A novel sandwich hybridization assay method and probe sets for cyanobacterial harmful algal bloom detection

A novel, rapid (2.5 h) sandwich hybridization assay (SHA) method for the detection of harmful cyanobacterial genera has been developed based on oligonucleotide hybridization to genus-specific sequences within the 16S ribosomal RNA. Unlike previously-developed 96-well plate SHA formats, this modification does not involve a robotic processor to maneuver reagents across the plate. Rather, it requires manual pipetting each reagent sequentially into the same well and washing the 96-well plate between each addition, thereby increasing sample throughput. Another key difference is the colorimetric reporter molecule. Yet, it enables specific detection of Planktothrix, Raphidiopsis and the Anabaena-Dolichospermum-Aphanizomenon (ADA) clade of the Nostocales. As these are the most common planktonic bloom-forming cyanobacteria, the method can be used to assess potential threats posed by these taxa, specifically cyanotoxins that are associated with particular bloom-forming taxa. The assays detected target groups and standard curves were created from cultured cyanobacteria biomass. Here, the SHA method, calibration, and the field performance of the assay on cyanobacterial bloom biomass is described. Linear standard curves were established for each assay, with a Planktothrix and Raphidiopsis quantification range of 10-500 mm³ L^-1 100 μL homogenate^-1 and a range of 50-500 mm³ L^-1 100 μL homogenate^-1 for the ADA clade assay. In environmental samples from three Ohio waterbodies, the assays detected the target genus in a mixed cyanobacterial community. This new SHA protocol will be highly beneficial to both management and research communities because it considerably broadens the capability to rapidly detect multiple cyanobacterial genera that cause persistent toxic blooms.