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Molecular lateral chromatography detection method based on CRISPR system and detection test strip

A lateral chromatography, detection test paper technology, applied in analytical materials, measuring devices, instruments, etc., can solve the problems of reducing the sensitivity of the system, difficult to develop multi-index combined detection, etc., to achieve improved specificity, convenient operation, and improved appearance. effect of concentration

Pending Publication Date: 2021-11-23
固安北吉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For weaker false positives, a feasible improvement method designed by the inventor team of the present application is to change the interpretation method, that is, it is stipulated that the far-end band is weaker than the near-end band and is negative, but this will greatly reduce the sensitivity of the system. Moreover, the interpretation of colorimetry has raised the requirements for users.
[0010] In addition, it is difficult to develop multi-indicator joint detection with the existing competition method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1 Labeled probe lateral flow chromatography test strip

[0064] The lateral flow chromatography test paper is sequentially provided with a sample pad, a binding pad, an NC membrane and a water-absorbing pad on the base plate; the Cas12 protein and immobilized labeled probes are provided at the sample pad. The Cas12 protein contains a guide sequence that can target the novel coronavirus ORF1ab gene, and the labeled probe is a nucleic acid sequence containing two small molecule markers, FAM and Digoxigenin. The fixed end is labeled with biotin; the binding pad is provided with an anti-FAM antibody-red latex microsphere complex; the NC membrane is provided with a detection line and a quality control line, and the detection line is coated with an anti-digoxigenin antibody. The quality control line was coated with goat anti-mouse antibody.

[0065] The preparation method of the present embodiment labeled probe lateral flow chromatography test strip:

[0066] (1)...

Embodiment 2

[0072] Embodiment 2 Labeled probe lateral flow chromatography test strip

[0073] The lateral flow chromatography test paper is sequentially provided with a sample pad, a binding pad, an NC membrane and a water-absorbing pad on the base plate; the Cas12 protein and immobilized labeled probes are provided at the sample pad. The Cas12 protein is provided with a guide sequence that can target the human mucosal protein MUC5a, and the labeled probe is a nucleic acid sequence labeled with two small molecule markers, DNP and TAMRA. For the convenience of probe immobilization, one end of the probe is labeled with Biotin; the binding pad is provided with anti-DNP antibody-red latex microsphere complex, IgY antibody-red latex microsphere complex; the NC membrane is provided with a detection line and a quality control line, and the detection line is coated with There is an anti-TRMRA antibody, and the quality control line is coated with a rabbit anti-chicken IgY antibody.

[0074] The p...

Embodiment 3

[0082] Embodiment 3 labeled probe lateral flow chromatography test strip

[0083] The lateral flow chromatography test paper is provided with a sample pad, an NC membrane and a water-absorbing pad in sequence on the bottom plate; the Cas12 protein and immobilized labeled probes are provided at the sample pad. The Cas12 protein is provided with a guide sequence that can target the human mucosal protein MUC5a, and the labeled probe is a colloidal gold-labeled anti-TRMRA antibody. For the convenience of probe immobilization, one end of the probe is labeled with biotin; the NC A detection line and a quality control line are arranged on the membrane, the detection line is coated with TRMRA-BSA whole antigen, and the quality control line is coated with rabbit anti-chicken IgY antibody.

[0084] The preparation method of the present embodiment labeled probe lateral flow chromatography test strip:

[0085] (1) Synthesize the labeled probe, send the labeled probe sequence T (biotin-la...

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Abstract

The invention relates to the technical field of molecular detection, in particular to a molecular lateral chromatography detection method based on a CRISPR system and a detection test strip, according to the method, lateral chromatography visual detection is realized by arranging Cas protein and a fixed labeled probe on lateral chromatography test paper or accessories, and multi-index joint detection can also be realized. The labeled probe contains a nucleic acid probe and a marker which are connected with each other, the Cas protein contains a guide sequence capable of targeting to-be-detected molecules, and when the to-be-detected molecules are contained in the detection sample, the Cas protein is activated to cut the nucleic acid probe in the labeled probe, so that the marker originally fixed on the lateral chromatography test paper or accessory is released. According to the invention, the labeled probe is fixed, the labeled substance is released after being cut, and detection is carried out through a sandwich method or capture method test strip, so that the detection specificity is improved, and the false positive problem in the prior art is avoided.

Description

technical field [0001] The invention relates to the technical field of molecular detection, and specifically relates to a CRISPR system-based molecular lateral chromatography detection method and a detection test strip. Background technique [0002] CRISPR molecular detection is a new type of molecular detection technology first developed by Zhang Feng's team at the Broad Institute in 2017. The main principle is that after the Cas protein contacts and binds to the targeted fragment, its indifferent trans-cleavage function is activated and will cut The characteristics of all nearby nucleic acid sequences, a detection method to determine whether the target fragment exists by detecting the cleavage of the probe sequence (Gootenberg, Jonathan, S, et al. Nucleic acid detection with CRISPR-Cas13a / C2c2.[J]. Science, 2017.). [0003] In 2019, Zhang Feng's team combined the CRISPR method with lateral flow test paper to launch the SHERLOCK technology (Kellner M J, Koob J G, Gootenber...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/543
CPCG01N33/558G01N33/54306
Inventor 李诗焱
Owner 固安北吉生物科技有限公司
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