Application of loop-mediated isothermal amplification combined with lateral flow dipstick to rapid and sensitive detection of Alexandrium catenella.

Abstract

Alexandrium catenella is one of the globally distributed toxic marine microalgae to cause paralytic shellfish poisoning that poses a great threat to marine fisheries, economy, and public health. Development of efficient and sensitive methods for accurate identification of A. catenella to minimize its damage is therefore necessary. In this study, a novel method referred to as loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) (LAMP-LFD) was established for rapid and sensitive detection of A. catenella. The internal transcribed spacer (ITS) gene of A. catenella was cloned for sequencing and used as target for LAMP-LFD. Three sets of LAMP primers (AcLF1, AcLF2, and AcLF3) targeting the ITS were successfully designed, among which AcLF2 displaying the best performance was used in the subsequent tests. A specific LFD probe targeting the amplification region of AcLF2 was further designed. The LAMP-LFD detection system was established and the amplification conditions were optimized. Cross-reactivity tests with common marine microalgae showed that the LAMP-LFD was exclusively specific for A. catenella. The detection limits of LAMP-LFD for A. catenella genomic DNA and the plasmid containing the ITS were 4.63 × 10-4 ng μL-1 and 1.26 × 104 copies μL-1, displaying a sensitivity that is 10 times higher than that of SYBR Green I assay and 100 times higher than that of conventional PCR, respectively. Finally, the practicability of LAMP-LFD was confirmed by test with spiked samples. LAMP-LFD revealed a detection limit of approximately 0.1 cell mL-1, which was 100 times more sensitive than conventional PCR. The optimized LAMP-LFD protocol can be completed within 75 min. Therefore, the established LAMP-LFD is a specific, sensitive, and rapid method that is possibly applicable to the field monitoring of A. catenella.