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
- 290 Downloads
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.
KeywordsHABs Molecular probes MIDTAL Microarray RNA Prymnesium
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).
- Edvardsen B, Paasche E (1998) Bloom dynamics and physiology of Prymnesium and Chrysochromulina. In: Anderson DM, Cembella AD, Hallegraeff GM (eds) Physiological ecology of harmful algal blooms. Springer-Verlag, Berlin, Heidelberg, Germany, pp 198–208Google Scholar
- Guillard RR, Ryther JH (1962) Studies of marine planktonic diatoms: I. cyclotella nana hustedt, and detonula confervacea (cleve) gran. Can J Microbiol 8:229–239Google Scholar
- Lewis J, Medlin LK, Raine R (2012) MIDTAL (Microarrays for the Detection of Toxic Algae): a protocol for a successful microarray hybridisation and analysis. Koeltz, GermanyGoogle Scholar
- Lim EL, Amaral LA, Caron DA, Delong EF (1993) Application of rRNA-based probes for observing marine nanoplanktonic protists. Appl Environ Microbiol 59:1647–1655Google Scholar
- McCoy GR, Raine R, Medlin LK, et al. (2012) Field testing for toxic algae with a microarray: initial results from the MIDTAL project. ICHA14 Conference Proceedings Crete 2010Google Scholar
- McCoy GR, Kegel JU, Touzet N, Fleming GTA, Medlin LK, Raine R (2014a) An assessment of RNA content in Prymnesium parvum, Prymnesium polylepis, cf. Chattonella sp. and Karlodinium veneficum under varying environmental conditions for calibrating an RNA microarray for species detection. FEMS Microbiol Ecol. doi: 10.1111/1574-6941.12277 Google Scholar
- McDermott G, Raine R (2010) Settlement bottle method for quantitative phytoplankton analysis. In: Karlson B, Cusack C, Bresnan E (eds) Microscopic and molecular methods for quantitative phytoplankton analysis. IOC of UNESCO, Paris, pp 21–24Google Scholar
- Moestrup Ø (1994) Economic aspects: ‘blooms’, nuisance species, and toxins. In: Green JC and Leadbeater BSC (ed) The haptophyte algae, Systematics Association Special Volume No. 51, Clarendon Press, Oxford, pp 265-285Google Scholar
- Scholin CA and Anderson DM (1998) Detection and quantification of HAB species using antibody and DNA probes: progress to date and future research objectives. In: Regura B, Blanko J, Fernandez ML and Wyatt T (eds). Harmful algae. Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO. pp. 253–257Google Scholar
- Smayda TJ (1990) Novel and nuisance phytoplankton blooms in the sea. In: Graneli E, Sundstrom B, Edler L, Anderson DM (eds) Evidence for a global epidemic, In Toxic marine phytoplankton. Elsevier, New York, pp 29–40Google Scholar
- Thondsen J (1978) Preservation and storage. In: Sournia A (ed) Phytoplankton manual. UNESCO, Paris, p 69–74Google Scholar