Fluorescence Biosensor based on Cell-Free Transcription System for Specie-specific DNA Sequences Detection and Seahorse Products Identification

Abstract

Seahorse product faces rampant counterfeiting due to its high value and low production. The determination of speciesspecific 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 had been designed and applied for seahorse product authenticity identification. A toehold-mediated triplex DNA complex had 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 by a magnetic easily since which 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 systems. 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 changing of 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.