• Development & implementation of the Phytotest project

      Kavanagh, S.; Brennan, C.; Lyons, C.; Chamberlain, T.; Salas, R.; Moran, S.; Silke, J.; Maher, M. (Marine Institute, 2008)
      Phytotest is a 3-year research and development project funded through the Marine Institute Strategic Research Programme in Advanced Technologies as part of the National Development plan 2000-2006. The project is a collaboration between the National Diagnostics Centre at NUI Galway and the MI and involves the development of real-time PCR assays for Dinophysis and Pseudo-nitzschia species that are important in Irish waters. In the current final phase of the project, the real-time PCR assays are being transferred to the MI to support the phytoplankton monitoring service.
    • Molecular methods for monitoring harmful algal bloom species

      Keady, E.; Maher, M. (Marine Institute, 2009)
      Shellfish production can be adversely affected by the presence of harmful microalgae (HABs). Toxins produced by Dinophysis, Alexandrium and Pseudo-nitzschia species can accumulate in shellfish and have the potential to cause serious human illness. In order to satisfy EU legislative requirements pertaining to the production and export of shellfish (EC Hygiene Regulations 2004, No. 853/2004 and No. 854/2004, which replaced the EU Shellfish Hygiene Directive 91/492/EEC in January 2006), monitoring the presence of harmful algal species and biotoxins in coastal waters is performed by EU member states. Routine microscopic monitoring methods are unable to identify certain toxic species, in particular, Alexandrium and Pseudo-nitzschia spp. Electron microscopy is required for species identification and this technique cannot be integrated into a routine monitoring programme. Molecular techniques utilise unique sequence signatures within microorganism genomes for species specific identification. Molecular methods applied for the identification and quantification of HAB species include Fluorescent in-situ hybridisation (FISH) and in-vitro amplification based methods, in particular, real-time PCR.
    • Nucleic acid tests for toxic phytoplankton in Irish waters-phytotest: Marine Strategic RTDI project AT/04/02/02 - research update

      Maher, M.; Kavanagh, S.; Brennan, C.; Moran, M.; Salas, R.; Lyons, J.; Silke, J. (Marine Institute, 2007)
      The Phytotest project is a 3 year collaborative project funded through the Marine Strategic Programme in Advanced Technologies as part of the National Development plan 2000-2006. The project partners include the National Diagnostics Centre at NUI Galway and MI. The overall objective of the project is the development of nucleic acid tests (molecular methods) for the identification of key toxic phytoplankton species in Irish waters. In the final year of the programme the aim is to transfer the molecular methods developed in the project into MI to support their monitoring service. Currently, the monitoring for phytoplankton species in Irish waters is performed by light microscopy which can easily identify some plankton species based on distinctive morphological traits. Other species in particular, Pseudonitzschia spp. and Alexandrium spp. cannot be identified to species level by light microscopy. Identification of these species requires more sophisticated microscopic techniques such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM). These techniques cannot easily be integrated into a routine testing environment. Molecular methods utilise unique information contained within an organism’s genome in order to identify it. This genetic information can be exploited in a range of molecular test platforms enabling microorganisms to be identified to species level. Additionally, there has been a major drive towards the development of highly automated platforms to support molecular tests for high-throughput testing in routine laboratory settings.
    • Real-time PCR detection of Dinophysis species in Irish coastal waters

      Kavanagh, S.; Brennan, C.; O’Connor, L.; Moran, S.; Salas, R.; Lyons, J.; Silke, J.; Maher, M. (Springer Verlag, 2010)
      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.