Development and validation of a quantitative qPCR assay for detecting Natterjack toad (Epidalea calamita) eDNA samples

Marina Reyne; Amanda M. Naaum; Ferdia Marnell; Neil Reid; Sarah J. Helyar

doi:10.1007/s12686-021-01199-3

Development and validation of a quantitative qPCR assay for detecting Natterjack toad (Epidalea calamita) eDNA samples

Marina Reyne, Amanda M. Naaum, Ferdia Marnell, Neil Reid, Sarah J. Helyar

Research output: Contribution to journal › Article › peer-review

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Abstract

The Natterjack toad (Epidalea calamita) is the rarest amphibian species in Ireland, regionally Red-Listed as Endangered. We applied an eDNA approach to detect species presence in breeding pond water samples. We developed a species-specific qPCR assay targeting the cytochrome c oxidase subunit I (COI). The assay was tested in silico, in vitro (DNA extracted from tissue) and in vivo (DNA extracted from water samples). Water samples were collected from five ponds with known Natterjack toad presence or absence to validate the sensitivity and specificity of the assay. The assay was shown to be highly specific to the Natterjack toad and tested positive only against toad tissue samples and eDNA samples from ponds with known species presence. We believe this method can be used for rapid assessment of species occurrence.

Original language	English
Journal	Conservation Genetics Resources
Early online date	04 Mar 2021
DOIs	https://doi.org/10.1007/s12686-021-01199-3
Publication status	Published - 2021

Keywords

eDNA
Environmental DNA
pond
presence/absence
surveys
Cytochrome oxidase I
Epidalea calamita
Quantitative PCR

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1007/s12686-021-01199-3Licence: CC BY

Development and validation of a quantitative qPCR assay for detecting Natterjack toad (Epidalea calamita) eDNA samples
Copyright 2021 the authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
Final published version, 667 KBLicence: CC BY

Conservation of an endangered amphibian: The case of the Natterjack toad (Epidalea calamita) in Ireland
Reyne, M. (Author), Reid, N. (Supervisor), Helyar, S. (Supervisor) & Emmerson, M. (Supervisor), Jul 2021
Student thesis: Doctoral Thesis › Doctor of Philosophy

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abstract = "The Natterjack toad (Epidalea calamita) is the rarest amphibian species in Ireland, regionally Red-Listed as Endangered. We applied an eDNA approach to detect species presence in breeding pond water samples. We developed a species-specific qPCR assay targeting the cytochrome c oxidase subunit I (COI). The assay was tested in silico, in vitro (DNA extracted from tissue) and in vivo (DNA extracted from water samples). Water samples were collected from five ponds with known Natterjack toad presence or absence to validate the sensitivity and specificity of the assay. The assay was shown to be highly specific to the Natterjack toad and tested positive only against toad tissue samples and eDNA samples from ponds with known species presence. We believe this method can be used for rapid assessment of species occurrence.",

keywords = "eDNA, Environmental DNA, pond, presence/absence, surveys, Cytochrome oxidase I, Epidalea calamita, Quantitative PCR",

author = "Marina Reyne and Naaum, {Amanda M.} and Ferdia Marnell and Neil Reid and Helyar, {Sarah J.}",

year = "2021",

doi = "10.1007/s12686-021-01199-3",

language = "English",

journal = "Conservation Genetics Resources",

issn = "1877-7252",

publisher = "Springer Verlag",

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TY - JOUR

T1 - Development and validation of a quantitative qPCR assay for detecting Natterjack toad (Epidalea calamita) eDNA samples

AU - Reyne, Marina

AU - Naaum, Amanda M.

AU - Marnell, Ferdia

AU - Reid, Neil

AU - Helyar, Sarah J.

PY - 2021

Y1 - 2021

N2 - The Natterjack toad (Epidalea calamita) is the rarest amphibian species in Ireland, regionally Red-Listed as Endangered. We applied an eDNA approach to detect species presence in breeding pond water samples. We developed a species-specific qPCR assay targeting the cytochrome c oxidase subunit I (COI). The assay was tested in silico, in vitro (DNA extracted from tissue) and in vivo (DNA extracted from water samples). Water samples were collected from five ponds with known Natterjack toad presence or absence to validate the sensitivity and specificity of the assay. The assay was shown to be highly specific to the Natterjack toad and tested positive only against toad tissue samples and eDNA samples from ponds with known species presence. We believe this method can be used for rapid assessment of species occurrence.

AB - The Natterjack toad (Epidalea calamita) is the rarest amphibian species in Ireland, regionally Red-Listed as Endangered. We applied an eDNA approach to detect species presence in breeding pond water samples. We developed a species-specific qPCR assay targeting the cytochrome c oxidase subunit I (COI). The assay was tested in silico, in vitro (DNA extracted from tissue) and in vivo (DNA extracted from water samples). Water samples were collected from five ponds with known Natterjack toad presence or absence to validate the sensitivity and specificity of the assay. The assay was shown to be highly specific to the Natterjack toad and tested positive only against toad tissue samples and eDNA samples from ponds with known species presence. We believe this method can be used for rapid assessment of species occurrence.

KW - eDNA

KW - Environmental DNA

KW - pond

KW - presence/absence

KW - surveys

KW - Cytochrome oxidase I

KW - Epidalea calamita

KW - Quantitative PCR

U2 - 10.1007/s12686-021-01199-3

DO - 10.1007/s12686-021-01199-3

M3 - Article

SN - 1877-7252

JO - Conservation Genetics Resources

JF - Conservation Genetics Resources

ER -

Development and validation of a quantitative qPCR assay for detecting Natterjack toad (Epidalea calamita) eDNA samples

Abstract

Keywords

UN SDGs

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Student theses

Conservation of an endangered amphibian: The case of the Natterjack toad (Epidalea calamita) in Ireland

Cite this