scholarly journals Numerous new mitogenomic sequences and multiple new environmental DNA markers for invasive bighead and silver carp (Hypophthalmichthys nobilis and H. molitrix) populations in North America

2014 ◽  
Author(s):  
Richard F. Lance ◽  
Heather L. Farrington ◽  
Christine E. Edwards ◽  
Xin Guan ◽  
Matthew R. Carr ◽  
...  
Keyword(s):  
Genetic Markers ◽  
North American ◽  
Complete Mitochondrial Genome ◽  
Silver Carp ◽  
Environmental Dna ◽  
Invasion Front ◽  
Specificity And Sensitivity ◽  
Introduced Range ◽  
Hypophthalmichthys Nobilis ◽  
Species Specific

Invasive Asian bighead and silver carp (Hypophthalmichthys nobilis and H. molitrix) pose a substantial threat to North American waterways. Recently, environmental DNA (eDNA), the use of species-specific genetic assays to detect the DNA of a particular species in a water sample, has gained recognition as a tool for tracking the invasion front of these species toward the Great Lakes. The goal of this study was to develop new species-specific conventional PCR (cPCR) and quantitative (qPCR) markers for detection of these species in North American waterways. We first generated complete mitochondrial genome sequences from 33 bighead and 29 silver carp individuals collected throughout their introduced range. These sequences were aligned with other common and closely related species to identify potential eDNA markers. We then field-tested these genetic markers for species-specificity and sensitivity in environmental samples. Newly developed markers performed well in field trials, had low false positive rates and had comparable sensitivity compared to current markers. The new markers developed in this study greatly expand the number of species-specific genetic markers available to track the invasion front of bighead and silver carp, and can be used to improve the resolution of these assays. Additionally, the use of the qPCR markers developed in this study may reduce sample processing time and cost of eDNA monitoring for these species.

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 Variation in inhibitor effects on qPCR assays and implications for eDNA surveys

2021 ◽  
Author(s):  
Richard Lance ◽  
Xin Guan
Keyword(s):  
Silver Carp ◽  
Environmental Dna ◽  
Hypophthalmichthys Molitrix ◽  
Leaf Extracts ◽  
Closely Related Species ◽  
Exogenous Dna ◽  
Hypophthalmichthys Nobilis ◽  
Polymerase Chain ◽  
High Concentrations ◽  
Positive Controls

Aquatic environmental DNA (eDNA) surveys are sometimes impacted by polymerase chain reaction (PCR) inhibitors. We tested varying concentrations of different inhibitors (humic, phytic, and tannic acids; crude leaf extracts) for impacts on quantitative PCR (qPCR) assays designed for eDNA surveys of bighead and silver carp (Hypophthalmichthys nobilis and Hypophthalmichthys molitrix). We also tested for inhibition by high concentrations of exogenous DNA, hypothesizing that DNA from increasingly closely related species would be increasingly inhibitory. All tested inhibitors impacted qPCR, though only at very high concentrations — likely a function, in part, of having used an inhibitor-resistant qPCR solution. Closer phylogenetic relatedness resulted in inhibition at lower exogenous DNA concentrations, but not at relatively close phylogenetic scales. Inhibition was also influenced by the qPCR reporter dye used. Importantly, different qPCR assays responded differently to the same inhibitor concentrations. Implications of these results are that the inclusion of more than one assay for the same target taxa in an eDNA survey may be an important countermeasure against false negatives and that internal positive controls may not, in the absence of efforts to maximize inhibition compatibility, provide useful information about the inhibition of an eDNA assay.

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.

Variation in inhibitor effects on qPCR assays and implications for eDNA surveys

2020 ◽  
Vol 77 (1) ◽  
pp. 23-33 ◽  
Author(s):  
Richard F. Lance ◽  
Xin Guan
Keyword(s):  
Silver Carp ◽  
Environmental Dna ◽  
Hypophthalmichthys Molitrix ◽  
Leaf Extracts ◽  
Closely Related Species ◽  
Exogenous Dna ◽  
Hypophthalmichthys Nobilis ◽  
Polymerase Chain ◽  
High Concentrations ◽  
Positive Controls

Aquatic environmental DNA (eDNA) surveys are sometimes impacted by polymerase chain reaction (PCR) inhibitors. We tested varying concentrations of different inhibitors (humic, phytic, and tannic acids; crude leaf extracts) for impacts on quantitative PCR (qPCR) assays designed for eDNA surveys of bighead and silver carp (Hypophthalmichthys nobilis and Hypophthalmichthys molitrix). We also tested for inhibition by high concentrations of exogenous DNA, hypothesizing that DNA from increasingly closely related species would be increasingly inhibitory. All tested inhibitors impacted qPCR, though only at very high concentrations — likely a function, in part, of having used an inhibitor-resistant qPCR solution. Closer phylogenetic relatedness resulted in inhibition at lower exogenous DNA concentrations, but not at relatively close phylogenetic scales. Inhibition was also influenced by the qPCR reporter dye used. Importantly, different qPCR assays responded differently to the same inhibitor concentrations. Implications of these results are that the inclusion of more than one assay for the same target taxa in an eDNA survey may be an important countermeasure against false negatives and that internal positive controls may not, in the absence of efforts to maximize inhibition compatibility, provide useful information about the inhibition of an eDNA assay.

