assay could provide resource managers with an effective means for surveying and monitoring redside shiner populations.

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Improving reliability in environmental DNA detection surveys through enhanced quality control

Marine and Freshwater Research ◽

10.1071/mf15349 ◽

2017 ◽

Vol 68 (2) ◽

pp. 388 ◽

Cited By ~ 15

Author(s):

Elise M. Furlan ◽

Dianne Gleeson

Keyword(s):

Quality Control ◽

Native Species ◽

Conservation Management ◽

Environmental Dna ◽

Positive Control ◽

False Negatives ◽

Method Error ◽

Target Dna ◽

Species Specific ◽

Australian Freshwater

Species-specific environmental DNA (eDNA) surveys are increasingly being used to infer species presence in an environment. Current inadequacies in quality control increase concern for false negatives, which can have serious ramifications for both the management of invasive species and the conservation of native species. eDNA surveys involve a multi-step process to sample, capture, extract and amplify target DNA from the environment. We outline various positive control options and show that many of the commonly used controls are capable of detecting false negatives arising during the amplification stage only. We suggest a secondary, generic primer, designed to co-amplify endogenous DNA sampled during species-specific eDNA surveys, constitutes a superior positive control to monitor method success throughout all stages of eDNA analysis. We develop a species-specific European carp (Cyprinus carpio) assay and a generic fish assay for use as an endogenous control for eDNA surveys in Australian freshwater systems where fish are known to be abundant. We use these assays in a multiplex on eDNA samples that are simultaneously sampled, captured, extracted and amplified. This positive control allows us to distinguish method error from informative non-amplification results, improving reliability in eDNA surveys, which will ultimately lead to better informed conservation management decisions.

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The use of environmental DNA metabarcoding and quantitative PCR for molecular detection of marine invasive non-native species associated with artificial structures

Biological Invasions ◽

10.1007/s10530-021-02672-8 ◽

2021 ◽

Author(s):

Laura M. Gargan ◽

Paul R. Brooks ◽

Siobhan R. Vye ◽

Joseph E. Ironside ◽

Stuart R. Jenkins ◽

...

Keyword(s):

Quantitative Pcr ◽

Native Species ◽

Molecular Detection ◽

Environmental Dna ◽

Artificial Structures ◽

Dna Metabarcoding

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Application of environmental DNA for monitoring and management of aquatic biological invasions: Emerging trends and advancements towards best practice

ARPHA Conference Abstracts ◽

10.3897/aca.4.e65476 ◽

2021 ◽

Vol 4 ◽

Author(s):

Emily Chen

Keyword(s):

Invasive Species ◽

Experimental Design ◽

Native Species ◽

Best Practice ◽

Environmental Dna ◽

Extraction Methods ◽

Indigenous Species ◽

Recent Emergence ◽

Species Specific ◽

Sampling Procedures

Introduction Aquatic Invasive Species (AIS) are a growing concern for global biodiversity as humans continue to accelerate the transport of non-indigenous species beyond their natural range. These species may possess traits that allow them to thrive in new environmental conditions such as non-selective feeding and high reproductive output, causing ecological harm through competition with native species for limited local resources. Consequently, environmental DNA (eDNA) has come to the forefront of AIS management in recent years as a promising method to detect or monitor invasive species using rapid and non-invasive sampling to complement traditional surveying. As eDNA’s potential is explored and beginning to be adopted for a variety of applications around the world, it is increasingly important to synthesize the trends in field and laboratory protocols from different working groups to establish guidelines that will allow greater comparability between studies and improve experimental design. Methodology and Results This meta-analytic study collated and reviewed information from previously published eDNA studies that targeted AIS in freshwater and marine environments to recognize current patterns in sampling techniques, laboratory protocols, and potential geographic or taxonomic biases. A total of 492 records from 192 full-text articles were used in the analysis, composed of 408 species-specific and 84 metabarcoding records. With regards to sampling procedures, many studies were not explicit enough for true replicability, lacking critical information such as the volume of filtered water and details of storage conditions. There was no observable trend for eDNA extraction methods in either species-specific or metabarcoding approaches, with choice of extraction method being mostly arbitrary among laboratories as well as influenced by the recent emergence of dedicated commercial kits . Discussion This analysis revealed a wide variety of choices for collecting and processing eDNA samples, so it is recommended that there should be some sort of standardized workflow diagram or decision tree for every stage of the experimental design in order for researchers to determine what approaches best meet their research objectives. There is also a clear need for improving metadata reporting guidelines; although the relevance of some criteria depends on the goals and limitations of specific projects, there should be a standardized minimum set of parameters to be reported for each eDNA study, from environmental variables to decontamination practices to PCR conditions. This will increase consistency and transparency through all stages of eDNA research, which is key to collectively improving methodologies and moving forward in this field.

