scholarly journals A workflow for the relative quantification of multiple fish species from oceanic water samples using environmental DNA (eDNA) to support large-scale fishery surveys

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257773
Author(s):  
Ana Ramón-Laca ◽  
Abigail Wells ◽  
Linda Park
Keyword(s):  
Dna Extraction ◽  
Marine Fish ◽  
Fish Species ◽  
Water Samples ◽  
Large Scale ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
The United States ◽  
Qpcr Assay ◽  
Multiplex Qpcr

While the number of published marine studies using environmental DNA (eDNA) has increased substantially in recent years, marine fish surveys are still scarce. To examine the potential for eDNA to support marine fisheries monitoring surveys, we optimized an eDNA isolation method, developed a multispecies assay and tested it on eDNA samples collected along the Pacific coast of the United States. Four commercial DNA extraction kits that exploit the capability of the nucleic acids binding a solid phase (two using a silica matrix and two magnetic beads) as well an organic separation method were tested. A species-specific multiplex qPCR assay was developed and tested to simultaneously target Pacific hake (Merluccius productus), Pacific lamprey (Entosphenus tridentatus) and eulachon (Thaleichthys pacificus). The specificity of the assay was tested in silico, in vitro and in natura. Environmental DNA isolation using phenol:chloroform:isoamyl purification with a phase lock was optimized and yielded the highest amount of total and target DNA and was used to extract 46 marine water samples for the detection of the three species of interest. The multiplex qPCR assay used in the quantification process was also optimized to provide convenience and accuracy. Pacific hake was present in 44% of the eDNA samples while the other two species were absent. Here, we present a complete workflow for the simultaneous detection and quantification of multiple marine fish species using eDNA. This workflow supports large-scale at-sea sampling efforts with preservation at ambient temperatures and has demonstrated DNA extraction efficiency and reliability. The multiplex qPCR assay is shown to be sensitive and specific for the purposes of simultaneously monitoring the relative abundance of multiple targeted fish species.

scholarly journals A Simple Technique for the Identification of Environmental DNA (eDNA ) in the Water Samples

2021 ◽  
pp. 77-80
Author(s):  
Chitra K. Y.
Keyword(s):  
Water Samples ◽  
Large Scale ◽  
Dna Isolation ◽  
Simple Technique ◽  
Environmental Dna ◽  
Water Bodies ◽  
Easy Method ◽  
Selective Staining ◽  
Generation Sequencing ◽  
Dark Pink

The environmental DNA(eDNA) is the DNA that is shed by the organisms in their environment by different ways viz. , mucous, faeces, skin, eggs, sperms and also when these organisms die due to natural death or disease. The eDNA will persist for several days. Identification of eDNA is a useful method of determining the organisms present in an aquatic environment like amphibians, reptiles, fishes , insects and larval forms of some of these organisms. By analysing the e-DNA it is possible to monitor the species distribution in water bodies like lakes and ponds simply by collecting a sample of water. The technique can be applied for the survey of the water bodies on a large scale for the genomic, taxonomic as well as pollutional studies. The DNA isolation procedures that are available are laborious and time consuming. Therefore, during the present study, a simplified method was devised i. e. , isolation of eDNA with ethanol after which Feulgen stain was applied to identify and confirm it, as it is an easy method before proceeding to work with the isolated eDNA using other techniqnies for further studies. The Feulgen method is used for the selective staining and the localisation of the DNA in the tissues but is adopted during the present study for the water samples for quick identification of eDNA. The smear of eDNA stained with Feulgen showed dark pink or magenta colour under the microscope where it was concentrated but stained lightly when dispersed and fragmented as observed in the present study. Further studies of the isolated eDNA are in progress in our laboratory for quantifying and sequencing eDNA using latest techniques like next generation sequencing for the identification of fish species in the lakes.

scholarly journals Environmental DNA (eDNA) metabarcoding surveys extend the range of invasion for non-indigenous freshwater species in Eastern Europe.

2021 ◽  
Author(s):  
Gert-Jan Jeunen ◽  
Tatsiana Lipinskaya ◽  
Helen Gajduchenko ◽  
Viktoriya Golovenchik ◽  
Michail Moroz ◽  
...  
Keyword(s):  
Surface Water ◽  
Water Samples ◽  
False Negative ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Reference Database ◽  
Indigenous Species ◽  
Freshwater Species ◽  
Traditional Approaches ◽  
Surface Water Samples

