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Nucleic acid aptamer capable of specifically recognizing florfenicol and florfenicol amine and application of nucleic acid aptamer

A technology of nucleic acid aptamer and florfenicol amine, applied in color/spectral characteristic measurement, biochemical equipment and methods, material inspection products, etc., can solve the problem of difficult to repeat production, antibody preparation requires experimental animals, processing process Complicated problems, to achieve the effect of wide application range, low cost and high specificity

Active Publication Date: 2022-03-25
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the detection methods of florfenicol and florfenicol amine are mainly immunoassay and gas chromatography-mass spectrometry; immunoassay is suitable for on-site detection, but the preparation of antibodies requires experimental animals, which is difficult to be reproduced ; Gas chromatography-mass spectrometry can be used for confirmatory detection, but the pre-treatment process is complicated and derivatization is required

Method used

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  • Nucleic acid aptamer capable of specifically recognizing florfenicol and florfenicol amine and application of nucleic acid aptamer
  • Nucleic acid aptamer capable of specifically recognizing florfenicol and florfenicol amine and application of nucleic acid aptamer
  • Nucleic acid aptamer capable of specifically recognizing florfenicol and florfenicol amine and application of nucleic acid aptamer

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Experimental program
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Effect test

Embodiment 1

[0055] Example 1. Screening of nucleic acid aptamers that can specifically recognize florfenicol amide

[0056] 1. Synthesize the random ssDNA library and primers shown in the following sequence

[0057] 5'-GCTGTGTGACTCCTGCAA-N 43 -GCAGCTGTATCTTGTCTCC-3'

[0058] Upstream primer: 5'-GCTGTGTGACTCCTGCAA-3'

[0059] Downstream primer: 5'-PHO-GGAGACAAGATACAGCTGC-3'

[0060] Among them, "N 43 "Indicates a sequence formed by connecting 43 arbitrary nucleotide bases. The library and primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0061] The library and primers were stored at -20°C in stock solutions with a concentration of 100 μM prepared from TE buffer (PH=8.0: 10 mmol / L Tris-HCl, 1 mmol / LEDTA) respectively.

[0062] 2. MB-SELEX screening specific nucleic acid aptamers

[0063] figure 1 It is the covalent coupling (A) and MB-SELEX screening flow chart (B) of tosyl magnetic beads and florfenicol amine provided by the embodiments of the present disclosu...

Embodiment 2

[0097] Embodiment 2, using the short-chain nucleic acid aptamer SEQ ID No.2 obtained after truncation to establish a method for detecting Florfenicol

[0098] Colloidal gold with a particle size of about 20nm was prepared by sodium citrate reduction method. 40μl 1μM nucleic acid aptamer was reacted with 50μl colloidal gold in the dark for 30min, and then a series of concentrations (0.00128ng / mL, 0.032ng / mL, 0.032ng / mL, 0.8ng / mL, 4ng / mL, 100ng / mL, 500ng / mL) of florfenicol, react in the dark at 37°C for 1h, and measure the absorbance value (A650 / A520) by a microplate reader. Take A650 / A520 as the ordinate, and the florfenicol concentration as the abscissa, so as to realize the sensitive detection of florfenicol. The results are as follows: Figure 7 shown. The method has a good linear range in the Florfenicol concentration of 0.00128~500ng / mL, and the minimum detection limit that can detect Florfenicol is 0.00128ng / mL, and the linear regression equation is y=0.0098ln(x)+0.7197...

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Abstract

The invention provides a nucleic acid aptamer capable of specifically recognizing florfenicol and florfenicol amine and application of the nucleic acid aptamer. The sequence of the nucleic acid aptamer is shown as SEQ ID NO. 1, or the sequence of the nucleic acid aptamer is shown as SEQ ID NO. 2. Compared with a protein antibody, the nucleic acid aptamer has higher affinity specificity, is free of immunogenicity, can be chemically synthesized, is small in molecular weight and stable in property, can be specifically combined with florfenicol and florfenicol amine, takes florfenicol and florfenicol amine as targets, and can be used for detecting florfenicol and florfenicol amine. The nucleic acid aptamer can realize qualitative and quantitative rapid detection of florfenicol and florfenicol amine, is low in cost, high in specificity and wide in application range, and can be used for constructing various biosensors for detecting florfenicol and / or florfenicol amine based on the nucleic acid aptamer.

Description

technical field [0001] The disclosure relates to the field of biotechnology, in particular to a nucleic acid aptamer capable of specifically recognizing florfenicol and florfenicol amine and its application. Background technique [0002] At present, the detection methods of florfenicol and florfenicol amine are mainly immunoassay and gas chromatography-mass spectrometry; immunoassay is suitable for on-site detection, but the preparation of antibodies requires experimental animals, which is difficult to be reproduced ; Gas chromatography-mass spectrometry can be used for confirmatory detection, but the pre-treatment process is complex and derivatization is required. . [0003] Therefore, there is an urgent need for a detection technology that is easy to operate and capable of detecting florfenicol and florfenicol amine with high efficiency and high sensitivity. Contents of the invention [0004] In view of this, the purpose of the present disclosure is to propose a nuclei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/53G01N33/58G01N33/543G01N21/31
CPCC12N15/115G01N33/5308G01N33/587G01N33/54346G01N21/31C12N2310/16Y02A50/30
Inventor 王向红刘若冰史明慧桑亚新刘敏轩崔琦琦
Owner HEBEI AGRICULTURAL UNIV.
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