Journal
HARMFUL ALGAE
Volume 8, Issue 2, Pages 196-211Publisher
ELSEVIER
DOI: 10.1016/j.hal.2008.05.003
Keywords
Bead array; Harmful Algal Bloom; Luminex; Simultaneous molecular detection
Categories
Funding
- NSF-NIEHS Oceans
- Human Health Center Program [0432368, P50 ES12736]
- MERHAB
- MERHAB Cruise
- NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES [P50ES012736] Funding Source: NIH RePORTER
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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. (C) 2008 Elsevier B.V. All rights reserved.
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