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, L. E.
AU - Hitchcock, G. L.
AU - Rein, K. S.
AU - Sinigalliano, C. D.
AU - Fell, J. W.
N1 - Funding Information:
Funding was provided in part by the NSF-NIEHS Oceans and Human Health Center Program (NSF 0432368 and NIEHS P50 ES12736). Participation in the MERHAB Cruise was courtesy of Dr. Gary Kirkpatrick, Mote Marine Laboratory, Sarasota, FL, USA. Cell counts for the Pine Island Sound samples were provided courtesy of V. Palubok (Mote Marine Laboratory) and for the MERHAB Cruise samples courtesy of R. Pigg.[SS]
PY - 2009/1
Y1 - 2009/1
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
UR - http://www.scopus.com/inward/record.url?scp=57649244971&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=57649244971&partnerID=8YFLogxK
U2 - 10.1016/j.hal.2008.05.003
DO - 10.1016/j.hal.2008.05.003
M3 - Article
AN - SCOPUS:57649244971
VL - 8
SP - 196
EP - 211
JO - Harmful Algae
JF - Harmful Algae
SN - 1568-9883
IS - 2
ER -