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Nucleic acid aptamer for specifically identifying di (2-ethyl) hexyl phthalate and screening method and application of nucleic acid aptamer

A technology of phthalic acid two and nucleic acid aptamers, which is applied in the field of biomedicine and can solve the problem of few reports on phthalate plasticizers and nucleic acid aptamers, increasing cost, complexity and difficulty, and affinity There are still problems to be improved to achieve the effects of high affinity, low synthesis cost and high sensitivity

Active Publication Date: 2019-09-20
HUBEI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are many nucleic acid aptamers obtained by in vitro screening technology, and many of them have achieved online rapid and high-sensitivity detection, but there are few reports on the specific recognition of phthalate plasticizer nucleic acid aptamers
Comparative document 1: the application number is "CN201610949635.5", the title of the invention is "a phthalate plasticizer single-stranded DNA nucleic acid aptamer and its screening and characterization method and electrochemical sensor", the target molecule needs Pre-modification increases the cost, complexity and difficulty of the experiment
And the affinity (affinity constant up to 100+5nM) still needs to be improved; the detection range of nucleic acid aptamers is 160ppt~1.6ppm, and the minimum detection limit is also high

Method used

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  • Nucleic acid aptamer for specifically identifying di (2-ethyl) hexyl phthalate and screening method and application of nucleic acid aptamer
  • Nucleic acid aptamer for specifically identifying di (2-ethyl) hexyl phthalate and screening method and application of nucleic acid aptamer
  • Nucleic acid aptamer for specifically identifying di (2-ethyl) hexyl phthalate and screening method and application of nucleic acid aptamer

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Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Screening of nucleic acid aptamers that specifically recognize di(2-ethyl)hexyl phthalate

[0037]1, synthesize the random single-stranded DNA library shown in Table 1, Biotin primers, prepare SA magnetic beads (streptavidin-modified magnetic beads) purchased from thermofisher company.

[0038] Table 1

[0039]

[0040] 2. Incubate the target substance DEHP with ssDNA:

[0041] Binding buffer for ssDNA library (0.1g CaCl 2 , 0.2g KCl, 0.2g KH 2 PO 4 , 0.1g MgCl 2 .6H 2 O, 8g NaCl, 1.15g Na 2 HPO 4 , 1L) was dissolved, and Biotin primers were added (the molar ratio to the library was 2:1) for slow denatured renaturation. Add to SA magnetic beads (beads were washed 4 times with binding buffer before use) and immobilized for 30-45min to determine the immobilization efficiency. Wash 6 times with binding buffer, add 100-200 μL of DEHP with a final concentration of 100 μM, and incubate at room temperature for 60-90 min.

[0042] 3. Screen and isolate ss...

Embodiment 2

[0059] The secondary structure of embodiment 2 nucleic acid aptamers

[0060] 1. A nucleic acid aptamer having the nucleotide sequence shown in SEQ ID NO.1

[0061] The secondary structure of the nucleic acid aptamer having the nucleotide sequence shown in SEQ ID NO.1 was analyzed using the M-fold platform. The results showed that the secondary structure (4A) of the full-length sequence (80bp) had a prominent loop, and the Gibbs free energy dG=-19.81, indicating that the structure had high stability. Its secondary structure is Figure 4 shown.

[0062] 2. A nucleic acid aptamer having the nucleotide sequence shown in SEQ ID NO.2

[0063] The secondary structure of the nucleic acid aptamer having the nucleotide sequence shown in SEQ ID NO.2 was analyzed using the M-fold platform. The results show that the sequence (58bp) secondary structure (4B) has a prominent loop after removing 11 bases before and after the constant region, and the Gibbs free energy dG=-17.56, showing th...

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Abstract

The invention provides a nucleic acid aptamer for specifically identifying di (2-ethyl) hexyl phthalate. The nucleic acid aptamer is one of the following nucleotide sequences: a nucleotide sequence as shown in SEQ ID NO. 1 and a nucleotide sequence as shown in SEQ ID NO. 2. The invention also provides a screening method of the nucleic acid aptamer and application thereof. The nucleic acid aptamer can rapidly and conveniently detect di (2-ethyl) hexyl phthalate, has high specificity, and has no cross reaction with a di (2-ethyl) hexyl phthalate analogue; the affinity is high, and the affinity constant is 2.26 + / - 0.06 nM; the sensitivity is high: the linear detection range is 7.629 pg / mL<-2> mu g / mL, and the lowest detection limit (LOD) of the di (2-ethyl) hexyl phthalate is 0.103 pg / mL.

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to a nucleic acid aptamer specifically recognizing bis(2-ethyl)hexyl phthalate, a screening method and application thereof. Background technique [0002] Bis(2-ethylhexyl) phthalate, also known as dioctyl phthalate, DOP, DEHP, dioctyl phthalate, chemical formula (molecular formula) is C 24 h 38 o 4 , is the ester compound formed by phthalic acid and 2-ethylhexanol. DEHP is one of the plasticizers used in a large amount and widely used. It can cause liver tumors in animals and is considered to be one of the possible carcinogens for humans. Therefore, the rapid monitoring of DEHP is of great significance to the protection of human health. [0003] At present, the methods for detecting DEHP are divided into two categories: instrumental detection technology and rapid detection technology. Among them, instrument detection technology includes: high performance liquid chromatograp...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12Q1/6806G01N33/53
CPCC12N15/115C12Q1/6806G01N33/5308C12N2310/16C12Q2531/113Y02A50/30
Inventor 刘细霞侯建军陆琪袁秋雪
Owner HUBEI NORMAL UNIV
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