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Visualized rapid nucleic acid testing method based on CRISPR-Cas12a system and application

A nucleic acid and nucleic acid probe technology, which can be used in biochemical equipment and methods, and the determination/inspection of microorganisms, and can solve problems such as limiting rapid detection.

Active Publication Date: 2020-06-05
HUAZHONG AGRI UNIV
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  • Summary
  • 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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  • Visualized rapid nucleic acid testing method based on CRISPR-Cas12a system and application
  • Visualized rapid nucleic acid testing method based on CRISPR-Cas12a system and application
  • Visualized rapid nucleic acid testing method based on CRISPR-Cas12a system and application

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 the target nucleic acid molecule; 2) Cas12a enzyme digestion reaction: adding a non-fluorescent luminescent group at one end and a fluorescence quenching group at the other end to the system. The specific ssDNA fluorescent reporter molecule, after the Cas12 / sgRNA complex binds to the target DNA, stimulates the cleavage activity of the Cas12a protein on the ssDNA-reporter, thereby generating a free fluorescent luminescent group and emitting detectable fluorescence. In order to screen the base modification method that can enhance the signal intensity of fluorescence visualization detection, the combination method is: 5' fluorescent group-NNNNNNNNNNNNNN-3' quench...

Embodiment 2

[0175] Example 2: A specific PCR primer pair and sgRNA targeting the p72 gene were designed for the 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). Use the primer3 software to design primer pairs for amplifying the p72 gene (Table 2); for the PCR amplification region, use the CRISPR-offinder software (https: / / sourceforge.net / projects / crispr-offinder-v1-2 / ) to design 5 primers sgRNA, PAM is TTTV, using sgRNA empty vector (pUC57-T7-sgRNA) as a template to design primer pairs for in vitro transcription (Table 2). The experimental steps for nucleic acid detection using CRISP-Cas12 are as follows:

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

Embodiment 3

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

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

[0206] (1) PCR amplification: The pMD18T-p72 plasmid was diluted 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 of different dilutions as templates, and p72-PCR-F and p72-PCR-R as primers, PCR amplification (Table 1). The specific steps are that the reaction system is 50 μL, including ...

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Abstract

The invention provides a visualized rapid nucleic acid testing method based on a CRISPR-Cas12a system and an application. Specially, a fluorescent or naked eye visualized detection system for detecting target nucleic acid molecules comprises (a) Cas12a protein; (b) sgRNA which leads the Cas12a protein to be specifically bound to the target nucleic acid molecules; and (c) a nucleic acid probe whichcontains a detectable marker, wherein when the nucleic acid probe is cut by a Cas12a protein bypass, a detectable signal is generated. The detection system and method have the advantages of being rapid, sensitive, high in specificity, visualized through naked eyes and suitable for rapid field detection and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a visual detection method and application of target nucleic acid molecules. Background technique [0002] Rapid detection of target nucleic acids is very important for clinical diagnosis and biotechnology applications, especially 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 disea...

Claims

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Application Information

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Patent Type & Authority Applications(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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