Universal rapid RNA-based quantification of toxigenic Alexandrium species (Dinophyceae) using quantitative recombinase polymerase amplification

Harmful algal blooms caused by toxigenic Alexandrium species pose recurrent risks to coastal ecosystems and public health, yet current monitoring approaches rely on microscopy and laboratory-based toxin analysis with limited capacity for rapid, functional early warning. Here, we present a universal quantitative reverse-transcriptase recombinase polymerase amplification (qRT-RPA) assay targeting the sxtA4 transcript, an essential gene in saxitoxin biosynthesis. The RPA chemistry is isothermal with a low running temperature, making it suitable for portable, on-site testing. The assay was designed against a conserved sxtA4 region and validated using synthetic templates, isolated amplicons and total RNA from multiple Alexandrium species. Using Alexandrium minutum as a reference, the assay achieved a limit of detection below 103 synthetic RNA copies and 0.1 ng total RNA per reaction, with uniform amplification kinetics across species and a runtime of less than 15 minutes. The assay selectively detected sxtA4 transcripts from toxigenic Alexandrium strains, showed no cross-reactivity with non-target phytoplankton, and retained its LoD in complex RNA matrices. These results establish qRT-RPA as a rapid, RNA-based, functionally informative molecular tool that provides a foundation for portable, early-warning monitoring of toxigenic Alexandrium blooms.