We have established an assay that relies on aptamer and isothermal amplification for the tetrodotoxin (TTX)detection. The method uses triple cycle amplification (strand displacement amplification combined with catalytic hairpin assembly) and fluorescent reporter as an output signal. Free TTX and cDNA compete for binding to aptamer-modified magnetic beads. The cDNA collected by magnetic separation then used as a primer to trigger triple cycle amplification to obtain more ssDNA. The ssDNA combined with the reporter probe, and the original quenched fluorescence can be recovered. In addition, a linear relationship between fluorescence spectrum and different target concentrations is revealed. This method allows TTX to be detected by fluorometry with a detection limit as low as 0.265 pg mL-1. It was applied to clams and shellfish, achieving recoveries ranging from 100% to 107.33% and 99.67%-116.67%, respectively. The results were consistent with the commercial TTX ELISA kit. This assay is highly sensitive, reliable and has a good specificity. Therefore, it provides a better alternative to the standard method for quantitative detection of TTX.
Keywords: Aptamer; CHA; Fluorescent reporter; SDA; TTX; Triple cycle amplification.
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