For Publishers
DiscoveryMetadataPeer reviewHostingPublishing
For Researchers
JoinPublishReviewCollect
Register
Blog
About
Search
Advanced search
My ScienceOpen
Search
For Publishers
For Researchers
Blog
About
4
views
4
references
 Top references
cited by
2
    
0 reviews
 Review
0
comments
 Comment
0
recommends
+1 Recommend
0 collections
    0
    shares
      436
      similar
       All similar
      • Record: found
      • Abstract: found
      • Article: not found

      Quantitative PCR Assays for Detecting Loach Minnow ( Rhinichthys cobitis) and Spikedace ( Meda fulgida) in the Southwestern United States

      research-article

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Loach minnow ( Rhinichthys cobitis) and spikedace ( Meda fulgida) are legally protected with the status of Endangered under the U.S. Endangered Species Act and are endemic to the Gila River basin of Arizona and New Mexico. Efficient and sensitive methods for monitoring these species’ distributions are critical for prioritizing conservation efforts. We developed quantitative PCR assays for detecting loach minnow and spikedace DNA in environmental samples. Each assay reliably detected low concentrations of target DNA without detection of non-target species, including other cyprinid fishes with which they co-occur.

          Related collections

          Most cited references4

          • Record: found
          • Abstract: found
          • Article: found
          Is Open Access

          Robust Detection of Rare Species Using Environmental DNA: The Importance of Primer Specificity

          Taylor Wilcox, Kevin S. McKelvey, Michael K. Young … (2013)
          Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design.
          Article 0 comments Cited 181 times – based on 0 reviews     Review now
            Bookmark
            • Record: found
            • Abstract: found
            • Article: found
            Is Open Access

            The Dual Challenges of Generality and Specificity When Developing Environmental DNA Markers for Species and Subspecies of Oncorhynchus

            Taylor Wilcox, Kellie Carim, Kevin S. McKelvey … (2015)
            Environmental DNA (eDNA) sampling is a powerful tool for detecting invasive and native aquatic species. Often, species of conservation interest co-occur with other, closely related taxa. Here, we developed qPCR (quantitative PCR) markers which distinguish westslope cutthroat trout (Oncorhynchus clarkii lewsi), Yellowstone cutthroat trout (O. clarkii bouvieri), and rainbow trout (O. mykiss), which are of conservation interest both as native species and as invasive species across each other’s native ranges. We found that local polymorphisms within westslope cutthroat trout and rainbow trout posed a challenge to designing assays that are generally applicable across the range of these widely-distributed species. Further, poorly-resolved taxonomies of Yellowstone cutthroat trout and Bonneville cutthroat trout (O. c. utah) prevented design of an assay that distinguishes these recognized taxa. The issues of intraspecific polymorphism and unresolved taxonomy for eDNA assay design addressed in this study are likely to be general problems for closely-related taxa. Prior to field application, we recommend that future studies sample populations and test assays more broadly than has been typical of published eDNA assays to date.
            Article 0 comments Cited 41 times – based on 0 reviews     Review now
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Exploiting extension bias in polymerase chain reaction to improve primer specificity in ensembles of nearly identical DNA templates.

              Erik Wright, L. Safak Yilmaz, Sri Ram … (2014)
              We describe a semi-empirical framework that combines thermodynamic models of primer hybridization with experimentally determined elongation biases introduced by 3'-end mismatches for improving polymerase chain reaction (PCR)-based sequence discrimination. The framework enables rational and automatic design of primers for optimal targeting of one or more sequences in ensembles of nearly identical DNA templates. In situations where optimal targeting is not feasible, the framework accurately predicts non-target sequences that are difficult to distinguish with PCR alone. Based on the synergistic effects of disparate sources of PCR bias, we used our framework to robustly distinguish between two alleles that differ by a single base pair. To demonstrate the applicability to environmental microbiology, we designed primers specific to all recognized archaeal and bacterial genera in the Ribosomal Database Project, and have made these primers available online. We applied these primers experimentally to obtain genus-specific amplification of 16S rRNA genes representing minor constituents of an environmental DNA sample. Our results demonstrate that inherent PCR biases can be reliably employed in an automatic fashion to maximize sequence discrimination and accurately identify potential cross-amplifications. We have made our framework accessible online as a programme for designing primers targeting one group of sequences in a set with many other sequences (http://DECIPHER.cee.wisc.edu).
              Article 0 comments Cited 29 times – based on 0 reviews     Review now
                Bookmark
                All references

