Development of a multiplex real-time qPCR assay for simultaneous enumeration of up to four marine toxic bloom-forming microalgal species

Eckford-Soper, Lisa K.; Daugbjerg, Niels

doi:10.1016/j.hal.2015.06.009

Cited by 39 publications

(13 citation statements)

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“…Here we used a hydrolysis probe for additional specificity and constrained the primers and probe design to allow for multiplexing qPCR by having the same annealing temperature (60°C). We propose that this approach permits greater efficiency in larger ecological studies when up to 4 species can be detected simultaneously (Eckford‐Soper and Daugbjerg ). However, our tests revealed that the primers‐probe set for P. verruculosa was species specific, whereas the primers‐probe set for P. farcimen gave positive results for both species.…”

Section: Discussionmentioning

confidence: 99%

“…The LSU rDNA sequences available for P. verruculosa from the North Sea (JG8) is 497 base pairs long, setting some limitations to the possibilities for species‐specific primer design when compared to P. farcimen for which 1,200 base pairs were available. Additionally we wanted to have an annealing temperature for our Pseudochattonella assay similar to that in other assays of ichthyotoxic microalgae in multiplex qPCR (Eckford‐Soper and Daugbjerg ). This added further constraints to the assay design for Pseudochattonella spp.…”

Section: Methodsmentioning

confidence: 99%

“…), Prymnesium parvum (Galluzzi et al. , Manning and Claire , Eckford‐Soper and Daugbjerg ), Pfiesteria spp. (Bowers et al.…”

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confidence: 99%

“…qPCR has been comprehensively tested and extensively used to identify and quantify a number of HAB species in natural marine samples including Alexandrium spp. (Erdner et al 2010, Toebe et al 2013, Eckford-Soper and Daugbjerg 2015b, Pseudonitzchia spp. (Fitzpatrick et al 2010), Prymnesium parvum (Galluzzi et al 2008, Manning and Claire 2010, Eckford-Soper and Daugbjerg 2015b, Pfiesteria spp.…”

mentioning

confidence: 99%

“…(Erdner et al 2010, Toebe et al 2013, Eckford-Soper and Daugbjerg 2015b, Pseudonitzchia spp. (Fitzpatrick et al 2010), Prymnesium parvum (Galluzzi et al 2008, Manning and Claire 2010, Eckford-Soper and Daugbjerg 2015b, Pfiesteria spp. (Bowers et al 2000, Lin et al 2006 Molecular studies of Pseudochattonella spp.…”

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confidence: 99%

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A quantitative real‐time PCR assay for identification and enumeration of the occasionally co‐occurring ichthyotoxic Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae) and analysis of variation in gene copy numbers during the growth phase of single and mixed cultures

Eckford-Soper

Daugbjerg

2016

Journal of Phycology

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The ichthyotoxic genus Pseudochattonella forms recurrent extensive blooms in coastal waters in Japan, New Zealand and Northern Europe. It comprises of two morphologically similar species, P. verruculosa and P. farcimen, which complicates visual species identification and enumeration of live and fixed material. Primers designed previously could not quantitatively distinguish species in mixed assemblages. To address this issue we developed two primer sets: one revealed itself to be genus specific for Pseudochattonella and the other species-specific for P. verruculosa. By subtracting cell estimates for P. verruculosa from combined results we could calculate cell numbers for P. farcimen. This approach has overcome the challenges posed by the very limited sequence availability and low gene variability between the two species. The qPCR assay was extensively tested for specificity, efficiency and sensitivity over an entire growth cycle in both single and mixed assemblages. Comparison of cell abundance estimates obtained by qPCR assay and microscopy showed no statistically significant difference until stationary and death phases. The assay was also tested on environmental samples collected during a small Pseudochattonella bloom in Denmark in March-April 2015. It was impossible to distinguish P. farcimen and P. verruculosa by light microscopy but qPCR showed both species were present. The two methods provided nearly identical cell numbers but the assay provided discrimination and enumeration of both species.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…), Prymnesium parvum (Galluzzi et al. , Manning and Claire , Eckford‐Soper and Daugbjerg ), Pfiesteria spp. (Bowers et al.…”

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confidence: 99%

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A quantitative real‐time PCR assay for identification and enumeration of the occasionally co‐occurring ichthyotoxic Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae) and analysis of variation in gene copy numbers during the growth phase of single and mixed cultures

Eckford-Soper

Daugbjerg

2016

Journal of Phycology

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Development of sensitive genotype‐specific quantitative polymerase chain reaction methods for detection of Phaeocystis globosa in the South China Sea

Niu

Liu

Zhang

et al. 2021

Limnology & Ocean Methods

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Since 1997, dense blooms of the alga Phaeocystis globosa have occurred in the South China Sea (SCS), and previous studies have revealed the co‐existence in this sea of two types of P. globosa cell characterized by different marker pigment profiles. However, owing to a lack of quantitative methods for the detection of different types of P. globosa cells, the dynamics of P. globosa blooms in the SCS are still poorly understood. In this study, on the basis of phylogenetic analysis of the mitochondrial atp8 (Mit atp8) gene and pigment profiles, the P. globosa strains from Pacific and Atlantic coastal waters were divided into four genotypes, of which Type I and Type IV strains co‐exist in the SCS. Furthermore, two genotype‐specific quantitative polymerase chain reaction (qPCR) methods were developed targeting the Mit atp8 gene for quantitative determinations of the densities of Type I and Type IV P. globosa cells. Both qPCR methods were reproducible, highly sensitive, and specific with a wide range of detection (from 30 to 1 × 108 cells L−1) for target P. globosa genotypes in the field, thereby facilitating the detection of low cell densities prior to bloom development, as well as high cell densities during bloom periods. Finally, the developed qPCR assays were successfully applied to determine the distribution patterns of Type I and Type IV P. globosa in the Beibu Gulf, a region of the SCS characterized by a high frequency of P. globosa blooms.

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Quantitative PCR for Monitoring the Plant Growth-Promoting Microalgae from Rice Fields

Rajendran,

Dharumadurai

2024

Methods and Protocols in Food Science

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Development of a multiplex real-time qPCR assay for simultaneous enumeration of up to four marine toxic bloom-forming microalgal species

Cited by 39 publications

References 29 publications

A quantitative real‐time PCR assay for identification and enumeration of the occasionally co‐occurring ichthyotoxic Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae) and analysis of variation in gene copy numbers during the growth phase of single and mixed cultures

A quantitative real‐time PCR assay for identification and enumeration of the occasionally co‐occurring ichthyotoxic Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae) and analysis of variation in gene copy numbers during the growth phase of single and mixed cultures

Development of sensitive genotype‐specific quantitative polymerase chain reaction methods for detection of Phaeocystis globosa in the South China Sea

Quantitative PCR for Monitoring the Plant Growth-Promoting Microalgae from Rice Fields

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