A detection method for miRNA based on click chemistry combined with CRISPR / Cas12a and its application

By combining click chemistry and CRISPR/Cas12a technology, the problems of poor specificity and long period of ultrashort sncRNA detection in the prior art are solved, and fast, simple and high specificity detection of ultrashort miRNAs are achieved.

CN118127127BActive Publication Date: 2025-03-25BODITAI (XIAMEN) BIOTECHNOLOGY CO LTD +1
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Patent Information

Application Number
CN202410150969.0
Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Filing Date
2024-02-02
Publication Date
2025-03-25
Estimated Expiration
2044-02-02

AI Technical Summary

Technical Problem

The prior art has problems with long detection cycles and poor specificity when detecting ultrashort sncRNAs less than 22 nucleotides, and it is particularly difficult to effectively detect ultrashort miRNAs such as miRNA-21.

Method used

Using a detection method based on click chemistry binding to CRISPR/Cas12a, the target miRNA is used as a template to make click chemistry ligation to form probe1-probe2; then crRNA1 and crRNA2 replace miRNA through strand replacement reactions, activate the trans cleavage ability of CRISPR/Cas12a, thereby cleaving reporter genes to generate fluorescent signals.

Benefits of technology

It realizes rapid and simple detection of ultra-short sncRNA, high specificity, no PCR amplification, short analysis time, simple operation, and can effectively detect miRNAs less than 22 nucleotides.

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Abstract

The present invention discloses a detection method and application of miRNA based on click chemistry combined with CRISPR / Cas12a, belonging to the field of biological detection technology. Taking miRNA (microRNA) as an example, first the target miRNA21 is used as a template to connect two DNA nucleic acid probes by click chemistry to form probe1-probe2. Subsequently, crRNA1 and crRNA2 replace miRNA21 by strand displacement reaction (SDR), and probe1-probe2 and crRNA1-crRNA2 activate the trans-cutting ability of CRISPR / Cas12a, thereby cutting the reporter gene to produce a fluorescent signal. The method in the present invention can detect ultrashort sncRNA. It has the advantages of good specificity, quick and easy, and has broad application prospects in the early diagnosis of cancer and the treatment of various diseases.
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Citation Information

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