Optimizing DNA extraction and quantitative PCR protocols for the efficient and precise quantification of the toxic...

In southwest Florida, Harmful Algal Blooms (HABs) caused by the dinoflagellate Karenia brevis occur nearly annually. Monitoring efforts typically depend on the enumeration of K. brevis, identified using morphological characters via light microscopy. Although this technique remains the standard, it requires taxonomic expertise, is not high-throughput, and may be biased towards cells displaying typical morphology. To overcome these limitations, we explore the use of a modified qPCR assay that targets the rbcL gene for monitoring K. brevis in water samples and marine sediments. We tested seven DNA extraction kits and found that Qiagen's DNeasy Plant Mini Kit yielded the most DNA and gave consistent results when working with membrane filters, but the DNeasy PowerSoil Kit returned better results when working with marine sediments. Both kits successfully prevented PCR inhibition. Using a synthetic gBlock we calculated an average of 252 ± 152 rbcL gene copies cell⁻¹ in cultured K. brevis and a LOD of 7.4 copies/reaction and a LOQ of 641 copies/reaction for the rbcL qPCR assay. Furthermore, we extend the use of this assay to a droplet digital PCR platform. We found positive detections of K. brevis DNA in marine sediments that correspond with recent HAB events and with signals of fewer than one cell per reaction. These positive detections reveal the presence of K. brevis DNA in marine sediments, raising the question of whether it resulted from free DNA, intact vegetative cells, or a resting stage (cells/cysts). Since cyst-like life-cycle stages have long been hypothesized to contribute critically to bloom dynamics, our results highlight the importance of further research on sediments, as this may hold the key to fully characterizing the life cycle of K. brevis and associated bloom dynamics.