Development of a Rapid Detection Method to Prorocentrum lima by Loop-Mediated Isothermal Amplification with Hydroxy Naphthol Blue.

Abstract

Prorocentrum lima, a widely distributed dinoflagellate known for its production of harmful biotoxins, poses a significant threat to humans, aquaculture, and marine ecosystems. As a result, the detection of this toxic alga in coastal waters has become an urgent research focus. In this study, a rapid, sensitive, and cost-effective detection method based on loop-mediated isothermal amplification (LAMP) was developed to identify P. lima. In this method, cell extracts of P. lima were diluted and used directly as templates for amplification, eliminating the need for nucleic acid purification and simplifying the detection process. Hydroxy naphthol blue (HNB) was incorporated into the reaction mix to facilitate result interpretation, enabling visual determination of the amplification outcome with the naked eye. The entire detection process, from DNA extraction to template amplification and product detection, could be completed within 80min using a simple constant temperature-control device. This LAMP-based detection method demonstrated excellent reliability, specificity, and a low detection limit of 5.87 cells/mL for DNA crude extract. The assay offered an efficient alternative to PCR for rapid detection of P. lima. By streamlining the detection process and offering a visual readout, this technique holds promise for efficient and routine monitoring of harmful algal species, benefitting both research efforts and environmental management strategies.