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.

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 Development of an Internal Control for Evaluation and Standardization of a Quantitative PCR Assay for Detection of Helicobacter pylori in Drinking Water

2007 ◽  
Vol 73 (22) ◽  
pp. 7380-7387 ◽  
Author(s):  
Keya Sen ◽  
Nancy A. Schable ◽  
Dennis J. Lye
Keyword(s):  
Helicobacter Pylori ◽  
Drinking Water ◽  
Quantitative Pcr ◽  
Water Samples ◽  
Sodium Thiosulfate ◽  
Qpcr Assay ◽  
E Coli ◽  
H Pylori ◽  
Labeled Probe ◽  
Treated Drinking Water

ABSTRACT Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.

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

Biology ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 899
Author(s):  
Muhammad Hilman Fu'adil Amin ◽  
Ji-Hyun Lee ◽  
Ah Ran Kim ◽  
Ju-Kyoung Kim ◽  
Chung-Il Lee ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Native Species ◽  
Limit Of Detection ◽  
Environmental Dna ◽  
Qpcr Assay ◽  
Spawning Season ◽  
Oncorhynchus Keta ◽  
Korean Waters ◽  
Quantitative Analyses ◽  
Species Specific

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.

scholarly journals Quantitative PCR assays to detect whales, rockfish, and common murre environmental DNA in marine water samples of the Northeastern Pacific

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0242689
Author(s):  
Elizabeth A. Andruszkiewicz ◽  
Kevan M. Yamahara ◽  
Collin J. Closek ◽  
Alexandria B. Boehm
Keyword(s):  
Quantitative Pcr ◽  
Water Samples ◽  
Environmental Dna ◽  
Humpback Whale ◽  
Environmental Water ◽  
Individual Species ◽  
Common Murre ◽  
Assay Design

Monitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. To obtain quantitative eDNA datasets for individual species, organism-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre, genus-specific for the shortbelly rockfish assay, and broadly whale-specific for the humpback whale assay, which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples.

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 Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

2014 ◽  
Vol 2014 ◽  
pp. 1-8
Author(s):  
Pricila da Silva Cunha ◽  
Heloisa B. Pena ◽  
Carla Sustek D’Angelo ◽  
Celia P. Koiffmann ◽  
Jill A. Rosenfeld ◽  
...  
Keyword(s):  
Real Time ◽  
Diagnostic Test ◽  
Quantitative Pcr ◽  
Target Genes ◽  
Cost Effective ◽  
Qpcr Assay ◽  
Deletion Syndrome ◽  
Comparative Genomic ◽  
Real Time Quantitative Pcr ◽  
Subtelomeric Deletion

Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36:PRKCZandSKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescentin situhybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR ofPRKCZandSKIis a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

VARIATION IN ANAL FIN RAY COUNT OF THE REDSIDE SHINER RICHARDSONIUS BALTEATUS (RICHARDSON)

1953 ◽  
Vol 31 (3) ◽  
pp. 211-225 ◽  
Author(s):  
C. C. Lindsey
Keyword(s):  
Morphological Characteristics ◽  
Introduced Populations ◽  
Fin Ray ◽  
Parent Stock ◽  
Different Populations ◽  
The Mean ◽  
Redside Shiner ◽  
Richardsonius Balteatus ◽  
Bodies Of Water ◽  
Different Parts

Anal fin rays were counted on 4766 specimens of Richardsonius balteatus from 61 localities in British Columbia. Individual counts varied from 10 to 21, and mean counts of different populations varied from 12.06 to 17.51. Significant differences in counts occurred between different bodies of water, between recently introduced populations and their parent stock, between different parts of the same lake, and between different year classes. Ray counts tended to be higher amongst females in populations with high over-all means, and higher amongst males in populations with low over-all means. A positive correlation was demonstrated between water temperatures recorded in the vicinity of developing fry and the mean numbers of anal rays produced. Within each latitudinal zone a similar correlation occurred between mean ray count and average air temperature during the spawning season, but data on 109 means of populations in U.S.A. and Canada indicated a tendency, probably genetic, towards production, at equivalent temperature, of higher ray count towards the northern end of the range. Loose correlations between anal ray count and certain other morphological characteristics suggest that these may be dependent on more or less common environmental factors but are not linked by direct causality.

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