A probe based on target-triggered dual signal amplification and its application

A dual-signal amplification and target technology, applied in the field of dual-signal amplification probes and their detection proteins, can solve the problems of high cost, long time, complicated operation, etc. The effect of false positive signal generation

Active Publication Date: 2018-04-24
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The second technical problem to be solved by the present invention is that the detection methods for proteins in the prior art are time-consuming, complicated to operate and high in cost, so as to provide a method for detecting protein molecules with simple operation and low cost

Method used

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  • A probe based on target-triggered dual signal amplification and its application
  • A probe based on target-triggered dual signal amplification and its application
  • A probe based on target-triggered dual signal amplification and its application

Examples

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Effect test

Embodiment 1

[0048] Example 1 Probe design based on target-triggered dual signal amplification

[0049] The probes for target-triggered dual signal amplification described in this example include: the first hairpin H1, the second hairpin H2 and the third hairpin H3, each of which has a sequence as shown in Table 1 below; The target protein is human thrombin.

[0050] Table 1 Probe sequences based on target-triggered dual signal amplification

[0051]

[0052] The first hairpin, the second hairpin and the third hairpin are all formed by the self-folding of the single-stranded linear molecule, and the complementary base pairing hybridization in the folded region, and the part of the double-stranded structure in the local region is the stem region, the part that does not form a double-strand structure and folds back is a ring region;

[0053] The first hairpin includes: an aptamer region I that can specifically recognize the target protein, and a first stem region II that hybridizes with...

Embodiment 2

[0068] Example 2 Detection of Target Protein Molecules at Different Concentrations Based on Target-Triggered Dual Signal Amplification Probes

[0069] The method for detecting target protein molecules based on target-triggered dual signal amplification probes of this embodiment includes the following steps:

[0070] First, take the solutions of the first hairpin, the second hairpin, and the third hairpin, heat them to 95° C., keep them for 5 minutes, then slowly lower them to room temperature, and set them aside. Take 10ml of dimethyl sulfoxide (DMSO) and 6.5mg of hemin to prepare 1mM hemin solution, and store it in a dark place at -20°C for future use. 4-Hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solution was prepared for use, and the buffer solution included: 25 mM HEPES, 200 mM NaCl, 20 mM KCl, 1% DMSO, pH 7.4.

[0071] Prepare Tris-HCl buffer for use, the buffer includes: 20mM Tris-HCl, 200mM NaCl, 20mM KCl, 2mM MgCl 2 , pH value is 7.4. Take human α-throm...

Embodiment 3

[0075] Example 3 Signal Amplification Verification Test of Probes Based on Target-Triggered Double Signal Amplification

[0076] In this example, the following solutions were prepared according to the method in Example 2, and numbered a, b, c, d in sequence:

[0077] Sample a: a solution in which the concentrations of the first hairpin H1, the second hairpin H2, and the third hairpin H3 are all 200 nM;

[0078] Sample b: a solution in which the concentrations of the first hairpin H1, the second hairpin H2, and the third hairpin H3 are all 200 nM, and the concentration of the exonuclease III is 20 U;

[0079] Sample c: a solution in which the concentrations of the first hairpin H1, the second hairpin H2, and the third hairpin H3 are all 200 nM, and the concentration of the target thrombin is 1 nM;

[0080] Sample d: a solution in which the concentrations of the first hairpin H1, the second hairpin H2, and the third hairpin H3 are all 200 nM, the concentration of the exonucleas...

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Abstract

The present invention provides a probe based on target-triggered dual signal amplification, the probe includes a first hairpin, a second hairpin and a third hairpin, the first hairpin, the second hairpin and the third hairpin Hairpins are all single-stranded linear molecules folded back on themselves, and the complementary bases in the folded region are hybridized. The part of the local region that forms a double-stranded structure is the stem region, and the part that does not form a double-stranded structure and folds back is a ring. Area. The probe of the present invention catalyzes the above-mentioned hairpin self-assembly through the target protein, and achieves signal amplification with the assistance of exonuclease III, and realizes the obvious amplification of the detection signal, thereby greatly improving the sensitivity of detection, and because it is in the detection process It does not rely on template replication, avoiding the problem of cross-contamination during the detection process, which leads to false positives, effectively preventing the generation of false positive signals, and greatly reducing background noise.

Description

technical field [0001] The invention belongs to the field of molecular bioinformatics, and in particular relates to a target-triggered double-signal amplification probe and a protein detection method and application thereof. Background technique [0002] The quantitative detection of protein plays a very important role in the field of biological research and drug diagnosis. However, since proteins are often at very low concentrations, highly sensitive analytical detection methods are often required to detect them. Therefore, it has always been the direction of people's research to detect protein molecules with high sensitivity, high selectivity, and simplicity. At present, enzyme-linked immunosorbent assay (ELISA) is the most commonly used method in the quantitative detection of proteins. Although this method can achieve the purpose of quantitative analysis, its detection sensitivity is still limited by the enzyme-substrate reaction. Trace protein molecules are detected, wh...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6876C12N15/11
Inventor 吴昊邹霈诸飞帆刘娅灵王洪勇吴军
Owner JIANGSU INST OF NUCLEAR MEDICINE
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