Development of a rapid screening test for Karenia mikimotoi by using loop-mediated isothermal amplification and lateral flow dipstick

Abstract

As a bloom-forming dinoflagellate, Karenia mikimotoi is toxic and globally distributed. Frequent outbreaks of K. mikimotoi blooms usually lead to the mass mortality of pelagic and benthic marine life, resulting in large economic losses. Therefore, the simple, rapid, and highly effective monitoring of K. mikimotoi is crucial to minimize the damage caused by this alga. This study aimed to establish a convenient, cost-effective, and efficient molecular detection technique through loop-mediated isothermal amplification (LAMP) combined with chromatographic lateral flow dipstick (LFD) for the daily monitoring and real-time detection of K. mikimotoi. The internal transcribed spacer (ITS) and large subunit (LSU) rDNA sequences of K. mikimotoi were used to design and screen an optimal primer (KmLF3) and a detection probe (KmLF3HP). The optimized LAMP conditions were as follows: dNTP concentration, 1.2 mM; betaine concentration, 1 M; ratio of the inner primer to the outer primer, 8:1; magnesium ion concentration, 8 mM; amplification temperature, 59 °C; and amplification time, 60 min. Cross-reactivity tests confirmed the specificity of LAMP–LFD. The detection limit of LAMP–LFD for K. mikimotoi genomic DNA was 1.70 × 10−4 ng μL−1, which is 100 times lower than that of conventional PCR. The detection limit of LAMP–LFD for the recombinant plasmid containing the LSU rDNA of K. mikimotoi was 6.21 × 103 copies μL−1, which was 10 times more sensitive than that of LAMP followed by agarose gel electrophoresis and SYBR Green I dye staining and 100 times lower than that of conventional PCR. The practicability of LAMP–LFD was validated by testing with simulated field-water samples, displaying a detection limit of 10−1 cells mL−1. In conclusion, the developed LAMP–LFD is a specific, sensitive, and accurate detection method and may be competent for the field detection of K. mikimotoi.