Development of multiplexed sandwich hybridization assay for in situ detection of freshwater harmful bloom-forming cyanobacterial genera

Freshwater cyanobacteria harmful algal blooms (CHABs) are being observed more frequently due to eutrophication, more robust monitoring programs and a warmer climate at the global level. Oftentimes, CHABs result in the production of toxins such as microcystins, anatoxins, cylindrospermopsins, and saxitoxins, that are harmful or fatal to humans and other animals. Quick and informed decisions are needed for better management of CHABs. To reduce the time between a suspected bloom and subsequent management decisions, a field-deployable and multiplexed sandwich hybridization assay (SHA) was developed to detect the prominent bloom-forming and potentially toxin-producing cyanobacterial genera. The initial targeted genera were Microcystis, Anabaena/Dolichospermum, Aphanizomenon, Planktothrix, Cylindrospermopsis, Lyngbya/Microseira, and Phormidium/Microcoleus. The key components of a typical SHA included a target-specific capture probe, a universal signal probe and a location-specific “zip-code” tag for multiplexing. We designed oligonucleotide capture probes based on the phylogenetic analysis of a curated database of more than 1400 sequences of cyanobacterial 16S rRNA genes to target their hypervariable regions. Initial testing of capture probe specificity utilized fluorescent in-situ hybridization (FISH) and probes with the best results were further tested in combination with a signal probe using streptavidin-coated plates. All testing results will be presented, and lessons learned will be discussed.