Related Experiment Video
Updated: Jul 17, 2025
Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications
Published on: November 5, 2020
Current status and topical issues on the use of eDNA-based targeted detection of rare animal species
Sofia Duarte1, Luara Simões1, Filipe O Costa1
1Centre of Molecular and Environmental Biology (CBMA) and ARNET-Aquatic Research Network, Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; Institute of Science and Innovation for Bio-Sustainability (IB-S), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Abstract:
Animal detection through DNA present in environmental samples (eDNA) is a valuable tool for detecting rare species, that are difficult to observe and monitor. eDNA-based tools are underpinned by molecular evolutionary principles, key to devising tools to efficiently single out a targeted species from an environmental sample. Here, we present a comprehensive review of the use of eDNA-based methods for the detection of targeted animal species, such as rare, endangered, or invasive species, through the analysis of 549 publications (2008-2022). Aquatic ecosystems have been the most surveyed, in particular, freshwaters (74 %), and to a less extent marine (14 %) and terrestrial systems (10 %). Vertebrates, in particular, fish (38 %), and endangered species, have been the focus of most of these studies, and Cytb and COI are the most employed markers. Among invertebrates, assays have been mainly designed for Mollusca and Crustacea species (21 %), in particular, to target invasive species, and COI the most employed marker. Targeted molecular approaches, in particular qPCR, have been the most adopted (75 %), while eDNA metabarcoding has been rarely used to target single or few species (approx. 6 %). However, less attention has been given in these studies to the effects of environmental factors on the amount of shed DNA, the differential amount of shed DNA among species, or the sensitivity of the markers developed, which may impact the design of the assays, particularly to warrant the required detection level and avoid false negatives and positives. The accuracy of the assays will also depend on the availability of genetic data and vouchered tissue or DNA samples from closely related species to assess both marker and primers' specificity. In addition, eDNA-based assays developed for a particular species may have to be refined for use in a new geographic area taking into account site-specific populations, as well as any intraspecific variation.
