Chemical modification-based S chain and Cas12a index amplification method

CN120665998AActive Publication Date: 2025-09-19THE FIRST AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIVERSITY
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Patent Information

Application Number
CN202510899265.8
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-07-01
Publication Date
2025-09-19
Estimated Expiration
2045-07-01

AI Technical Summary

Technical Problem

The existing CRISPR/Cas12a detection system has limited detection sensitivity under single-stranded DNA activation, which makes it difficult to meet the detection needs of clinical low-abundance targets. The complex pre-amplification procedure increases the complexity and cost of detection.

Method used

A chemically modified S chain is introduced, which includes thiophosphate-modified regions at both ends that are complementary to S-crRNA and an intermediate trans-splicing region. Cas12a is activated by a split activator to achieve exponential amplification of CRISPR/Cas12a, and the Cas12a trans-splicing region is regulated by thiophosphate modification.

Benefits of technology

It achieves ultrasensitive detection of low-abundance targets with a detection sensitivity of up to fM level. It is simple to operate, low-cost, does not require complex probe design, and has a fast response.

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Abstract

The invention discloses an S chain based on chemical modification and a Cas12a index amplification method, and belongs to the technical field of nucleic acid diagnos.The S chain based on chemical modification comprises a thiophosphoric acid modification area with the two ends capable of being complementary with S-crRNA and a middle trans-shear area, a 5 '-TTATT-3' sequence is used in a trans-shear region, and thiophosphoric acid modification is performed on thiophosphoric acid modification regions at two ends every 0-2 basic groups, so that Cas12a trans-shear is resisted. An existing CRISPR / Cas12a detection system is improved, regulation and control of a Cas12a trans-shear region are achieved by introducing a simple S chain and by means of thiophosphoric acid modification, exponential amplification of CRISPR / Cas12a is finally achieved by utilizing the characteristics of Cas12a activated by a split activator, ultra-sensitive detection of a low-abundance target can be achieved without additional target pre-amplification, the detection sensitivity can reach the fM level, and the method is suitable for large-scale popularization and application. The operation is simple, no complex probe design is needed, the reaction is rapid, and the cost is low.
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Citation Information

Patent Citations

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  • CRISPR-Cas detection system based on modified crRNA

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  • One-tube light-operated CRISPR / Cas nucleic acid detection method

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  • Method for synergistically activating CRISPR / Cas12a system based on T-type structure and application thereof

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  • Increasing the sensitivity of a crispr-cas nucleaic acid detection system

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