Dual-signal amplification probe, sensor, detection method and application

A dual signal amplification and detection sensor technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve problems such as aptamer sensors that are not combined to detect malathion

Active Publication Date: 2021-01-12
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, there are no reports of aptasensors combining exonuclease-assisted dual signal amplification with G-quadruplex / hemin DNase for the detection of malathion

Method used

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  • Dual-signal amplification probe, sensor, detection method and application
  • Dual-signal amplification probe, sensor, detection method and application
  • Dual-signal amplification probe, sensor, detection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093]Example 1 Design of aptamer probe S1-S2-S3 and hairpin probe H1

[0094]In this embodiment, aptamer probes S1-S2-S3 and hairpin probe H1 are designed, including:

[0095]The aptamer probe is self-assembled by at least 3 single-stranded sequences, the single-stranded sequence includes sequence S1, sequence S2 and aptamer sequence S3 for binding to the target; said sequence S1 is from 5'to 3' The end includes a sequence ST complementary to the aptamer sequence S3 and a first binding sequence in sequence; the sequence S2 sequentially includes a sequence ST complementary to the aptamer sequence S3 and a complementary sequence of the first binding sequence from the 5'end to the 3'end;

[0096]The hairpin probe includes a second binding sequence, a G-quadruplex sequence, and a sequence ST* that can be partially complementary to the sequence ST from the 5'end to the 3'end; the second binding sequence and the G-quadruplex The 3'end sequence of the body sequence is partially complementary; the ...

Embodiment 2

[0112]This example provides a method for preparing the aptamer probes S1-S2-S3 and hairpin probes H1, H2, and H3 designed in Example 1.

[0113]The preparation method of hairpin probe H1: Centrifuge the oligonucleotide powder H1 at 12000 rpm (Ebend centrifuge 5417R) for 5 minutes, and then dissolve it in 20mM Tris-HCl buffer (200mM NaCl, 20mM KCl, 2mM MgCl)2, pH 7.4) prepared 10μM stock solution. The H1 stock solution was then heated to 95°C for 10 minutes, and then slowly cooled to room temperature to form a hairpin structure. Hairpin probes H2 and H3 were prepared in the same way.

[0114]Preparation method of aptamer probe S1-S2-S3: Centrifuge the oligonucleotide powders S1, S2 and S3 at 12000rpm for 5 minutes, and then dissolve them in 20mM Tris-HCl buffer (pH 7.4) to obtain 100μM stock solution solution. Then 10 μL of sequence S1 (100 μM), 10 μL of sequence S2 (100 μM) and 20 μL of sequence S3 (100 μM) were added to 60 μL of 20 mM Tris-HCl buffer (pH 7.4) and incubated at 37° C. for ...

Embodiment 3

[0115]Example 3 Double signal amplification detection kit

[0116]This embodiment provides a dual signal amplification detection kit, which includes the aptamer probe S1-S2-S3 and the hairpin probe H1 in the first embodiment.

[0117]Further, the molar ratio of the aptamer probe S1-S2-S3 to the hairpin probe H1 is 2:3.

[0118]Furthermore, the ratio of the amount of exonuclease Exo I, exonuclease Exo I and aptamer probe S1-S2-S3 is 15:0.02, and the ratio is U / nmol.

[0119]Further, it also includes exonuclease Exo III, the ratio of the amount of exonuclease Exo III and hairpin probe H1 is 20:0.03, and the ratio is U / nmol.

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Abstract

The invention belongs to the field of biochemical analysis and detection, and particularly relates to a dual-signal amplification probe, a sensor, a detection method and application. The dual-signal amplification probe comprises an aptamer probe, wherein the aptamer probe is formed by self-assembling at least three single-stranded sequences, a sequence S1 comprises a sequence ST complementary to an aptamer sequence S3 and a first binding sequence, a sequence S2 comprises a sequence ST complementary to the aptamer sequence S3 and a complementary sequence of the first binding sequence, and the sequences include the aptamer sequence S3; and a hairpin probe, wherein the hairpin probe comprises a second binding sequence, a G-quadruplex sequence and a sequence ST* which can be partially complementary to the sequence ST, the second binding sequence is partially complementary to the G-quadruplex sequence, when the second binding sequence and a sequence ST' are partially complementary, a protruding tail end is formed at the 3' end of the sequence ST', and when the sequence ST* and the sequence ST are complementary, a flat tail end is formed at the 3' end of the sequence ST*. The dual-signalamplification probe adopts an exonuclease-assisted signal amplification method to detect a target, and is excellent in system stability, high in detection sensitivity and strong in specificity.

Description

Technical field[0001]The invention relates to the field of biochemical analysis and detection, in particular to a dual signal amplification probe, sensor, detection method and application.Background technique[0002]Organophosphorus pesticides are a type of organic compound pesticides containing phosphorus, such as the commonly used trichlorfon, dichlorvos, omethoate and malathion (Mal). In the family of organophosphorus pesticides, malathion is one of the most common commercial broad-spectrum pesticides and is widely used as a protective agent for the prevention and control of crop pests and diseases. Although malathion has huge economic benefits in food production, its threat to human health cannot be underestimated. According to reports, malathion can inhibit the activity of acetylcholinesterase (AChE), causing breathing difficulties, headaches and dizziness, and in severe cases, respiratory paralysis and death. China's National Food Safety Standard (GB 2763-2019) stipulates that t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6825C12N15/11
CPCC12Q1/6825C12Q2525/205C12Q2525/301C12Q2521/319C12Q2563/103Y02A50/30
Inventor 吴昊邹霈吴军王洪勇刘娅灵韩国庆
Owner JIANGSU INST OF NUCLEAR MEDICINE
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