Recombinase polymerase amplification combined with lateral flow dipstick assay for rapid and sensitive detection of the toxigenic harmful algae bloom species Pseudo-nitzschia multistriata
摘要
The diatom genus Pseudo-nitzschia includes many species that are cosmopolitan and can cause toxigenic harmful algal blooms (HABs). These blooms are capable of producing domoic acid (DA), a kainic acid-type neurotoxin that causes amnesic shellfish poisoning (ASP), which poses threats to marine animals and humans. It is thus important to develop rapid technologies that enable monitoring of toxigenic Pseudo-nitzschia species with high specificity and sensitivity to facilitate early warning. In this study, we developed a rapid assay for the toxigenic species P. multistriata, based on its dabA gene, which has been demonstrated to be a key gene in DA biosynthesis, combining recombinase polymerase amplification with lateral flow dipstick (RPA-LFD). The RPA-LFD assay is specific to P. multistriata and has high sensitivity, with a detection limit of 0.02 ng μL−1 for genomic DNA, a cell density detection limit of approximately 1 cells/reaction, and a detection limit of 3.36 × 101 copies μL−1 for recombinant plasmid DNA. The established RPA-LFD assay can complete the reaction in just 30 min at 37 °C, and the results can be directly visualized on the LFD within a short period of time, typically within 10 min. The practicality of this assay for on-site detection has also been verified in this study. The RPA-LFD assay with the high specific and sensitivity developed in this study will aid in predicting toxigenic P. multistriata blooms.