| Abstract |
Cyanobacteria of the Microcystis genus often form blooms in freshwater and estuarine environments, producing microcystin, a toxin harmful to animals and humans. The analysis of water samples for the detection of toxic Microcystis is usually time-consuming and not effective when the organisms are in pre-bloom concentrations, resulting in exposure of animals and humans to toxins. Thus, early detection of the presence of microcystin-producing Microcystis is crucial. In this work, we present a method based on loop-mediated isothermal amplification (LAMP) to detect the presence of Microcystis cells capable of producing microcystins. Specific primers were designed for the mcyJ gene (biosynthetic gene cluster mcy) and detection of LAMP products was visualized using different approaches, including a fluorescent dye in the reaction mix (pre-stained LAMP), the addition of a posteriori staining using SybrGreen (post-stained LAMP), colorimetric, and lateral flow reaction stripes. The limit of detection (LoD) of the LAMP assay was estimated based on DNA quantification by quantitative real-time PCR (qPCR). The pre-stained, post-stained and colorimetric assays applied to Microcystis NIES cultures achieved the same detection limit of 28 pg/μL of DNA corresponding to 1490 ± 620 toxic Microcystis cells mL-1, within 12.2 min, with 100% sensitivity. The lateral flow reached the lowest LoD, giving a positive signal at a DNA concentration of 2.8 pg/μL (equivalent to ca. 150 toxic Microcystis cells mL-1). The method was applied successfully to various natural water samples. The LAMPs can be optimized for local in-situ use, enabling rapid adaptive monitoring and management of aquatic ecosystems.
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