Application of real-time PCR for simultaneous identification and quantification of larval abalone

Vadopalas, Brent; Bouma, Josh V.; Jackels, Chemine R.; Friedman, Carolyn S.

doi:10.1016/j.jembe.2006.02.005

Cited by 54 publications

(45 citation statements)

References 38 publications

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“…The presence of one versus two nauplii, when added to 20 mg (wet weight) plankton samples was easily distinguished. This finding agrees with diagnostic studies of QPCR detection of three other zooplankters (Vadopalus et al 2006;Pan et al 2008;Wright et al 2009). Given this level of resolution, QPCR should be expected to provide some ecologically relevant information in a survey, perhaps allowing a single larva in a sample to be distinguished from dense aggregations.…”

Section: Discussionsupporting

confidence: 90%

“…"Real-time" QPCR formats involving automated monitoring of fluorescence were rapidly adopted in health-related applications (Bustin 2000;Ginzinger 2002), and offer an invaluable set of applications in aquatic biologyfor example, in measuring biodiversity and gene expression patterns in bacterioplankton (Brinkman et al 2003;Thompson et al 2005;Boström et al 2007), monitoring for toxic algal species in sediment and water (Popels et al 2003;Coyne and Cary 2005;Coyne et al 2005;Bowers et al 2006), or determining diet of zooplankters (Troedsson et al 2009;Töbe et al 2010). Recently, studies have been applied using real-time QPCR to measure biomass of specific larval invertebrates (Vadopalus et al 2006;Pan et al 2008;Wright et al 2009). …”

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confidence: 99%

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Experimental parameters affecting quantitative PCR of Artemia franciscana: a model for a marine zooplanktonic target in natural plankton samples

Mackie

Geller

2010

Limnology & Ocean Methods

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The brine shrimp Artemia franciscana was used as a model zooplankter to explore the range and accuracy of quantitative PCR (QPCR) in detecting a target species in plankton community DNA. Specific primers were designed in the 18S ribosomal RNA and mitochondrial cytochrome c oxidase subunit I genes and analyzed by TaqMan and SYBR Green I reporting systems. Assays were sensitive in detecting A. franciscana nauplii in DNA extractions from bulk plankton: a single nauplius was detected when added to 20 mg wet, packed plankton and distinguished from two added nauplii. Indeed, Artemia DNA diluted to 0.1 picograms per reaction (equivalent to 10−5 nauplii) was detectable. Artemia franciscana was detected without coamplification of other plankton, alleviating concern for errors caused by the presence of similar organisms in the plankton community. A natural plankton DNA sample and purified herring sperm DNA inhibited PCR above 100 ng reaction−1, but these samples could be diluted to eliminate inhibition. Because QPCR could detect 10−5 nauplii, dilution solves inhibition without sacrificing sensitivity. This sensitivity allows for analysis of a mass of plankton made large enough sufficient to include a target organism at low density. The newly hatched nauplius (DNA mass = 6. 1 ng) is a useful internal control in experiments examining plankton samples, providing a means of controlling for variations in DNA extraction and amplification efficiency in surveys for species of interest.

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Section: Discussionsupporting

confidence: 90%

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confidence: 99%

Experimental parameters affecting quantitative PCR of Artemia franciscana: a model for a marine zooplanktonic target in natural plankton samples

Mackie

Geller

2010

Limnology & Ocean Methods

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“…The most common application for using molecular tools is species identification, whereby a cryptic individual, pathogen, or a partly processed fish sample (Miller and Mariani, 2010) can be identified by a specific DNA-based assay. Species ID techniques can also be used in a quantitative manner; in marine systems example uses include quantification of fecal contamination at swimming beaches (Griffith and Weisberg, 2011) or for direct quantification of zooplankton that are otherwise difficult to identify (Vadopalas et al, 2006). The identification of species within a sample can now be taken a level further.…”

