Development and application of a quantitative real-time PCR assay for the globally invasive tunicate Styela clava

Gillum, Joanne; Jiménez, Laura; White, Daniel J.; Goldstien, Sharyn J.; Gemmell, Neil J.

doi:10.3391/mbi.2014.5.2.06

Cited by 9 publications

(5 citation statements)

References 23 publications

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“…Most targeted methods applied to aquatic systems have used qPCR (e.g., Takahara, Minamoto, & Doi, 2013;Gillum, Jimenez, White, Goldstien, & Gemmell, 2014;Sigsgaard et al, 2015;Wood et al, 2017). Quantitative PCR assays are relatively sensitive and specific, and results can be generated rapidly (<12 hr from DNA extraction to data).…”

mentioning

confidence: 99%

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Wood

Pochon

Laroche

et al. 2019

Molecular Ecology Resources

109

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Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R2 = 0.81, p < .001, biofouling R2 = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.

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mentioning

confidence: 99%

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Wood

Pochon

Laroche

et al. 2019

Molecular Ecology Resources

109

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“…Direct droplet digital PCR (direct-ddPCR) was conducted in an automated droplet generator (QX200™ Droplet Digital PCR System; BioRad, Hercules, CA, USA). Copy numbers (per µL) of the Cytochrome c oxidase subunit 1 (COI) gene were measured in all samples using primers and probes specific to S. spallanzanii ( Wood et al, 2017 ) and S. clava ( Gillum, 2014 ) and primers specific to B. neritina ( Kim et al, 2018 ) ( Table 2 ). To compare the results between the two ddPCR chemistries, both the hydrolysis probe (TaqMan, Carlsbad, CA, USA) and DNA binding dye (EvaGreen®, Taoyuan City, Taiwan) assays were utilized in this study.…”

Section: Methodsmentioning

confidence: 99%

Drop it all: extraction-free detection of targeted marine species through optimized direct droplet digital PCR

Scriver,

von Ammon,

Youngbull

et al. 2024

PeerJ

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Molecular biomonitoring programs increasingly use environmental DNA (eDNA) for detecting targeted species such as marine non-indigenous species (NIS) or endangered species. However, the current molecular detection workflow is cumbersome and time-demanding, and thereby can hinder management efforts and restrict the “opportunity window” for rapid management responses. Here, we describe a direct droplet digital PCR (direct-ddPCR) approach to detect species-specific free-floating extra-cellular eDNA (free-eDNA) signals, i.e., detection of species-specific eDNA without the need for filtration or DNA extraction, with seawater samples. This first proof-of-concept aquarium study was conducted with three distinct marine species: the Mediterranean fanworm Sabella spallanzanii, the ascidian clubbed tunicate Styela clava, and the brown bryozoan Bugula neritina to evaluate the detectability of free-eDNA in seawater. The detectability of targeted free-eDNA was assessed by directly analysing aquarium marine water samples using an optimized species-specific ddPCR assay. The results demonstrated the consistent detection of S. spallanzanii and B. neritina free-eDNA when these organisms were present in high abundance. Once organisms were removed, the free-eDNA signal exponentially declined, noting that free-eDNA persisted between 24–72 h. Results indicate that organism biomass, specimen characteristics (e.g., stress and viability), and species-specific biological differences may influence free-eDNA detectability. This study represents the first step in assessing the feasibility of direct-ddPCR technology for the detection of marine species. Our results provide information that could aid in the development of new technology, such as a field development of ddPCR systems, which could allow for automated continuous monitoring of targeted marine species, enabling point-of-need detection and rapid management responses.

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“…Further progress has been made with quantitative PCR (qPCR, also known as real-time PCR), one of the most promising molecular techniques for the detection of specific and sensitive targets [80]. In particular, assays have been developed for harmful species: dinoflagellates of the genus Alexandrium [84,85], sea squirts Styela clava [86], and Didemnum spp. [68], Amur River clam Potamocorbula amurensis [87], and Mediterranean fan worm Sabella spallanzanii [88].…”

Section: Molecular Methodologiesmentioning

confidence: 99%

Ballast Water Management in Ports: Monitoring, Early Warning and Response Measures to Prevent Biodiversity Loss and Risks to Human Health

Kraus

2023

JMSE

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Ballast water is recognised as successfully transporting non-native (potentially) invasive alien species and other harmful organisms (human pathogens and toxic phytoplankton) from one region to another. Global warming enables the successful adaptation of non-native species in new areas. The early detection of harmful species increases the likelihood that the response will be effective and cause less damage to biodiversity, ecosystems, economies and human health. Scientific evidence strongly points to the importance of prevention. In this context, this refers to continuous port monitoring, carried out with the aim of detecting harmful species soon after their introduction. The objectives of rapid detection are (a) early warning and prevention of further spread of harmful species through ballast water or natural circulation, and (b) a timely response through eradication or other appropriate strategies to reduce the number or spatial extent of introduced species. This paper provides guidance for the development of ballast water management in ports based on a literature review. Available and new methods for identifying marine species and best practises in port monitoring for the early detection of harmful species, as well as early warning and response measures following the introduction of species in ports, are presented and discussed.

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Development and application of a quantitative real-time PCR assay for the globally invasive tunicate Styela clava

Cited by 9 publications

References 23 publications

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Drop it all: extraction-free detection of targeted marine species through optimized direct droplet digital PCR

Ballast Water Management in Ports: Monitoring, Early Warning and Response Measures to Prevent Biodiversity Loss and Risks to Human Health

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