Development of an oligonucleotide microarray for the detection and monitoring of marine dinoflagellates.
Harmful Algal Blooms (HABs), mainly caused by dinoflagellates and diatoms, have great economic and sanitary implications. An important contribution for the comprehension of HAB phenomena and for the identification of risks related to toxic algal species is given by the monitoring programs. In the microscopy-based monitoring methods, harmful species are distinguished through their morphological characteristics. This can be time consuming and requires great taxonomic expertise due to the existence of morphologically close-related species. The high throughput, automation possibility and specificity of microarray-based detection assay, makes this technology very promising for qualitative detection of HAB species. In this study, an oligonucleotide microarray targeted to the ITS1-5.8S-ITS2 rDNA region of nine toxic dinoflagellate species/clades was designed and evaluated. Two probes (45-47 nucleotides in length) were designed for each species/clade to reduce the potential for false positives. The specificity and sensitivity of the probes were evaluated with ITS1-5.8S-ITS2 PCR amplicons obtained from 20 dinoflagellates cultured strains. Cross hybridization experiments confirmed the probe specificity; moreover, the assay showed a good sensitivity, allowing the detection of up to 2 ng of labeled PCR product. The applicability of the assay with field samples was demonstrated using net concentrated seawater samples, un-spiked or spiked with known amounts of cultured cells. Despite the general application of microarray technology for harmful algae detection is not new, a peculiar group of target species/clades has been included in this new-format assay. Moreover, novelties regarding mainly the probes and the target rDNA region have allowed sensitivity improvements in comparison to previously published studies.
Department of Biomolecular Sciences, Section of Biotechnology, University of Urbino, Fano (PU), Italy. luca.galluzzi@uniurb.it
Luca
L
Galluzzi
Alessandra
A
Cegna
Silvia
S
Casabianca
Antonella
A
Penna
Nick
N
Saunders
Mauro
M
Magnani
D016054:DNA, Protozoan / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D004141:Dinoflagellida / Q000235:genetics / Q000302:isolation & purification*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D012623:Seawater / Q000469:parasitology*; D012680:Sensitivity and Specificity
D016054:DNA, Protozoan; D021903:DNA, Ribosomal Spacer