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Real-time PCR Detection of Dinophysis Species in Irish Coastal Waters

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Abstract

Diarrhetic shellfish toxin-producing Dinophysis species occur in Irish coastal waters throughout the year. Dinophysis acuta and Dinophysis acuminata are the most commonly occurring species and are responsible for the majority of closures of Irish mussel farms. This study describes the development of a qualitative real-time polymerase chain reaction (PCR) assay for identification of D. acuta and D. acuminata in Irish coastal waters. DNA sequence information for the D1-D2 region of the large ribosomal sub-unit (LSU) was obtained, following single-cell PCR of D. acuta and D. acuminata cells isolated from Irish coastal locations. PCR primers and hybridization probes, specific for the detection of D. acuta, were designed for real-time PCR on the LightCycler™. The LightCycler™ software melt curve analysis programme determined that D. acuta was identified by a melt-peak at 61°C, while D. acuminata cells produced a melt peak at 48°C. The limit of detection of the real-time PCR assay was determined to be one to ten plasmid copies of the LSU D1-D2 target region for both species and one to five D. acuminata cells. Lugol's preserved water samples were also tested with the assay. The real-time PCR assay identified Dinophysis species in 100% of samples found to contain Dinophysis species by light microscopy and had a greater than 90% correlation with light microscopy for identification of D. acuta and D. acuminata in the samples. The assay can identify and discriminate D. acuta and D. acuminata at low numbers in Irish waters and has the potential to add value to the Irish phytoplankton monitoring programme.

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Acknowledgements

The study was carried out as part of the PHYTOTEST project, funded by the National Development Plan (NDP) Marine Institute Strategic Research Program-Advanced Technologies (Project No: AT04/02/02). The authors would like to express gratitude to the FRS, Marine Laboratory, Scotland, for the provision of samples containing Dinophysis cells that were used in this study.

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Authors and Affiliations

  1. Molecular Diagnostics Research Group, National University of Ireland, Galway, Ireland

    Siobhán Kavanagh, Claire Brennan, Louise O’Connor & Majella Maher

  2. Marine Institute, Rinville, Oranmore, Co, Galway, Ireland

    Siobhán Moran, Rafael Salas, Josephine Lyons & Joe Silke

Authors
  1. Siobhán Kavanagh
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  2. Claire Brennan
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  3. Louise O’Connor
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  4. Siobhán Moran
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  5. Rafael Salas
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  6. Josephine Lyons
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  7. Joe Silke
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  8. Majella Maher
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Corresponding author

Correspondence to Majella Maher.

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Supplementary Table 1

Marine Institute phytoplankton monitoring programme samples (25 ml) used in this study. Light microscopy results and real-time polymerase chain reaction assay results are included. a–lRefer to corresponding samples in Fig. 3 (DOC 96 kb)

Supplementary Table 2

Phytoplankton specificity panel assayed with the Dinophysis real-time polymerase chain reaction (PCR) assay. aDenotes phytoplankton species that were directly tested with the Dinophysis real-time PCR assay as single cell templates or DNA extracts of pure cultures. bDenotes phytoplankton species that were present in Marine Institute phytoplankton monitoring programme samples. These species were identified microscopically (DOC 60 kb)

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Kavanagh, S., Brennan, C., O’Connor, L. et al. Real-time PCR Detection of Dinophysis Species in Irish Coastal Waters. Mar Biotechnol 12, 534–542 (2010). https://doi.org/10.1007/s10126-009-9238-6

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  • DOI: https://doi.org/10.1007/s10126-009-9238-6

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