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Aptamer for specific recognition of amantadine and its application

An amantadine and aptamer technology, which is applied in the field of aptamers that specifically recognize amantadine, can solve the problems of natural structure damage and lack of the target, and achieves the effects of high sensitivity, high affinity and easy preparation.

Active Publication Date: 2022-05-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Almost all nucleic acid aptamers are screened through the exponential enrichment ligand evolution system (SELEX technology), while the screening of small molecule target aptamers in the traditional SELEX technology is less due to the lack of chemical Groups immobilized on supports, or even when small molecule targets can be immobilized, the native structure of the target is often disrupted during the screening process

Method used

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  • Aptamer for specific recognition of amantadine and its application
  • Aptamer for specific recognition of amantadine and its application
  • Aptamer for specific recognition of amantadine and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Synthesis of aptamers and capture probes

[0027] Aptamer sequences with random libraries and 5' biotinylated capture probes were synthesized by Shanghai Bioengineering Co., Ltd.

[0028] Aptamer sequence (SEQ ID NO.2):

[0029] 5'-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3'

[0030] Biotinylated capture probe (SEQ ID NO.3):

[0031] 5'-botin-GACCCTCTGTGCTGCT-3'.

Embodiment 2

[0032] Example 2: Fe 3 o 4 Preparation and coating of magnetic nanoparticles

[0033] 2.1 Preparation of aminated magnetic beads

[0034] One-step preparation of aminated magnetic beads using hot melt, the process is as follows: 1. Add 30mL of ethylene glycol, 1g of ferric chloride hexahydrate and 2g of sodium acetate into a round bottom flask in turn, shake well and add 6.50g of 1,6- Hexamethylenediamine; 2. Magnetically stir the above solution in a 50°C water bath until a uniform purple-red colloidal solution; 3. Transfer the solution to a 100mL polytetrafluoroethylene-lined autoclave, and heat it at 198°C 6h; 4. After the reaction kettle is cooled, remove the supernatant in the kettle under the action of an external magnetic field, add 100mL absolute ethanol to the kettle, stir and pour it into a beaker; 5. Remove the supernatant by magnetic separation, and then add 100mL of water for ultrasonic cleaning and magnetic separation. According to this method, wash three time...

Embodiment 3

[0037] Example 3: Screening of amantadine aptamer sequences

[0038] 3.1 Library fixation and screening

[0039] The capture-SELEX technology based on magnetic separation is used for circular screening, and the steps are as follows: before each round of screening, the library or secondary library is combined with the biotinylated capture probe at a molar ratio of 1:2, and a certain amount of After denaturation at 95°C for 10 minutes, incubate in a 37°C, 200rpm incubator for 2h; after the incubation, add avidinated aminated magnetic beads and incubate in an incubator at 37°C, 200rpm for 3h. Using the specific binding of biotin and avidin, the library or secondary library is immobilized on magnetic beads. After the incubation, the supernatant was kept, and the immobilization degree of the library was verified by UV spectrophotometry; the magnetic beads after the immobilized library were washed 5 times with a buffer solution, and the target amantadine was dispersed in the buffer...

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Abstract

The invention discloses an aptamer specifically recognizing amantadine and its application. The nucleotide sequence of the aptamer specifically recognizing amantadine of the present invention is shown in SEQ ID NO.1. The present invention utilizes the systematic evolution technology of library immobilized exponentially enriched ligands, takes amantadine as the target, screens and obtains an aptamer sequence with high affinity and strong specificity for amantadine, and the screened aptamer sequence The ligand sequence is highly sensitive, easy to prepare and easy to modify, and will be applied to the rapid detection of amantadine veterinary drug residues in food.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an aptamer specifically recognizing amantadine and its application. Background technique [0002] Amantadine is an amino derivative whose parent structure is saturated tricyclodecanylamine, usually exists in the form of hydrochloride, and can inhibit the replication of influenza A virus at a relatively low concentration. Long-term use of amantadine antiviral drugs can easily lead to drug resistance of influenza-like viruses and increase the possibility of influenza-like viruses mutating. The research on the metabolism of amantadine drugs shows that amantadine can be enriched in the body in the form of the original body, and the residual amantadine in livestock and poultry animals can enter the human body through the food chain, thus causing certain damage to human health . The Ministry of Agriculture of my country banned the use of amantadine antiviral drugs on pou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/53
CPCC12N15/115C12N15/1048G01N33/53C12N2310/16
Inventor 段诺吕辰王周平吴世嘉
Owner JIANGNAN UNIV
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