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A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Molecular Ecology Resources ◽

10.1111/1755-0998.13055 ◽

2019 ◽

Vol 19 (6) ◽

pp. 1407-1419 ◽

Cited By ~ 18

Author(s):

Susanna A. Wood ◽

Xavier Pochon ◽

Olivier Laroche ◽

Ulla Ammon ◽

Janet Adamson ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Quantitative Pcr ◽

Environmental Dna ◽

Specific Detection ◽

Chain Reaction ◽

Polymerase Chain ◽

Species Specific ◽

Digital Polymerase Chain Reaction

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Quantitative PCR Assay To MeasureAspergillus fumigatus Burden in a Murine Model of Disseminated Aspergillosis: Demonstration of Efficacy of Caspofungin Acetate

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.12.3474-3481.2001 ◽

2001 ◽

Vol 45 (12) ◽

pp. 3474-3481 ◽

Cited By ~ 144

Author(s):

J. C. Bowman ◽

G. K. Abruzzo ◽

J. W. Anderson ◽

A. M. Flattery ◽

C. J. Gill ◽

...

Keyword(s):

Disease Progression ◽

Quantitative Pcr ◽

Murine Model ◽

Dynamic Range ◽

Limit Of Detection ◽

Qpcr Assay ◽

Taqman Assay ◽

Poor Correlation ◽

Fungal Load ◽

Disseminated Aspergillosis

ABSTRACT Caspofungin acetate (MK-0991) is an antifungal antibiotic that inhibits the synthesis of 1,3-β-d-glucan, an essential component of the cell wall of several pathogenic fungi. Caspofungin acetate was recently approved for the treatment of invasive aspergillosis in patients who are refractory to or intolerant of other therapies. The activity of 1,3-β-d-glucan synthesis inhibitors against Aspergillus fumigatus has been evaluated in animal models of pulmonary or disseminated disease by using prolongation of survival or reduction in tissue CFU as assay endpoints. Because these methods suffer from limited sensitivity or poor correlation with fungal growth, we have developed a quantitative PCR-based (qPCR) (TaqMan) assay to monitor disease progression and measure drug efficacy. A. fumigatus added to naı̈ve, uninfected kidneys as either ungerminated conidia or small germlings yielded a linear qPCR response over at least 4 orders of magnitude. In a murine model of disseminated aspergillosis, a burden of A. fumigatus was detected in each of five different organs at 4 days postinfection by the qPCR assay, and the mean fungal load in these organs was 1.2 to 3.5 log10 units greater than mean values determined by CFU measurement. When used to monitor disease progression in infected mice, the qPCR assay detected an increase of nearly 4 log10 conidial equivalents/g of kidney between days 1 and 4 following infection, with a peak fungal burden that coincided with the onset of significant mortality. Traditional CFU methodology detected only a marginal increase in fungal load in the same tissues. In contrast, when mice were infected with Candida albicans, which does not form true mycelia in tissues, quantitation of kidney burden by both qPCR and CFU assays was strongly correlated as the infection progressed. Finally, treatment of mice with induced disseminated aspergillosis with either caspofungin or amphotericin B reduced the A. fumigatus burden in infected kidneys to the limit of detection for the qPCR assay. Because of its much larger dynamic range, the qPCR assay is superior to traditional CFU determination for monitoring the progression of disseminated aspergillosis and evaluating the activity of antifungal antibiotics against A. fumigatus.

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Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

Journal of Visualized Experiments ◽

10.3791/61825 ◽

2020 ◽

Author(s):

Katy E. Klymus ◽

Dannise V. Ruiz Ramos ◽

Nathan L. Thompson ◽

Catherine A. Richter

Keyword(s):

Quantitative Pcr ◽

Environmental Dna ◽

Pcr Assays ◽

Species Specific

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A quantitative PCR based environmental DNA assay for detecting Atlantic salmon (Salmo salar L.)

10.1101/226829 ◽

2017 ◽

Author(s):

Siobhán Atkinson ◽

Jeannette E.L. Carlsson ◽

Bernard Ball ◽

Damian Egan ◽

Mary Kelly-Quinn ◽

...

Keyword(s):

Atlantic Salmon ◽

Quantitative Pcr ◽

Salmo Salar ◽

Environmental Dna ◽

Coi Gene ◽

List Type ◽

The Pacific ◽

Invasive Method ◽

Species Specific ◽

A Minor

AbstractThe Atlantic salmon (Salmo salar L.) has worldwide ecological, cultural and economic importance. The species has undergone extensive decline across its native range, yet concerns have been raised about its invasive potential in the Pacific. Knowledge on the distribution of this species is vital for addressing conservation goals.This study presents an eDNA assay to detect S. salar in water samples, using quantitative PCR (qPCR) technology. Species-specific primers and a minor groove binding (MGB) probe were designed for the assay, based on the mitochondrial cytochrome oxidase I (COI) gene.The results of this study indicate that eDNA is a highly sensitive tool for detecting S. salar in situ, and could potentially provide an alternative, non-invasive method for determining the distribution of this species.

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