Visual rapid nucleic acid detection method and application based on CRISPR-Cas12a system

A technology of target nucleic acid and nucleic acid probe, which is applied in the field of visual detection of target nucleic acid molecules and can solve problems such as limiting rapid detection

Active Publication Date: 2022-05-27
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this method mainly relies on fluorescent signals to determine the concentration of target nucleic acid molecules in the detection sample, and relevant instruments and equipment are still required for detection, which limits the need for on-site rapid detection.

Method used

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  • Visual rapid nucleic acid detection method and application based on CRISPR-Cas12a system
  • Visual rapid nucleic acid detection method and application based on CRISPR-Cas12a system
  • Visual rapid nucleic acid detection method and application based on CRISPR-Cas12a system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0164] Example 1: Using exonuclease (Exonuclease I) to cut ssDNA-reporter to evaluate the influence of different base modification methods on the signal intensity of fluorescence detection

[0165] The nucleic acid detection experiment based on CRISPR-Cas12a technology is mainly divided into two steps: 1) Amplification of target nucleic acid molecules; 2) Cas12a digestion reaction: adding a non-fluorescent light-emitting group at one end and a fluorescence quenching group at the other end into the system The specific ssDNA fluorescent reporter molecule, after the Cas12 / sgRNA complex binds to the target DNA, excites the cleavage activity of Cas12a protein on ssDNA-reporter, thereby generating free fluorescent light-emitting groups and emitting detectable fluorescence. In order to screen the base modification method that can enhance the intensity of fluorescence visualization detection signal, the combination method is: 5' fluorophore-NNNNNNNNNNNN-3' quenching group, N represents...

Embodiment 2

[0175] Example 2: Design of specific PCR primer pair and sgRNA targeting p72 gene for nucleic acid detection of African swine fever

[0176] The p72 gene (MK333180) and genome (AY261366.1) sequences of African swine fever virus were downloaded from the NCBI database, respectively. Send the company to synthesize a partial fragment of the p72 gene and insert it into the cloning vector (pMD18T-p72). Primer pairs for amplifying the p72 gene were designed using primer3 software (Table 2); for PCR amplification regions, 5 primers were designed using CRISPR-offinder software (https: / / sourceforge.net / projects / crispr-offinder-v1-2 / ) sgRNA, PAM is TTTV, and the primer pair for in vitro transcription was designed with the sgRNA empty vector (pUC57-T7-sgRNA) as the template (Table 2). The experimental steps for nucleic acid detection using CRISP-Cas12 are as follows:

[0177] (1) PCR amplification of target gene fragments: configure 50 μL of PCR reaction solution, including 25 μL of Ex ...

Embodiment 3

[0204] Example 3: Assessing the sensitivity of the CRISPR-Cas12a-assisted nucleic acid visualization method for detection of African swine fever virus nucleic acid

[0205]In this example, to further evaluate the sensitivity of the CRISPR-Cas12a-assisted nucleic acid visualization method for detecting African swine fever virus nucleic acid, the pMD18T-p72 plasmids with different copy numbers were diluted as templates, and CRISPR-Cas12a nucleic acid visualization was performed after PCR amplification. The experimental process is as follows:

[0206] (1) PCR amplification: fold the pMD18T-p72 plasmid to 8×10 7 copy / µl, 8×10 6 copy / µl, 8×10 5 copy / µl, 8×10 4 copy / µl, 8×10 3 copy / µl, 8×10 2 copy / µl, 8×10 1 copies / µl, 4×10 1 copy / µl, 2×10 1 copy / µl, 8×10 0 Copy / microliter, etc., using plasmids with different dilutions as templates, p72-PCR-F and p72-PCR-R as primers, PCR amplification (Table 1). The specific steps are that the reaction system is 50 μL, of which Extaq Mix ...

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Abstract

The invention provides a visual rapid nucleic acid detection method and application based on the CRISPR-Cas12a system. Specifically, the detection system for detecting target nucleic acid molecules by fluorescence or naked-eye visualization of the present invention includes: (a) Cas12a protein; (b) sgRNA, which guides Cas12a protein to specifically bind to the target nucleic acid molecule; and ( c) a nucleic acid probe containing a detectable label, wherein a detectable signal is generated when the nucleic acid probe is bypassed by the Cas12a protein. The detection system and detection method of the present invention have the advantages of rapidity, sensitivity, high specificity, naked eye visualization, and suitability for rapid detection on site.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular, the invention relates to a visual detection method and application of target nucleic acid molecules. Background technique [0002] The rapid detection of target nucleic acid is very important for clinical diagnosis and biotechnology applications, especially in in vitro molecular diagnosis. With the rapid development of the nucleic acid molecule detection market, new demands are constantly being put forward for nucleic acid detection technology, especially for on-site rapid detection. Point-of-care testing (POCT) is a test 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 inspection, chronic disease monitoring, emergency counter-terrorism, disaster medical rescue, infectious disease monitoring, inspection and qua...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2563/107
Inventor 谢胜松赵书红李新云刘向东陶大刚刘佳佳徐兵荣聂雄伟
Owner HUAZHONG AGRI UNIV
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