Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of CRISPR cascade nucleic acid detection system and its detection method and application

A detection system and detection method technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, resistance to vector-borne diseases, etc., can solve the problems of sample loss, amplification deviation, false positive risk, etc., and achieve easy operation , short detection time and low cost

Active Publication Date: 2022-05-20
SHENZHEN UNIV
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some substantial problems still exist in these reported nucleic acid amplification-based detection techniques, including sample loss due to incomplete reverse transcription steps, amplification bias due to error-prone sequence duplication, and amplification bias due to Risk of false positives due to sub-contamination

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of CRISPR cascade nucleic acid detection system and its detection method and application
  • A kind of CRISPR cascade nucleic acid detection system and its detection method and application
  • A kind of CRISPR cascade nucleic acid detection system and its detection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] 1. Selection of target genome

[0081] Use NCBI to find the target sequence, use known information such as gene name or gene ID, and associate it with the corresponding Reference Sequence (RefSeq), so as to obtain the target sequence information, and finally use NCBI Blast to perform homology comparison to find the conserved sequence .

[0082] 2. Activity verification of Cas12 enzyme

[0083] Take the Lbcas12a protein as an example:

[0084] Activity verification of LbCas12a protein: design and synthesize a ssDNA probe labeled with a fluorescent group and a quencher group (specifically 5'-FAM-TTTTTT-BHQ1-3'), and prepare a reaction system based on the CRISPR-Cas system, For real-time fluorescence verification of trans-cleavage of LbCas12a.

[0085] The specific reaction system includes (30μL system): 60nM Lbcas12a, 60nM target guide 12-crRNA, 3× Cas buffer, 3μM ssDNA probe, 40nM target sequence, 1U / μL Murine RNase Inhibitor.

[0086] React at a constant temperature...

Embodiment 2

[0101] In order to further verify the feasibility of the nucleic acid detection method described in the embodiments of the present invention, a novel coronavirus (SARS-CoV-2) was selected for nucleic acid detection. It should be noted that the examples are for illustration only, and do not constitute any limitation to the protection scope of the present invention.

[0102] 1. Design the target guide RNA (13-crRNA) targeting the SARS-CoV-2 genome

[0103] The 13-crRNA targets the forward sequence of the SARS-CoV-2 genome, which is derived from the E genome of SARS-CoV-2. The forward sequence of 13-crRNA is: 5'-GAGACCACCCCAAAAAUGAAGGGGACUAAAAACCAAGACUCACGUUAACAAUAUUGCAGCA-3'.

[0104] The nucleic acid of the sample to be tested is synthesized by a third-party synthesis company. The selected fragment is the E gene fragment of SARS-CoV-2 (NCBI Reference Sequence: NC_045512.2), and its forward sequence is: 5'-UGCUGCAAUAUUGUUAACGUGAGUCUUG-3'.

[0105] 2. Prepare detection reaction...

Embodiment 3

[0112] In order to further evaluate the detection sensitivity of the nucleic acid detection method described in the embodiments of the present invention, the E gene fragment of SARS-CoV-2 is used as a target, and a series of serial dilutions (1 amol / L, 10 amol / L, 10 fmol / L, 10 pmol / L, 10nmol / L; Note: amol / L is 10 -18 mol / L), with no target SARS-CoV-2 genome as a negative control, nucleic acid detection was performed. The detection method and reagents are the same as in Example 2.

[0113] The result is as Figure 6 As shown, it can be seen that the CRISPR cascade nucleic acid detection method provided by the embodiment of the present invention can realize the detection of the E gene of SARS-Cov-2 as low as 1aM.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

A CRISPR cascaded nucleic acid detection system and its detection method and application provided by the present invention utilize the activity of Cas12 enzyme and Cas13 enzyme, and 13-crRNA complementary to the target nucleic acid sequence to be detected guides the Cas13 enzyme to specifically bind to the target nucleic acid sequence to be detected , triggering the cascade auxiliary probe to be trans-cleaved by the Cas13 enzyme, the released Trigger ssDNA can be complementary to the 12-crRNA base pair, triggering the fluorescent detection probe to be trans-cleaved by the Cas12 enzyme, and then generate a detectable signal. The detection method has the characteristics of target-specific binding and non-specific cutting activity. The application of this method can directly detect single-stranded RNA targets and multiple detection sites corresponding to the targets, relying on the CRISPR-Cas system cascade to achieve "aM level (10 ‑18 mol / L)” nucleic acid detection, which has extremely high sensitivity; moreover, this method has high flexibility in the design of the detection target, the time required for detection is short, no amplification steps are required, the cost is low, and the operation is simple, which can realize the “ Nucleic acid in-result out” closed-tube detection.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a CRISPR cascade nucleic acid detection system and its detection method and application. Background technique [0002] The rapid detection of nucleic acid is very important in clinical diagnosis and biotechnology, especially in in vitro molecular diagnosis. With the rapid development of the nucleic acid molecular detection market, new requirements are constantly being put forward for nucleic acid detection technology, especially new requirements for on-site rapid detection. On-site rapid testing (point-of-care testing, POCT) is a testing method that is carried out at the sampling site and uses portable analytical instruments and supporting reagents to quickly obtain test results. On-site rapid detection POCT technology is currently widely used in clinical testing, chronic disease monitoring, emergency anti-terrorism, disaster medical rescue, infectious disease monitoring, ins...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6816
CPCC12Q1/68C12Q2521/327Y02A50/30
Inventor 刘翼振张奕滨
Owner SHENZHEN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com