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.

scholarly journals qPCR assays for the detection of two western North American freshwater mussel species, Anodonta nuttalliana and Anodonta oregonensis, from environmental DNA

2018 ◽  
Author(s):  
Torrey W. Rodgers ◽  
Karen E. Mock
Keyword(s):  
North America ◽  
Threatened Species ◽  
Freshwater Mussel ◽  
Environmental Dna ◽  
Western North America ◽  
Mussel Species ◽  
Specificity And Sensitivity ◽  
Pcr Assays ◽  
Species Specific ◽  
Management And Conservation

AbstractWe developed species-specific quantitative PCR assays for the detection of two freshwater mussel species native to the western North America, Anodonta nuttalliana and Anodonta oregonensis, from environmental DNA. These species have experienced dramatic declines over the last century, and are currently threatened in many portions of their range. Improved tools for detecting and monitoring these species are needed. Species-specificity and sensitivity of the assays was empirically tested in the lab, and both assays were also validated with field collected eDNA samples. We found that the assays we designed are species-specific, sensitive, and are effective for detecting Anodonta nuttalliana and Anodonta oregonensis from environmental DNA samples collected from streams and rivers. These assays will aid in the detection, monitoring, management, and conservation of these threatened species.

Identification for adulteration of beef with chicken based on single primer-triggered isothermal amplification

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Jiao Chen ◽  
Pansong Zhang ◽  
Haixia Wang ◽  
Yanjing Shi
Keyword(s):  
Limit Of Detection ◽  
Chicken Meat ◽  
Isothermal Amplification ◽  
Work Flow ◽  
Acid Extraction ◽  
Specificity And Sensitivity ◽  
Total Dna ◽  
Meat Species ◽  
Species Specific ◽  
Single Primer

Abstract Adulteration of beef with cheap chicken has become a growing problem worldwide. In this study, a quick, single primer-triggered isothermal amplification (SAMP) combined with a fast nucleic acid extraction method was employed to detect the chicken meat in adulterated beef. Chicken from adulterated beef was identified using the chicken species-specific primer designed according to the Gallus gallus mitochondrial conserved sequences. Our SAMP method displayed good specificity and sensitivity in detecting chicken and beef meat DNA–the limit of detection (LOD) of SAMP is 0.33 pg/μL of chicken and beef total DNA and 2% w/w chicken meat in beef. The whole work flow from DNA extraction to signal detection can be finished within 1 h, fulfilling the requirement of on-site meat species identification.

scholarly journals Multiplex End-Point PCR for the Detection of Three Species of Ophiosphaerella Causing Spring Dead Spot of Bermudagrass

Plant Disease ◽  
2019 ◽  
Vol 103 (8) ◽  
pp. 2010-2014 ◽  
Author(s):  
J. Francisco Iturralde Martinez ◽  
Francisco J. Flores ◽  
Alma R. Koch ◽  
Carla D. Garzón ◽  
Nathan R. Walker
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Temperature Cycling ◽  
Molecular Technique ◽  
Correct Identification ◽  
Internal Transcribed Spacer Region ◽  
Spring Dead Spot ◽  
End Point ◽  
Specificity And Sensitivity ◽  
Species Specific

A multiplex end-point polymerase chain reaction (PCR) assay was developed for identifying the three-fungal species in the genus Ophiosphaerella that cause spring dead spot (SDS), a devastating disease of bermudagrass. These fungi are difficult to identify by morphology because they seldom produce pseudothecia. To achieve species-specific diagnosis, three pairs of primers were designed to identify fungal isolates and detect the pathogen in infected roots. The internal transcribed spacer region, the translation elongation factor 1-α, and the RNA polymerase II second-largest subunit were selected as targets and served as templates for the design of each primer pair. To achieve uniform melting temperatures, three to five random nucleotide extensions (flaps) were added to the 5′ terminus of some of the designed specific primers. Temperature cycling conditions and PCR components were standardized to optimize specificity and sensitivity of the multiplex reaction. Primers were tested in multiplex on DNA extracted from axenic fungal cultures and from field-collected infected and uninfected roots. A distinct amplicon was produced for each Ophiosphaerella sp. tested. The DNA from Ophiosphaerella close relatives and other common bermudagrass pathogens did not amplify during the multiplex assay. Metagenomic DNA from infected bermudagrass produced species-specific amplicons while DNA extracted from noninfected roots did not. This multiplex end-point PCR approach is a sensitive and specific molecular technique that allows for correct identification of SDS-associated Ophiosphaerella spp. from field-collected roots.

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