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Sulfadimidine aptamer screening method, kit and application

A technology of sulfamethazine nucleic acid and sulfamethazine, applied in biochemical equipment and methods, biological testing, material inspection products, etc., can solve the problems of long experimental period, expensive chromatography, cumbersome processing process, etc., and achieve screening The number of rounds is short, the detection operation is simple, and the target is not fixed.

Active Publication Date: 2020-09-15
CHONGQING NORMAL UNIVERSITY
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, methods such as high-performance liquid chromatography, chromatography-mass spectrometry, and enzyme-linked immunosorbent assay are mostly used for the detection of sulfamethazine drugs. Although these methods have high selectivity and sensitivity, chromatography The use of relatively expensive analytical instruments and professional technicians is required, especially the sample pretreatment process is cumbersome, the extraction efficiency of low-level target substances is low, the cost of sample detection is high, there is matrix interference, and it is difficult to achieve rapid detection.
However, ELISA requires the preparation of antibodies, while the preparation of pharmaceutical antibodies requires the synthesis of complete antigens and immunization of animals. The experiment cycle is long, and the biological activity is easily inactivated due to the influence of various factors.

Method used

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  • Sulfadimidine aptamer screening method, kit and application
  • Sulfadimidine aptamer screening method, kit and application
  • Sulfadimidine aptamer screening method, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. GO-SELEX process for screening nucleic acid aptamers of sulfamethazine

[0023] 1) Library denaturation treatment: Take 500nM library (5'-FAM-GACAGGCA GGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3'; N is a random sequence) and place it in a metal bath at 95°C for 10 minutes, then quickly place it on ice for 10 minutes, take out Afterwards, equilibrate at room temperature for 25 minutes in the dark, so that a large number of ssDNA in the library can be folded to form complex and diverse three-dimensional structures. And measure the fluorescence value.

[0024] 2) The first round of screening: Take 7 μL of sulfamethazine solution (4 μg / mL), add it to 200 μL Lib, and incubate at 25 ° C and 250 rpm in the dark for 1 hour, ssDNA self-adaptively forms a three-dimensional structure, and a part of ssDNA forms The three-dimensional structure can combine with sulfamethazine to form ssDNA-sulfamethazine complex. Add a certain proportion of GO and incubate on a shaker at 25°C and 25...

Embodiment 2

[0051] The mensuration of embodiment 2 actual sample sulfamethazine

[0052] Milk and egg samples purchased from local supermarkets were previously analyzed by HPLC and confirmed to be free of SMZ compounds. A 10 ml milk sample was taken and first centrifuged at 14,000 rpm at 4°C for 20 minutes. The supernatant was diluted to 100 mL with binding buffer, and filtered through a 0.22-μm microporous membrane. For egg samples, after mixing and homogenizing well, add 2 g of egg samples to the centrifuge tube. Then add 4mL ethyl acetate and shake for 3min. After centrifugation at 5000×g for 5 min at room temperature, the supernatant was dried in a water bath at 80°C or in nitrogen, and diluted to 500 μL with binding buffer. In order to evaluate the recovery rate of the established graphene oxide-based fluorescent aptamer sensor, samples were added with different concentrations of sulfamethazine (2.0, 10.0, 25.0, 50.0 and 100.0 ng / mL), and the constructed aptamer The sensor detect...

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Abstract

The invention provides a sulfadimidine nucleic acid aptamer screening method and a sulfadimidine nucleic acid aptamer detection kit. The kit comprises a fluorescence aptamer sensor based on graphene oxide, wherein the fluorescence aptamer sensor based on graphene oxide comprises an aptamer marked by an FAM fluorophore and graphene oxide, wherein the sequence of the sulfadimidine aptamer marked bythe FAM fluorophore is No 1s recorded in a nucleotide sequence table. The invention discloses an application of the sulfadimidine fluorescence detection kit in quantitative detection of sulfadimidinein test samples of milk or eggs.

Description

technical field [0001] The invention relates to the fields of biotechnology and food antibiotic residue detection. More specifically, it relates to a sulfamethazine nucleic acid aptamer screening method, kit and application. Background technique [0002] Sulfamethazine, the chemical name is 2-(p-aminobenzenesulfonamido)-4,6-dimethylpyrimidine, which is a common sulfonamide drug and a broad-spectrum antibacterial agent. In addition to being used in clinical treatment, It can also be used as a veterinary drug and feed additive, and is widely used in the feeding of food-derived animals. After entering the animal body through various administration routes, it can be transferred to animal-derived foods such as meat, eggs, and milk. If used improperly, it is easy to produce residues in animal tissues and animal-derived foods, enter the human body through the food chain, and cause harm to human health. And it is discharged into the external environment through animal feces and ot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/115G01N33/53G01N21/64
CPCC12N15/1048C12N15/115G01N33/5308G01N21/6428C12N2310/16G01N2021/6432Y02A50/30
Inventor 乐涛寇启明孙琦吴平
Owner CHONGQING NORMAL UNIVERSITY
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