Multiplex real-time PCR enables the simultaneous detection of environmental DNA from freshwater fishes: a case study of three exotic and three threatened native fishes in Japan

Jo, Toshiaki; Fukuoka, Arisa; Uchida, Keiji; Ushimaru, Atushi; Minamoto, Toshifumi

doi:10.1007/s10530-019-02102-w

Cited by 28 publications

(26 citation statements)

References 69 publications

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“…One way to test the eDNA method's validity is to directly compare it with traditional monitoring methods, as pioneered by Thomsen et al (2012). Many comparative studies have been conducted (e.g., Fernández et al, 2019;Ficetola et al, 2015;Hinlo et al, 2017;Jo et al, 2020;Pilliod et al, 2013;Rice et al, 2018;Wilcox et al, 2016), encompassing a variety of traditional method types, shown in Table 1. However, there have been few attempts to synthesize the results of comparative analyses.…”

Section: Introductionmentioning

confidence: 99%

Meta‐analysis shows that environmental DNA outperforms traditional surveys, but warrants better reporting standards

Fediajevaite

Priestley

Arnold³

et al. 2021

Ecology and Evolution

132

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Section: Introductionmentioning

confidence: 99%

Meta‐analysis shows that environmental DNA outperforms traditional surveys, but warrants better reporting standards

Fediajevaite

Priestley

Arnold³

et al. 2021

Ecology and Evolution

132

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“…Primers/probe sets used in this study. Notes: Primers/probe sets were taken from Jo et al (2020) except for Msa_P2, which was newly developed for the present study (we changed only the fluorescence dye at the end of 5'). All primers/probe sets targeted mitochondrial cytochrome b genes of the target species.…”

Section: Multiplex Real-time Pcr For Edna Detection In Reservoirsmentioning

confidence: 99%

“…In the present study, we verified the utility of eDNA analysis as a tool in monitoring representative invasive fish species (bluegill, largemouth bass, and smallmouth bass) in reservoirs in Japan. Here, we used the multiplex real-time PCR approach, which simultaneously detects target eDNA using multiple species-specific primers/ probe sets labeled with different fluorescent dyes, which could reduce the analysis cost and detect the eDNA of several species with higher sensitivity than multiple single PCR runs (Jo et al 2020). First, due to its huge surface area and water depth, eDNA detection rates in reservoirs may vary depending on sampling location.…”

Section: Introductionmentioning

confidence: 99%

Utility of environmental DNA analysis for effective monitoring of invasive fish species in reservoirs

Ikeda

Fukuoka

et al. 2021

Ecosphere

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Dam-mediated biological invasions are a serious problem all over the world. Once established in reservoirs, the invasive species have catastrophic impacts on the river ecosystems downstream, and thus, rapid monitoring of invasive species is an urgent issue for the effective removal of them and the conservation management of native ecosystems. Here, we verified the utility of environmental DNA (eDNA) analysis as a tool to effectively monitor three invasive fish species (bluegill, largemouth bass, and smallmouth bass) in reservoirs using multiplex real-time PCR. First, to determine the optimal sampling location and season to detect eDNA from these species, we analyzed the eDNA in water samples from shore and offshore sites in three reservoirs all year around. We found that eDNA detection rates either did not differ between sampling locations or were higher for shore than offshore sites. In addition, eDNA detection rates were higher in spring (breeding season of target species) and/or summer than winter. Second, we extensively surveyed the distribution of the three species in 30 reservoirs in Japan using eDNA analysis. Consequently, a single eDNA-based surveillance in summer allowed to match approximately 90% of the presence/absence of the invasive fish species known from 27 yr of administrative capture-based surveillances. Given these results, we recommend collecting the replicated water samples from shore sites in summer or the breeding season for the effective detection of invasive fish eDNA in reservoirs. Our eDNA assays with multiplex real-time PCR enable the rapid and sensitive monitoring of invasive fish distribution in reservoirs.

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“…An optimal solution for many applications would preserve the sensitivity, specificity, and simple interpretation of single‐species approaches, but allow for efficient, parallel analysis for detection of multiple taxa. Some studies have sought to accomplish this goal through nested PCR, where DNA is initially enriched across a broad taxonomic group, followed by targeted PCR of those amplicons for individual taxa (Stoeckle, Das, & Charlop‐Powers, 2018) or by simply multiplexing qPCR assays (Jo, Fukuoka, Uchida, Ashimaru, & Minamoto, 2020; Pilliod, Goldberg, Arkle, & Waits, 2013; Tsuji et al, 2018). Here, we test a related approach, known as high‐throughput qPCR (HT‐qPCR), with which large numbers (e.g., thousands) of individual reactions are run in parallel using microfluidic or assay‐printed plates to assay samples simultaneously across different primer–probe sets.…”

Section: Introductionmentioning

confidence: 99%

Parallel, targeted analysis of environmental samples via high‐throughput quantitative PCR

et al. 2020

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Environmental DNA (eDNA) sampling in aquatic environments is a unique tool in that the taxonomic coverage represented by a single sample is large, with virtually all members of the local biota potentially represented (Hauck, Weitemeir, Penaluna, Garcia, & Cronn, 2019). The obvious potential of eDNA sampling to revolutionize monitoring, bioassessment, and ecological research has been pointed out repeatedly in recent years (Cristescu & Hebert, 2018; Deiner et al., 2017). However, accessing taxonomically broad biodiversity information from environmental samples is nontrivial. The available eDNA analysis methods present an apparent tradeoff between taxonomic scope and reliability of detection. One end of the spectrum is represented by single-taxon methods (e.g., taxon-specific quantitative or digital PCR). Properly designed taxon-specific assays are free from cross-amplification with other taxa and can reliably detect target DNA at low concentrations (e.g., <10 copies per reaction; Klymus et al., 2019). These approaches necessarily have a narrow taxonomic scope, but also tend to be more sensitive for rare species detection than multi-taxa approaches (Harper et al., 2018;

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Multiplex real-time PCR enables the simultaneous detection of environmental DNA from freshwater fishes: a case study of three exotic and three threatened native fishes in Japan

Cited by 28 publications

References 69 publications

Meta‐analysis shows that environmental DNA outperforms traditional surveys, but warrants better reporting standards

Meta‐analysis shows that environmental DNA outperforms traditional surveys, but warrants better reporting standards

Utility of environmental DNA analysis for effective monitoring of invasive fish species in reservoirs

Parallel, targeted analysis of environmental samples via high‐throughput quantitative PCR

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