Section: Molecular Biology Techniquesmentioning

confidence: 99%

A Review of the Tools Used for Marine Monitoring in the UK: Combining Historic and Contemporary Methods with Modeling and Socioeconomics to Fulfill Legislative Needs and Scientific Ambitions

et al. 2017

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Marine environmental monitoring is undertaken to provide evidence that environmental management targets are being met. Moreover, monitoring also provides context to marine science and over the last century has allowed development of a critical scientific understanding of the marine environment and the impacts that humans are having on it. The seas around the UK are currently monitored by targeted, impact-driven, programmes (e.g., fishery or pollution based monitoring) often using traditional techniques, many of which have not changed significantly since the early 1900s. The advent of a new wave of automated technology, in combination with changing political and economic circumstances, means that there is currently a strong drive to move toward a more refined, efficient, and effective way of monitoring. We describe the policy and scientific rationale for monitoring our seas, alongside a comprehensive description of the types of equipment and methodology currently used and the technologies that are likely to be used in the future. We contextualize the way new technologies and methodologies may impact monitoring and discuss how whole ecosystems models can give an integrated, comprehensive approach to impact assessment. Furthermore, we discuss how an understanding of the value of each data point is crucial to assess the true costs and benefits to society of a marine monitoring programme.

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“…Becker et al 2007). Thus, there has been a recent focus on developing molecular techniques to improve identification of larvae (Goffredi et al 2006, Livi et al 2006, Vadopalas et al 2006, Le Goff-Vitry et al 2007a, Pradillon et al 2007). However, while these techniques more accurately identify targeted species in the plankton, they either sacrifice the ability to quantify plankton or still require time-consuming sorting.…”

Section: Introductionmentioning

confidence: 99%

“…Organisms can be quantified if individually processed, but this is still extremely time-consuming, as it requires manually pre-sorting samples using morphological features and then extracting and amplifying the DNA of each individual. Quantitative PCR (qPCR) provides quantification of targeted species within a batch-processed plankton sample, but the accuracy of this method is complicated by a number of factors, including variation in the size of larvae (and hence gene copy number) and possible presence of PCR inhibitors in the plankton sample (Vadopalas et al 2006, Pan et al 2008.…”

Section: Introductionmentioning

confidence: 99%

FISH-CS—a rapid method for counting and sorting species of marine zooplankton

Henzler¹,

Hoaglund

Gaines

2010

Mar. Ecol. Prog. Ser.

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Understanding population dynamics in marine species has long been hindered by the inherent difficulties of studying species in which all or part of the life cycle is planktonic. Plankton sample processing is laborious and, due to morphological similarity between disparate taxa, often identifies zooplankton only to higher taxonomic levels. As a consequence, many scientific issues that require identification to species level are impossible to explore adequately. Several in situ hybridization protocols show promise for identifying marine larvae by color-coding them with taxon-specific, dye-labeled DNA probes. We adapted these protocols and coupled them with recent cell sorting technology to rapidly and accurately identify bivalve larvae from diverse plankton samples. We developed probes for 2 bivalve taxa: Musculista senhousia and the species complex Mytilus edulis/galloprovincialis/trossulus. Coupled fluorescence in situ hybridization and cell sorting (FISH-CS) separated M. galloprovincialis larvae from both oyster Crassostrea gigas larvae and from a mixed plankton/M. galloprovincialis sample. The number of false positives and false negatives was assessed by a PCR assay. Our FISH-CS method is robust to plankton autofluorescence and can be easily adapted to work with nearly any planktonic species or life stage of appropriate size. A green fluorescent DNA probe allows identification of mussel Mytilus galloprovincialis larvae in a plankton sample, despite autofluorescence in the plankton.

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Application of real-time PCR for simultaneous identification and quantification of larval abalone

Cited by 54 publications

References 38 publications

Experimental parameters affecting quantitative PCR of Artemia franciscana: a model for a marine zooplanktonic target in natural plankton samples

Experimental parameters affecting quantitative PCR of Artemia franciscana: a model for a marine zooplanktonic target in natural plankton samples

A Review of the Tools Used for Marine Monitoring in the UK: Combining Historic and Contemporary Methods with Modeling and Socioeconomics to Fulfill Legislative Needs and Scientific Ambitions

FISH-CS—a rapid method for counting and sorting species of marine zooplankton

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