Broad-spectrum organophosphorus pesticide aptamer as well as method and application thereof

An organophosphorus pesticide and aptamer technology, applied in biochemical equipment and methods, material testing products, instruments, etc., can solve the problems of wide detection range and low detection sensitivity, and achieve the effect of low synthesis cost and improved affinity

Active Publication Date: 2020-08-28
XIHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The purpose of the present invention is to provide a broad-spectrum organophosphorus pesticide aptamer and its method and application, which solves the p

Method used

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  • Broad-spectrum organophosphorus pesticide aptamer as well as method and application thereof
  • Broad-spectrum organophosphorus pesticide aptamer as well as method and application thereof
  • Broad-spectrum organophosphorus pesticide aptamer as well as method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] A broad-spectrum organophosphorus pesticide aptamer, the nucleotide sequence of the aptamer is the sequence shown in SEQ.ID NO7 (S4-29), and the sequence is shown in Table 1 below.

experiment example 1

[0041] Binding free energy of experiment example 1 aptamer

[0042] Such as figure 1 As shown, it is the secondary structure diagram of the nucleotide sequence of the aptamer of Example 1 and S4-54 of the present invention and Comparative Example 1. The 3D structure of the aptamer model is obtained through the secondary structure, and the RNA and DNA are completed. Structural transformations. Optimize the structure of pesticides (isocarbophos, omethoate, methamidophos and profenofos), and dock the optimized ligand (pesticide structure) with the aptamer to obtain the three-dimensional structure of the complex ligand-ligand structure.

[0043]The binding free energy was obtained after molecular docking, as shown in Table 2. Taking the existing aptamers S4-54 and S24 as comparisons, it can be seen that the aptamers S4-16, S4-21 and S4-24 in Comparative Example 1 The binding ability of aptamer S4-54 is worse than that of the existing aptamer S4-54, which is higher than that of S...

experiment example 2

[0046] Experimental Example 2 Affinity analysis of aptamers

[0047] The affinity of the aptamers S4-16, S4-21, S4-24 and S4-29 of the present invention and the existing aptamer S4-54 was analyzed, and the aptamers were labeled with the fluorescent group FAM at the 5' end, specifically as follows:

[0048] First, with binding buffer (50mmol / L Tris, 50mmol / L NaCl, 10mmol / L KCl and 10mmol / L MgCl 2 , pH 8.0) dilute the aptamer to a certain concentration, denature at 90°C for 5min, and immediately ice-bath. Next, use the binding buffer to prepare a high-concentration pesticide solution, the pesticide is isocarbophos, profenofos, phorate or omethoate, use the high-concentration pesticide solution as the highest concentration of the ligand, and make a sample volume of 10 μL 16 samples were prepared using a 1:1 serial dilution strategy in . Then, 10 μL of aptamer solution was added to each sample. All samples were reacted at room temperature in the dark for 30 min, and loaded to ...

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PUM

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Abstract

The invention discloses a broad-spectrum organophosphorus pesticide aptamer as well as a method and application thereof. The nucleotide sequence of the aptamer is as shown in SEQ. ID NO. 7. The aptamer can recognize various organophosphorus pesticides, the affinity is improved compared with that of an existing aptamer, the S4-29 only has 29 bases, the sequence is short, the synthesis cost is low,construction of a sensor is facilitated, the aptamer can be applied to organophosphorus pesticide detection, the concentration range of the detected pesticide is 0.001-100 nM. Moreover, the concentrations of phorate, omethoate and profenofos have a good linear relationship with signal values, and the detection limits are 0.0016 nM, 0.0013 nM and 0.00125 nM respectively.

Description

technical field [0001] The invention relates to an aptamer, in particular to a broad-spectrum organophosphorus pesticide aptamer, method and application. Background technique [0002] Organophosphorus pesticides are currently the most widely used pesticides in my country. They can be divided into four categories: highly toxic, highly toxic, moderately toxic, and lowly toxic. It can also cause harm if a large amount enters the body. Therefore, in order to ensure the health of the human body, it is of great significance to establish an efficient, convenient and quick detection method for organophosphorus pesticide residues. [0003] Aptamer is a new type of detection element developed in the 1990s. It is composed of DNA or RNA, forms a specific three-dimensional structure through folding, and recognizes the spatial conformation of the target molecule. It has high affinity and high specificity. Aptamers are used in the detection of organophosphorus pesticides. Organophosphorus...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/53
CPCC12N15/115C12N2310/16G01N33/5308
Inventor 黄玉坤陶璇陈祥贵车振明陈鹏飞王力均杨潇
Owner XIHUA UNIV
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