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CRISPR cascade nucleic acid detection system and detection method and application thereof

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 amplification deviation, false positive risk, sample loss, etc., and achieve easy operation , low cost and short detection time

Active Publication Date: 2022-02-18
SHENZHEN UNIV
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  • 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

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  • CRISPR cascade nucleic acid detection system and detection method and application thereof
  • CRISPR cascade nucleic acid detection system and detection method and application thereof
  • CRISPR cascade nucleic acid detection system and detection method and application thereof

Examples

Experimental program
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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.

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Abstract

The invention provides a CRISPR cascade nucleic acid detection system as well as a detection method and the application thereof. According to invention, the activity of Cas12 enzyme and Cas13 enzyme is utilized, 13-crRNA complementary with a to-be-detected target nucleic acid sequence guides the Cas13 enzyme to be specifically combined with the to-be-detected target nucleic acid sequence, a cascade auxiliary probe is triggered to be subjected to trans-cleavage by the Cas13 enzyme, released Trigger ssDNA can be complementarily paired with a 12-crRNA base, a fluorescence detection probe is triggered to be subjected to trans-cleavage by the Cas12 enzyme, and then a detectable signal is generated. The detection method has the characteristics of specific target binding and non-specific target cleavage activity. By applying the method, a single-stranded RNA target and a plurality of detection sites corresponding to the target can be directly detected, aM-level (10-18 mol / L) nucleic acid detection can be realized without amplification by virtue of cascade of a CRISPR-Cas system, and extremely high sensitivity is obtained; and moreover, the method is high in design flexibility of a detection target, relatively short in detection time, free of an amplification step, low in cost and simple and convenient to operate, and closed tube detection of nucleic acid in-result out can be realized.

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

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Application Information

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IPC IPC(8): C12Q1/6816
CPCC12Q1/68C12Q2521/327Y02A50/30
Inventor 刘翼振张奕滨
Owner SHENZHEN UNIV