Active environmental DNA (eDNA) surveillance through species-specific amplification has shown increased sensitivity in the detection of non-indigenous species (NIS) compared to traditional approaches. When many NIS are of interest, however, active surveillance decreases in cost- and time-efficiency. Passive surveillance through eDNA metabarcoding takes advantage of the complex DNA signal in environmental samples and facilitates the simultaneous detection of multiple species. While passive eDNA surveillance has previously detected NIS, comparative studies are essential to determine the ability of eDNA metabarcoding to accurately describe the range of invasion for multiple NIS versus alternative approaches. Here, we surveyed twelve sites, covering nine rivers across Belarus for NIS with three different techniques, i.e., an ichthyological, hydrobiological, and eDNA survey, whereby DNA was extracted from 500 mL surface water samples and amplified with two 16S rRNA primer assays targeting the fish and macro-invertebrate biodiversity. Nine non-indigenous fish and ten non-indigenous sediment-living macro-invertebrates were detected by traditional surveys, while seven NIS eDNA signals were picked up, including four fish, one aquatic and two sediment-living macro-invertebrates. Passive eDNA surveillance extended the range of invasion further north for two invasive fish and identified a new NIS for Belarus, the freshwater jellyfish Craspedacusta sowerbii. False-negative detections for the eDNA survey could be attributed to (i) preferential amplification of aquatic over sediment-living macro-invertebrates from surface water samples and (ii) an incomplete reference database. The evidence provided in this study recommends the implementation of both molecular-based and traditional approaches to maximize the probability of early detection of non-native organisms.

scholarly journals Environmental DNA (eDNA) Metabarcoding in the Fish Market and Nearby Seafood Restaurants in Taiwan Reveals the Underestimation of Fish Species Diversity in Seafood

Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1132
Author(s):  
Hung-Tai Lee ◽  
Cheng-Hsin Liao ◽  
Te-Hua Hsu
Keyword(s):  
Species Diversity ◽  
Species Identification ◽  
Fish Species ◽  
Large Scale ◽  
Environmental Dna ◽  
Universal Primers ◽  
Similar Species ◽  
Operational Taxonomic Units ◽  
Fish Market ◽  
Fish Species Identification

Seafood, especially in traditional food Taiwan, is rarely sourced from a fixed species and routinely from similar species depending on their availability. Hence, the species composition of seafood can be complicated. While a DNA-based approach has been routinely utilized for species identification, a large scale of seafood identification in fish markets and restaurants could be challenging (e.g., elevated cost and time-consuming only for a limited number of species identification). In the present study, we aimed to identify the majority of fish species potentially consumed in fish markets and nearby seafood restaurants using environmental DNA (eDNA) metabarcoding. Four eDNA samplings from a local fish market and nearby seafood restaurants were conducted using Sterivex cartridges. Nineteen universal primers previously validated for fish species identification were utilized to amplify the fragments of mitochondrial DNA (12S, COI, ND5) of species in eDNA samples and sequenced with NovaSeq 6000 sequencing. A total of 153 fish species have been identified based on 417 fish related operational taxonomic units (OTUs) generated from 50,534,995 reads. Principal Coordinate Analysis (PCoA) further showed the differences in fish species between the sampling times and sampling sites. Of these fish species, 22 chondrichthyan fish, 14 Anguilliformes species, and 15 Serranidae species were respectively associated with smoked sharks, braised moray eels, and grouper fish soups. To our best knowledge, this work represents the first study to demonstrate the feasibility of a large scale of seafood identification using eDNA metabarcoding approach. Our findings also imply the species diversity in traditional seafood might be seriously underestimated and crucial for the conservation and management of marine resources.

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

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.

Evaluation of alternative DNA extraction processes and real-time PCR for detecting Cryptosporidium parvum in drinking water

2015 ◽  
Vol 15 (6) ◽  
pp. 1295-1303
Author(s):  
Gina H. Kimble ◽  
Vincent R. Hill ◽  
James E. Amburgey
Keyword(s):  
Drinking Water ◽  
Real Time ◽  
Dna Extraction ◽  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Real Time Pcr ◽  
Extraction Procedure ◽  
The United States ◽  
Detection Sensitivity ◽  
Spin Column

USEPA Method 1623 is the standard method in the United States for the detection of Cryptosporidium in water samples, but quantitative real-time polymerase chain reaction (qPCR) is an alternative technique that has been successfully used to detect Cryptosporidium in aqueous matrices. This study examined various modifications to a commercial nucleic acid extraction procedure in order to enhance PCR detection sensitivity for Cryptosporidium. An alternative DNA extraction buffer allowed for qPCR detection at lower seed levels than a commercial extraction kit buffer. In addition, the use of a second spin column cycle produced significantly better detection (P = 0.031), and the volume of Tris–EDTA buffer significantly affected crossing threshold values (P = 0.001). The improved extraction procedure was evaluated using 10 L of tap water samples processed by ultrafiltration, centrifugation and immunomagnetic separation. Mean recovery for the sample processing method was determined to be 41% using microscopy and 49% by real-time PCR (P = 0.013). The results of this study demonstrate that real-time PCR can be an effective alternative for detecting and quantifying Cryptosporidium parvum in drinking water samples.

