A new sandwich hybridization assay method to identify and quantify Microcystis spp.

Cyanobacteria are responsible for the largest number of harmful algal blooms (HABs) worldwide. HABs caused by the genus Microcystis are health threats because they often occur within close proximity to humans and produce phycotoxins such as microcystins that can contaminate drinking water and recreational areas. Molecular techniques enable accurate and rapid (similar to 1 h) HAB detection that facilitates monitoring, "early warnings" of blooms, and corresponding management responses. Sandwich hybridization assay (SHA), the technique considered here, directly (no amplification) identifies and quantifies plankton species using ribosomal RNA (rRNA)-targeted oligonucleotides. This project focused on the development of a new SHA method for the detection of Microcystis (16S rRNA) using laboratory cultures. Assay calibration curve and limits of detection were determined using Microcystis aeruginosa, though signal intensity differed significantly (p < 0.05) between three species (M. aeruginosa, Microcystis botrys, Microcystis wesenbergii). SHA results for M. aeruginosa raised under three light intensities (40, 60, and 100 mu mol photons m(-2) s(-1)) and two temperatures (25 degrees C and 32 degrees C) were greatest at the highest irradiance for both temperatures, but otherwise variable, yielding an overall significant (p < 0.05) interaction between light and temperature. The 32 degrees C treatment also resulted in significantly lower (p < 0.05) Photosystem II quantum efficiency (F-v/F-m) and microcystins concentrations per light intensity. Spiked field experiments showed that SHA signal was not significantly affected (p > 0.05) by a mixed phytoplankton assemblage. The assay speed, wide detection range, and specificity indicate that this method has promise for additional field studies and HAB monitoring.