Extracellular vesicle detection technology based on aptamer hairpin-type triggered super-branch rolling ring amplification

Abstract

The invention discloses an extracellular vesicle detection technology based on aptamer hairpin-type triggered super-branch rolling ring amplification. An aptamer hairpin part is firstly designed by taking an extracellular vesicle surface protein as a target, and a corresponding padlock probe, a connecting sequence and a second primer are designed; an annular template is prepared by hybridization of the padlock probe and the connection sequence; after the aptamer hairpin part is incubated with extracellular vesicles, a hairpin structure is opened so as to be combined with the annular template,the super-branch rolling ring amplification is triggered under the condition that the second primer is added, and a large amount of length gradient double-stranded nucleic acid is formed; a strong fluorescence signal can be generated in combination with a nucleic acid dye SYBR Green I, and the intensity of the fluorescent signal and the extracellular vesicle concentration are positively correlated, so that the target extracellular vesicles can be quantitatively detected according to the fluorescence intensity, and the free-state aptamer hairpin part does not need to be cleaned based on the fact that the free-state aptamer hairpin part is not able to trigger amplification. The extracellular vesicle detection technology provided by the invention is simple and high in specificity, the extracellular vesicles are free of cleaning, and high sensitivity is achieved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to an extracellular vesicle detection technology based on aptamer hairpin triggering hyperbranched rolling circle amplification. Background technique [0002] Extracellular vesicles (Extracellular Vesicles, EVs) are membrane-derived vesicles released by cells, which are mainly divided into exosomes (30-200nm), microvesicles (200-1000nm) and apoptotic bodies ( 50-5000nm) 3 subpopulations. Extracellular vesicles carry and transmit important signaling molecules, forming a new intercellular information transmission system, which is closely related to various disease processes including tumorigenesis, invasion and metastasis. Using extracellular vesicles as the target of liquid biopsy has the advantages of non-invasiveness, abundant content, stable existence, real-time detection, and comprehensive information. [0003] Rolling circle amplification is an isothermal...

AI Technical Summary

Problems solved by technology

[0004] The currently reported extracellular vesicle detection technology has some problems in terms of simplicity and sample processing. For example, the detection step needs to be washed repeatedly, resulting in the loss of EVs, low detection sensitivity and poor repeatability, which cannot meet the needs of clinical detection.

[0005] The method of triggering rolling circle amplification by aptamer hairpin can eliminate the steps of repeated cleaning to remove excess aptamer hairpin, simplify the operation steps, shorten the detection time, and improve the repeatability of the method. It has been used in the detection of miRNA in laboratories. However, there is no report for the detection of extracellular vesicles

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