Multiple simultaneous detection of Harmful Algal Blooms (HABs) through a high throughput bead array technology, with potential use in phytoplankton community analysis

G. Scorzetti, L. E. Brand, G. L. Hitchcock, K. S. Rein, C. D. Sinigalliano, J. W. Fell

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

As an alternative to traditional, morphology-based methods, molecular techniques can provide detection of multiple species within the HAB community and, more widely, the phytoplankton community in a rapid, accurate and simultaneous qualitative analysis. These methods require detailed knowledge of the molecular diversity within taxa in order to design efficient specific primers and specific probes able to avoid cross-reaction with non-target sequences. Isolates from Florida coastal communities were sequence-analyzed and compared with the GenBank database. Almost 44% of the genotypes obtained did not match any sequence in GenBank, showing the existence of a large and still unexplored biodiversity among taxa. Based on these results and on the GenBank database, we designed 14 species-specific probes and 4 sets of specific primers. Multiple simultaneous detection was achieved with a bead array method based on the use of a flow cytometer and color-coded microspheres, which are conjugated to the developed probes. Following a parallel double PCR amplification, which employed universal primers in a singleplex reaction and a set of species-specific primers in multiplex, detection was performed in a cost effective and highly specific analysis. This multi-format assay, which required less than 4 h to complete from sample collection, can be expanded according to need. Up to 100 different species can be identified simultaneously in a single sample, which allows for additional use of this method in community analyses extended to all phytoplankton species. Our initial field trials, which were based on the 14 species-specific probes, showed the co-existence and dominance of two or more species of Karenia during toxic blooms in Florida waters.

Original languageEnglish (US)
Pages (from-to)196-211
Number of pages16
JournalHarmful Algae
Volume8
Issue number2
DOIs
StatePublished - Jan 1 2009

Keywords

  • Bead array
  • Harmful Algal Bloom
  • Luminex
  • Simultaneous molecular detection

ASJC Scopus subject areas

  • Aquatic Science
  • Plant Science

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