Rapid and sensitive detection of Amphidinium carterae by loop-mediated isothermal amplification combined with a chromatographic lateral-flow dipstick

摘要

Frequent outbreaks of toxic algal blooms devastate marine ecosystems, marine fisheries, and public health. Monitoring toxic algae is crucial to reduce losses caused by imminent algal blooms. However, traditional detection techniques relying on morphological examination are tedious and time-consuming. Therefore, the development of convenient strategies to detect toxin-producing microalgae is necessary. In this study, a novel method for the rapid, sensitive detection of Amphidinium carterae by loop-mediated isothermal amplification (LAMP) combined with a chromatographic lateral-flow dipstick (LFD) was established. The partial internal transcribed spacer gene was PCR amplified, cloned, and sequenced to design four LAMP primers and a detection probe for A. carterae detection. The LAMP detection conditions were optimized, and the optimum parameters were determined to be the following: dNTP concentration, 1.2 mM; betaine concentration, 1.2 M; magnesium ion concentration, 8 mM; ratio of inner primer to outer primer, 8:1; amplification temperature, 59 degrees C; and amplification time, 60 min. The specificity of LAMP-LFD was confirmed by cross-reactivity tests with other algal species. LAMP-LFD was 100 times more sensitive than regular PCR and similarly sensitive as LAMP and SYBR Green I. LAMP-LFD can be completed within 70 min and did not require special detection equipment. The convenience of the established LAMP-LFD assay was further validated by tests with simulated field-water samples. In conclusion, the developed LAMP-LFD assay can be used as a reliable and simple method of detecting A. carterae.