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

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.

Fig. 1

LSU D1D2 rRNA phylogenetic stick tree comprising CCMP specimen vouchers, FL waters isolates and related GenBank sequences. The tree was constructed with neighbor joining analysis. Bootstrap values are reported on branches when higher than 50%. Clades are shown to indicate their orientation within lineages (see Figs. 2–5).

Fig. 2

Karenia, Karlodinium, Akashiwo and Gymnodinium clusters and unrelated Gymodinium species as from Fig. 1.

Fig. 3

Amphidinium, Coolia, Heterocapsa/Cachonina and Protoceratium clusters as from Fig. 1.

Fig. 4

Alexandrium, Fragilidium, Ceratium, Gonyaulax and Scrippsiella clusters as from Fig. 1.

Fig. 5

Prorocentrum 1, 2 and 3 clusters as from Fig. 1.

Fig. 6

Probe singleplex with different strains of the same target (Heterosigma akashiwo).

Fig. 7

Probe singleplex with culture dilutions of the same target (Prorocentrum micans).

Fig. 8

Probe multiplex with 14 probes against single-species samples.

Fig. 9

Probe and target multiplex with 9 probes and 2-species samples containing uniform DNA amounts for each species.

Fig. 10

Minigel electrophoresis showing different size amplicons generated by specific primers sets.

Fig. 11

Probe and target multiplex with 7 probes and 4-species amplicons generated with species specific primers. The first two samples were originated from a PCR DNA mix with random amount of DNA for each of the 4 species. For the second two samples a cell mix containing a random number of cells for each species was extracted and amplified.

Fig. 12

Probe multiplex on environmental samples collected in Florida waters from Pine Island Sound in July 2006.

Fig. 13

Probe multiplex on environmental samples collected off Charlotte Harbor in October 2006 (MERHAB Cruise).