eDNA-based detection of the invasive crayfish Pacifastacus leniusculus in streams with a LAMP assay using dependent replicates to gain higher sensitivity

Porco, David; Hermant, Sylvie; Purnomo, Chanistya Ayu; Horn, Mario; Marson, Guy; Colling, Guy

doi:10.1038/s41598-022-10545-w

Cited by 8 publications

(4 citation statements)

References 42 publications

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“…In addition to fish tissue samples, we used water samples from aquarium tanks that contained multiple local marine species to simulate a natural environmental sample. Our assay showed a reliable and highly sensitive detection as low as 0.002 ng per reaction of the mitochondrial COI gene, comparable to other studies using LAMP as their detection method for eDNA in aquatic environments (49-51). This is approximately equivalent to 25 gene copies per reaction, which is within the usual LoD for LAMP and other quantitative DNA amplification methods, such as qPCR and qRPA.…”

Section: Discussionsupporting

confidence: 78%

Development of a quantitative colorimetric LAMP assay for fast and targeted molecular detection of the invasive lionfishPterois milesfrom environmental DNA

Hartle-Mougiou,

Gubili,

Xanthopoulou

et al. 2023

Preprint

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The Mediterranean basin has seen an increased influx of invasive species since the Suez Canal expansion in 2015. The invasive lionfish species,Pterois miles, has rapidly established new populations in the Eastern Mediterranean Sea, impacting local fish biodiversity. Here, we have developed a new, fast (< 35 min) molecular approach to detect and quantifyP. milesenvironmental DNA (eDNA) in combination with a portable device for field-based analysis. Using a species-specific real-time colorimetric loop-mediated isothermal amplification (qcLAMP) for the cytochrome oxidase subunit 1 (COI) gene, we demonstrate a high sensitivity with a limit of detection of 0.002 ng DNA per reaction, equivalent to only 50 copies of the COI gene. The assay is specific to the target in the presence of closely related and co-occurring species, and it is quantitative over five orders of magnitude. We validated the assay using aquarium water samples and further demonstrated its utility on natural eDNA samples collected from locations around the island of Crete whereP. mileshad been sighted.P. mileswas indeed detected in three out of nine locations, two nature reserves and a closed bay. Lack of detection in the remaining locations suggest that populations are still at a low density. We also demonstrate the feasibility ofP. mileseDNA qualitative detection directly from the filter used to collect eDNA-containing particles, completely omitting DNA extraction. Overall, we present a new approach for fast and targeted eDNA quantification. The developed LAMP assay together with the quantitative real-time colorimetric detection approach open new possibilities for monitoring invasiveP. milesin the field.

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Section: Discussionsupporting

confidence: 78%

Development of a quantitative colorimetric LAMP assay for fast and targeted molecular detection of the invasive lionfishPterois milesfrom environmental DNA

Hartle-Mougiou,

Gubili,

Xanthopoulou

et al. 2023

Preprint

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show abstract

“…Our assay showed a reliable and highly sensitive detection as low as 0.002 ng per reaction of the mitochondrial COI gene, comparable to other studies using LAMP as their detection method for eDNA in aquatic environments (Williams et al, 2017;Kageyama et al, 2022;Porco et al, 2022). This is approximately equivalent to 25 gene copies per reaction, which is within the usual LoD for LAMP and other quantitative DNA amplification methods, such as qPCR and qRPA.…”

Section: Lamp Optimization For Target Detection In Edna Samplessupporting

confidence: 79%

Development of a quantitative colorimetric LAMP assay for fast and targeted molecular detection of the invasive lionfish Pterois miles from environmental DNA

Hartle-Mougiou,

Gubili,

Xanthopoulou

et al. 2024

Front. Mar. Sci.

