Molecular detection of harmful algal blooms (HABs) using locked nucleic acids and bead array technology

Abstract

Harmful algal bloom (HABs) events are a threat to public health, marine life, commercial fisheries, tourism, recreation, and the environment. These outbreaks are known to decrease water quality by increasing the biological O 2 demand, which can cause die-offs of marine life (Figley et al. 1979). Expanded spatial distribution of HABs can cover over a thousand kilometers and are triggered by the effects of winds, currents, storms, and ship ballast activities (Tester and Steidinger 1997;Doblin et al. 2004;Bolch and Salas 2007). Increases in HAB frequency and duration are probably fueled by changes in sea surface temperature, salinity, grazing, turbulence, irradiance, or increases in nutrient loads derived from run off from agricultural and urban areas (Reynolds 1989;Sterner 1989;Thomas and Gibson 1990; Anderson et al. 2002;Xiaonan and Wei 2004).HABs are formed by species of phytoplankton or cyanobacteria, which can produce toxins that can be transferred through marine food webs, affecting consumers at higher trophic levels including fish, shellfish, marine mammals, and humans (Sellner et al. 2003;Smayda 1997 AbstractHarmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid-modified oligonucleotides (LNA) and Mirus Label IT ® nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5¢ terminal labeling technique, the chemicallabeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions.

Karenia: The biology and ecology of a toxic genus

“…This bead array method is based on color coded microspheres, which are conjugated to probes designed for each single species (Scorzetti et al, 2009). Recently the same technology was used with locked-nuclei acid modified capture probes for the detection of K. brevis and K. mikimotoi (Diaz et al, 2010). This flow cytometry-based technology has the potential for detection and quantification of multiple (up to 100) RNA targets simultaneously (Scorzetti et al, 2009;Diaz et al, 2010).…”

Approaches for the detection of harmful algal blooms using oligonucleotide interactions

“…Higher affinity and fluorescent signals were obtained from the LNA, with the results being more significant when the assay was performed at temperatures above 53°C. This research allowed an integration between LNA probes, Luminex technology and direct chemical labelling (Mirys Label IT) for simple identification and quantification of K. brevis and K. mikimotoi [111].…”

Molecular Techniques for the Detection of Organisms in Aquatic Environments, with Emphasis on Harmful Algal Bloom Species

“…Hybridization of the target is detected using a reporter molecule (e.g., phycoerythrin coupled to streptavidin). Multiplexed, bead-based arrays that employ flow cytometric detection of color-coded fluorescent bead populations have been developed as a microarray for toxic algal species [ 90 , 91 ]. Luminex-based detection strategies are still considered preliminary research and development efforts.…”