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

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.

scholarly journals Multiplex qPCR Assay for Direct Detection and Quantification of Colletotrichum truncatum, Corynespora cassiicola, and Sclerotinia sclerotiorum in Soybean Seeds

Plant Disease ◽  
2020 ◽  
Vol 104 (11) ◽  
pp. 3002-3009
Author(s):  
Maísa Ciampi-Guillardi ◽  
Juliana Ramiro ◽  
Maria Heloisa Duarte de Moraes ◽  
Marina Coan Goldoni Barbieri ◽  
Nelson S. Massola
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Internal Control ◽  
Direct Detection ◽  
Simultaneous Detection ◽  
Plant Diseases ◽  
Qpcr Assay ◽  
Soybean Seeds ◽  
Corynespora Cassiicola ◽  
Colletotrichum Truncatum ◽  
Multiplex Qpcr

Precise diagnosis of plant diseases is one of the most effective tools to minimize yield losses. Colletotrichum truncatum, Corynespora cassiicola, and Sclerotinia sclerotiorum are common soilborne pathogens that affect soybeans all over the world. We developed a multiplex quantitative real-time polymerase chain reaction (qPCR) assay to simultaneously detect and quantify the three pathogens in soybean seeds and to survey their occurrence in the main soybean production areas in Brazil. Species-specific primers and probes for C. truncatum and C. cassiicola were designed based on GAPDH and TEF1 genes, respectively, to be combined with qPCR detection of S. sclerotiorum previously reported. The multiplex qPCR assay was successful in the simultaneous detection of C. truncatum, C. cassiicola, and S. sclerotiorum, along with a host internal control. The four pathogens were detected and quantified in artificially and naturally infested soybean seeds, even in the lowest incidence level tested of 0.0625% or 1 infected seed out of 1,599 healthy ones. From 81 seed samples tested, C. truncatum was the most frequently detected pathogen and with higher incidence levels (0.25 to 0.125%), followed by S. sclerotiorum and C. cassiicola, both with lower incidence levels (0.125 to 0.0625%). Together, the results evidenced the high sensitivity of the multiplex qPCR assay, indicating its usefulness for a quick and reliable diagnosis of soybean diseases in seeds.

scholarly journals Next Day Legionella PCR: a highly reliable negative screen for Legionella in the built environment

2020 ◽  
Vol 18 (3) ◽  
pp. 345-357
Author(s):  
Katherine E. Fisher ◽  
Leah P. Wickenberg ◽  
Lesley F. Leonidas ◽  
Anna A. Ranz ◽  
Michelle A. Habib ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Water Samples ◽  
Traditional Culture ◽  
Culture Method ◽  
The United States ◽  
Extraction Methods ◽  
Waterborne Pathogen ◽  
Legionnaires Disease ◽  
Dna Extraction Methods ◽  
Validation Testing

Abstract The opportunistic, waterborne pathogen Legionella caused 9,933 cases of Legionnaires' disease in 2018 in the United States (CDC.gov). The incidence of Legionnaires' disease can be reduced by maintaining clean building water systems through water management programs (WMPs). WMPs often include validation testing to confirm the control of bacteria, but the traditional culture method for enumerating Legionella requires 10–14 days to obtain results. A rapid DNA extraction developed by Phigenics and a real-time PCR negative screen for the genus Legionella provided results the day after sampling. This study evaluated the Next Day Legionella PCR (Phigenics, LLC) compared with the traditional culture method (ISO 11731) on 11,125 building water samples for approximately 1 year. Two DNA extraction methods (Methods 1 and 2) were compared. The negative predictive value (NPV) of the Next Day Legionella PCR in comparison to traditional culture for Method 1 was 99.95%, 99.92%, 99.85%, and 99.17% at >10, >2, >1, and >0.1 CFU/ml limits of detection, respectively. The improved DNA extraction (Method 2) increased the NPV to 100% and 99.88% at >1 and >0.1 CFU/ml, respectively. These results demonstrate the reliability of the genus-level Legionella PCR negative screen to predict culture-negative water samples.

scholarly journals Effects of large-scale poultry farms on aquatic microbial communities: A molecular investigation

2006 ◽  
Vol 4 (1) ◽  
pp. 77-86 ◽  
Author(s):  
Joseph A. Ringbauer ◽  
Joseph B. James ◽  
Fred J. Genthner
Keyword(s):  
Water Samples ◽  
Bacterial Communities ◽  
Pathogenic Bacteria ◽  
Large Scale ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Late Summer ◽  
The United States ◽  
Sampling Location ◽  
Poultry Production ◽  
Poultry Farms

The effects of large-scale poultry production operations on water quality and human health are largely unknown. Poultry litter is frequently applied as fertilizer to agricultural lands adjacent to large poultry farms. Run-off from the land introduces a variety of stressors into the surface waters including nutrients, antimicrobials and pathogenic bacteria. The Delaware, Maryland and Virginia (Delmarva) Peninsula has the highest concentration of broiler chickens per farm acre in the United States and provides an ideal location for studying the effects of stressors from poultry farms. We investigated potential effects by characterizing shifts in the structure of aquatic bacterial communities. DNA was isolated from microorganisms in water samples from streams and rivers at varying distances from, or having different frequencies of, litter applications. Fingerprints of 16S rDNA amplicons from bacteria in water samples collected during late summer 2001 to late spring 2002 were produced by denaturing gradient gel electrophoresis (DGGE). A statistical analysis of multiple fingerprints from each sampling location demonstrated that each site harboured a bacterial community significantly different from the communities at other sites. Similarly, the bacterial communities from each sampling time differed significantly from communities at other sampling times. Most importantly, a competitive, library-based analysis showed time of sampling (month) had a greater effect on community structure than did location.

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