Environmental Science and Pollution Research

, Volume 22, Issue 13, pp 9704–9716 | Cite as

Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples

  • Gary R. McCoy
  • Nicolas Touzet
  • Gerard T. A. Fleming
  • Robin Raine
Research Article

Abstract

The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5′ end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland.

Keywords

HABs Molecular probes MIDTAL Microarray RNA Prymnesium 

Notes

Acknowledgments

The authors would like to acknowledge the assistance of Simon Kennedy (Killary Fjord Shellfish), Sarah Cosgrove and Annette Wilson during the sampling survey period. We would also like to acknowledge the continuous guiding support from all the MIDTAL project partners. This work was funded through the EU 7th Framework Programme (FP7-ENV-2007-1-MIDTAL-201724).

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Gary R. McCoy
    • 1
  • Nicolas Touzet
    • 1
    • 2
  • Gerard T. A. Fleming
    • 1
  • Robin Raine
    • 1
  1. 1.The Ryan InstituteNational University of Ireland, GalwayGalwayIreland
  2. 2.School of Science, Centre for Environmental Research, Innovation and SustainabilityInstitute of Technology SligoSligoIreland

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