A fluorescence biosensor based on a cell-free transcription system for species-specific DNA sequence detection and seahorse product identification

Seahorse products face rampant counterfeiting due to their high value and low production. The determination of species-specific DNA sequences is a key factor in identifying species. A conserved genetic fragment in the seahorse genome has been identified as a specific marker. In this study, a novel cell-free transcription system-based fluorescence biosensor has been designed and applied for seahorse product authenticity identification. A toehold-mediated triplex DNA complex has been designed. Within this complex, a single-stranded DNA (ssDNA) component is designed to hybridize with two other incomplete template strands, forming a complete aptamer transcription template. The target seahorse DNA, which exhibits greater complementarity with the other two strands in the triplex complex, can displace the ssDNA upon its presence and trigger a strand displacement reaction. The released ssDNA can be removed using magnetic beads easily since it is modified with biotin. The absence of ssDNA in the system prevented the formation of the complete transcription template, which inhibited the recognition by T7 RNA polymerase and reduced subsequent RNA transcription through the cell-free transtcription system. The resultant reduction in RNA production diminishes its binding to fluorescent aptamers, leading to a decrease in the detected fluorescence signal. Under optimized assay conditions, the fluorescence signal difference exhibited a linear correlation with the logarithm of seahorse DNA concentration over the range of 150 pM to 35 nM, achieving a limit of detection (LOD) of 65 pM. The proposed system has good reproducibility and selectivity, and has been successfully applied to seahorse product identification.