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Specificity of LSU rRNA-targeted oligonucleotide probes for Pseudo-nitzschia species tested through dot-blot hybridisation

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Abstract

In the scope of the development of a microarray PhyloChip for the detection of toxic phytoplankton species, we designed a large series of probes specific against targets in the nuclear large subunit (LSU) rRNA of a range of Pseudo-nitzschia species and spotted these onto the microarray. Hybridisation with rRNA extracted from monoclonal cultures and from plankton samples revealed many cross-reactions. In the present work, we tested the functionality and specificity of 23 of these probes designed against ten of the species, using a dot-blot procedure. In this case, probe specificity is tested against the target region in PCR products of the LSU rRNA gene marker region blotted on nitrocellulose filters. Each filter was incubated with a species-specific oligoprobe. Eleven of the tested probes showed specific responses, identifying seven Pseudo-nitzschia species. The other probes showed non-specific responses or did not respond at all. Results of dot-blot hybridisations are more specific than those obtained with the microarray approach and the possible reasons for this are discussed.

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Acknowledgments

LB and MVR were funded by the FP7-funded EU consortium MIDTAL (Microarray Detection of Toxic Algae, http://www.midtal.com, contract number 201724). The SZN services “Gestione Ambiente ed Ecologia Costiera” and “Taxonomic Identification of Mediterranean Phytoplankton” are acknowledged for plankton sampling and identification, respectively. Carmen Minucci (SZN) is thanked for DNA extraction and Francisco Rodríguez, Centro Oceanográfico de Vigo of the Instituto Español de Oceanografía, for providing the P. hasleana strain B528. Pasquale de Luca and Linda Medlin provided advice for manuscript improvement.

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  1. Jixin Chen

    Present address: The State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, Xiamen, Fujian, People’s Republic of China

Authors and Affiliations

  1. Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy

    Lucia Barra, Maria Valeria Ruggiero & Wiebe H. C. F. Kooistra

  2. The Laboratory, Marine Biological Association, Citadel Hill, Plymouth, PL1 2 PB, UK

    Jixin Chen

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  1. Lucia Barra
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  2. Maria Valeria Ruggiero
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  3. Jixin Chen
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  4. Wiebe H. C. F. Kooistra
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Correspondence to Wiebe H. C. F. Kooistra.

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Barra, L., Ruggiero, M.V., Chen, J. et al. Specificity of LSU rRNA-targeted oligonucleotide probes for Pseudo-nitzschia species tested through dot-blot hybridisation. Environ Sci Pollut Res 21, 548–557 (2014). https://doi.org/10.1007/s11356-013-1953-x

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