Dual-output toehold-mediated strand displacement amplification for sensitive homogeneous electrochemical detection of specie-specific DNA sequences for species identification.

The determination of specie-specific DNA sequences is a key factor for identification of animal species and detection of meat adulteration. Herein, a simple homogeneous electrochemical biosensor was developed for sensitive detection of specie-specific DNA sequences from meat products based on high efficient and specific dual-output toehold-mediated strand displacement (TMSD). After incubation with target DNA, large amount of methylene blue (electrochemical signal molecule) labeled probes (MB-P) were released from preformed DNA duplex structures by the process of dual-output TMSD amplification. The free MB-P could be further digested by Exonuclease I, and the enzymatic products contain little negative charge could diffuse to the surface of indium tin oxide electrode, generating significantly electrochemical signal. As a result, the designed biosensor showed a broad dynamic range from 0.01 pM to 100 pM, with a low detection limit of 8.2 fM, and ideal selectivity and reproducibility. Meanwhile, the approach exhibited acceptable accuracy for the detection of specie-specific DNA sequences, and possessed the potential application for the identification of animal species from meat products.