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The Mediterranean basin has faced an increased influx of invasive species since the Suez Canal expansion in 2015. The invasive lionfish species, Pterois miles, has rapidly established new populations in the Eastern Mediterranean Sea, impacting local fish biodiversity. Here, we have developed a new, fast (< 35 min) molecular approach to detect and quantify P. miles environmental DNA (eDNA) in combination with a portable device for field-based analysis. Using a species-specific real-time colorimetric loop-mediated isothermal amplification (qcLAMP) for the cytochrome oxidase subunit 1 (COI) gene, we demonstrate a high sensitivity with a limit of detection of 0.002 ng DNA per reaction, equivalent to only 50 copies of the COI gene. The assay is specific to the target in the presence of closely related and co-occurring species, and it is quantitative over five orders of magnitude. We validated the assay using aquarium water samples and further demonstrated its utility on natural eDNA samples collected from locations around the island of Crete where P. miles had been sighted. P. miles was indeed detected in three out of nine locations, two nature reserves and a closed bay. Lack of detection in the remaining locations suggests that populations are still at a low density. We also demonstrate the feasibility of P. miles eDNA qualitative detection directly from the filter used to collect eDNA-containing particles, completely omitting DNA extraction. Overall, we present a new approach for fast and targeted eDNA quantification. The developed LAMP assay together, with the quantitative real-time colorimetric detection approach, open new possibilities for monitoring invasive P. miles in the field.

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“…On the contrary, while loop-mediated isothermal amplification facilitates eDNA on-site detection without bulky equipment (Mori & Notomi, 2009;Porco et al, 2022), it requires the design of four to six primer sets and operates at a high temperature (65°C), posing challenges for flexibility and field use (Kageyama et al, 2022;Kellner et al, 2019). In terms of crRNA design, the Cas13 crRNA design differs from the Cas12 by allowing targeting broad species without protospacer adjacent motifs (PAM) site constraints (Kellner et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Rapid, easy, sensitive, low‐cost and on‐site detection of environmental DNA and RNA using CRISPR‐Cas13

Yang,

Matsushita,

Xia

et al. 2024

Methods Ecol Evol

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Environmental DNA (eDNA) monitoring of species distribution has become a critical tool in ecology, conservation biology and fisheries for identifying the presence and distribution of diverse organisms, including important, threatened and invasive species. However, eDNA detection still has room for improvement in sensitivity, requiring time‐consuming real‐time polymerase chain reaction (qPCR) steps and costly machines. In this study, we report a CRISPR‐Cas13‐based method for rapid, easy, sensitive, low‐cost and on‐site detection of eDNA. The assay for the detection of common carp (Cyprinus carpio) and medaka (Oryzias latipes) nucleic acid employs a two‐step process, starting with recombinase polymerase amplification (RPA) and followed by cleavage using Cas13 nuclease, to effectively identify mitochondrial DNA or RNA. Our results showed that the Cas13‐based method has a higher sensitivity than the qPCR‐based method in detecting tiny amounts of eDNA. When combined with reverse transcription of environmental RNA (eRNA), our method increased detection sensitivity by approximately one order of magnitude. Cas13‐based detection could achieve on‐site detection of eDNA using a quick nucleic acid extraction solution and lateral flow strips. Cas13‐based detection of eDNA and eRNA requires minimal training efforts and can be performed in 1 h, without the need for centrifugation and qPCR machines. The portability of the Cas13‐based method, along with its accuracy and relative ease in designing primers and CRISPR RNA (crRNA), underscores its potential to broaden the application of eDNA and eRNA in various fields, including biodiversity conservation.

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eDNA-based detection of the invasive crayfish Pacifastacus leniusculus in streams with a LAMP assay using dependent replicates to gain higher sensitivity

Cited by 8 publications

References 42 publications

Development of a quantitative colorimetric LAMP assay for fast and targeted molecular detection of the invasive lionfishPterois milesfrom environmental DNA

Development of a quantitative colorimetric LAMP assay for fast and targeted molecular detection of the invasive lionfishPterois milesfrom environmental DNA

Development of a quantitative colorimetric LAMP assay for fast and targeted molecular detection of the invasive lionfish Pterois miles from environmental DNA

Rapid, easy, sensitive, low‐cost and on‐site detection of environmental DNA and RNA using CRISPR‐Cas13

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