                Author and article information

                Contributors
                Michael Hofreiter: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, CA USA )
                ISSN (Electronic): 1932-6203
                Publication date (Electronic): 1 September 2016
                Publication date Collection: 2016
                Volume: 11
                Issue: 9
                Electronic Location Identifier: e0162200
                Affiliations
                [1 ]United States Department of Agriculture, Forest Service, National Genomics Center for Wildlife and Fish Conservation, Rocky Mountain Research Station, Missoula, MT, United States of America
                [2 ]United States Department of Agriculture, Forest Service, Southwestern Region, Albuquerque, NM, United States of America
                University of York, UNITED KINGDOM
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: KJC YMP KSM MKY MKS.

                • Data curation: JCD.

                • Formal analysis: JCD.

                • Funding acquisition: YMP.

                • Investigation: JCD KJC YMP.

                • Methodology: JCD KJC YMP KSM MKY MKS.

                • Project administration: JCD KJC.

                • Resources: YMP MKS.

                • Supervision: JCD KJC KSM MKY MKS.

                • Validation: JCD.

                • Visualization: JCD KJC KSM MKY.

                • Writing – original draft: JCD KJC.

                • Writing – review & editing: JCD KJC YMP KSM MKY MKS.

                Author information
                http://orcid.org/0000-0002-6790-7841
                Article
                Publisher ID: PONE-D-16-16931
                DOI: 10.1371/journal.pone.0162200
                PMC ID: 5008727
                PubMed ID: 27583576
                SO-VID: 9ad47a70-2ffa-4647-849f-57be47b79157
                License:

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                Date received : 27 April 2016
                Date accepted : 18 August 2016
                Page count
                Figures: 0, Tables: 4, Pages: 7
                Funding
                Funded by: USDA Forest Service Southwestern Region
                Award Recipient :
                This work was completed through funding issued to Yvette M Paroz by the USDA Forest Service Southwestern Region with in-kind support from the USDA Forest Service Region 1. As the authors of this study, the funders did have a role in all aspects of the study and manuscript preparation.
                Categories
                Subject: Research Article
                Subject: Biology and Life Sciences
                Subject: Organisms
                Subject: Animals
                Subject: Vertebrates
                Subject: Fishes
                Subject: Biology and Life Sciences
                Subject: Molecular Biology
                Subject: Molecular Biology Techniques
                Subject: Artificial Gene Amplification and Extension
                Subject: Polymerase Chain Reaction
                Subject: Research and Analysis Methods
                Subject: Molecular Biology Techniques
                Subject: Artificial Gene Amplification and Extension
                Subject: Polymerase Chain Reaction
                Subject: Ecology and Environmental Sciences
                Subject: Species Colonization
                Subject: Invasive Species
                Subject: Biology and Life Sciences
                Subject: Ecology
                Subject: Ecosystems
                Subject: Deserts
                Subject: Ecology and Environmental Sciences
                Subject: Ecology
                Subject: Ecosystems
                Subject: Deserts
                Subject: Ecology and Environmental Sciences
                Subject: Terrestrial Environments
                Subject: Deserts
                Subject: Biology and Life Sciences
                Subject: Conservation Biology
                Subject: Endangered Species
                Subject: Ecology and Environmental Sciences
                Subject: Conservation Science
                Subject: Conservation Biology
                Subject: Endangered Species
                Subject: People and places
                Subject: Geographical locations
                Subject: North America
                Subject: United States
                Subject: Arizona
                Subject: Biology and Life Sciences
                Subject: Molecular Biology
                Subject: Molecular Biology Techniques
                Subject: Sequencing Techniques
                Subject: Sequence Analysis
                Subject: Sequence Alignment
                Subject: Research and Analysis Methods
                Subject: Molecular Biology Techniques
                Subject: Sequencing Techniques
                Subject: Sequence Analysis
                Subject: Sequence Alignment
                Subject: Research and analysis methods
                Subject: Extraction techniques
                Subject: DNA extraction
                Custom metadata
                Data Availability All relevant data are within the paper. All genetic data used in this study were obtained from GenBank and accession numbers have been provided within the paper.

                ScienceOpen disciplines: Uncategorized
                Data availability:
                ScienceOpen disciplines: Uncategorized

                Comments

                Comment on this article

                scite_

                Similar content436

                See all similar

                Cited by2

                See all cited by

                Most referenced authors169

                See all reference authors