Development of loop-mediated isothermal amplification combined with a chromatographic lateral-flow dipstick for rapid detection of Chattonella marina

Abstract

Harmful algal blooms caused by Chattonella marina recently have caused severe negative effect on coastal economy worldwide, with increased occurrence frequency and scale. It is therefore vital to establish new methods for rapid detection of this alga. In this study, the internal transcribed spacer (ITS) sequence was used as the target gene for molecular detection of C. marina. First, four loop-mediated isothermal amplification (LAMP) primers were designed based on the six regions of ITS, and the LAMP reaction system was established using these primers. Next, a probe was designed to detect the LAMP products by lateral-flow dipstick (LFD). Finally, a new method for rapid and sensitive detection of C. marina that is referred to as LAMP-LFD was established. The LAMP reaction system, amplification time, and amplification temperature were particularly optimized. The optimal parameters are as follows: Mg2+ concentration, 10 mM；dNTP concentration, 1.2 mM；ratio of internal primer concentration to outer primer concentration, 8:1；reaction time, 60 min；and reaction temperature, 60 °C. Both specificity and sensitivity were tested using the optimized LAMP reaction system in combination with LFD (LAMP-LFD). The established LAMP-LFD displayed good specificity and no cross reaction was detected with non-target algal species. The detection limit of LAMP-LFD was 3.4 × 10−4 ng μL−1 (3.4 × 10−4 ng per reaction) for the genomic DNA of target algae, and 1.3 copies μL−1 (1.3 copies per reaction) for the plasmid DNA containing the target ITS. Sensitivity tests using genomic DNA and plasmid DNA as templates consistently revealed that LAMP-LFD is 100 times more sensitive than regular PCR. The established LAMP-LFD was applied to analyze the simulated samples and the results showed that the detection limit of LAMP-LFD could reach 1 cell mL−1. LAMP-LFD also demonstrated good specificity and sensitivity in the analysis of natural samples. The whole procedure of LAMP-LFD could be completed within 1.5 h. Taken together, the LAMP-LFD assay developed here is characterized by simplicity, high specificity and sensitivity, and rapidity and therefore is promising for rapid detection of C. marina.