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 language | English |
|---|---|
| Pages (from-to) | 196-211 |
| Number of pages | 16 |
| Journal | Harmful Algae |
| Volume | 8 |
| Issue number | 2 |
| DOIs | |
| State | Published - Jan 1 2009 |
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Keywords
- Bead array
- Harmful Algal Bloom
- Luminex
- Simultaneous molecular detection
ASJC Scopus subject areas
- Aquatic Science
- Plant Science
Cite this
Multiple simultaneous detection of Harmful Algal Blooms (HABs) through a high throughput bead array technology, with potential use in phytoplankton community analysis. / Scorzetti, G.; Brand, Larry E; Hitchcock, G. L.; Rein, K. S.; Sinigalliano, C. D.; Fell, J. W.
In: Harmful Algae, Vol. 8, No. 2, 01.01.2009, p. 196-211.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Multiple simultaneous detection of Harmful Algal Blooms (HABs) through a high throughput bead array technology, with potential use in phytoplankton community analysis
AU - Scorzetti, G.
AU - Brand, Larry E
AU - Hitchcock, G. L.
AU - Rein, K. S.
AU - Sinigalliano, C. D.
AU - Fell, J. W.
PY - 2009/1/1
Y1 - 2009/1/1
N2 - 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.
AB - 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.
KW - Bead array
KW - Harmful Algal Bloom
KW - Luminex
KW - Simultaneous molecular detection
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U2 - 10.1016/j.hal.2008.05.003
DO - 10.1016/j.hal.2008.05.003
M3 - Article
VL - 8
SP - 196
EP - 211
JO - Harmful Algae
JF - Harmful Algae
SN - 1568-9883
IS - 2
ER -