Isolation-free digital multiplex detection method based on DNA walker and tdt enzyme amplification

Abstract

This invention relates to the field of biodetection technology, specifically to a non-isolated digital multiplex detection method based on a DNA walker and TdT enzyme amplification. The method first utilizes coding microspheres with capture elements coupled to their surface to specifically bind to target miRNAs or proteins in the sample; then, specific guide DNA (gDNA) corresponding to the target molecule is released through enzymatic digestion or thermal denaturation; the gDNA then... Pf Under the action of Ago endonuclease, the DNA walker on the surface of the encoded microspheres is activated, cyclically cutting the orbital strand to generate multiple DNA fragments with 3'-hydroxyl ends. Finally, TdT enzyme is used to extend fluorescently labeled nucleotides at the hydroxyl ends, achieving in-situ signal amplification, and digital quantitative analysis is performed on the fluorescence signal of the encoded microspheres. This invention achieves high-sensitivity detection of multiple targets in a single reaction without chamber isolation, with detection limits for each target molecule reaching the fM or fg / mL level, providing an effective technical means for the early screening of major diseases such as Alzheimer's.

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