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DNA aptamer specifically recognizing streptomycin and application of DNA aptamer

An aptamer and streptomycin technology, applied in DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of difficulty in antibody activity, long-term maintenance, and high price. The method is simple, easy to operate, and easy to use. Retouching and labeling, good stability effects

Active Publication Date: 2012-10-03
苏州医疗器械产业发展集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some companies in Europe and the United States have produced ELISA kits for the detection of antibiotic residues, but they are expensive, and the types of enzyme-labeled antibodies are limited, and only a few types of common antibiotic residues can be detected.
Since streptomycin is a small molecular compound and has no immunogenicity itself, it needs to be linked with a biological macromolecule such as a protein as a carrier antigenic determinant to stimulate animals to produce specific antibodies, and there are many limitations in production and application. Including: Antibiotic haptens with weak immunogenicity in antigen preparation are difficult to produce high-titer antibodies; heterologous antibodies produced in mice, rabbits and other animals may produce non-specific reactions (false positives) during use; antibody preparation costs are high , Time-consuming and labor-intensive, the clones are not easy to preserve; the quality varies between batches; the activity of the antibody is difficult to maintain for a long time, sensitive to temperature, and prone to irreversible denaturation
These have largely limited the application of immunoassay methods in the detection of streptomycin

Method used

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  • DNA aptamer specifically recognizing streptomycin and application of DNA aptamer
  • DNA aptamer specifically recognizing streptomycin and application of DNA aptamer
  • DNA aptamer specifically recognizing streptomycin and application of DNA aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031]Example 1. Immobilization of streptomycin on the surface of epoxy-modified magnetic beads

[0032] The schematic diagram of the coupling of streptomycin and magnetic beads is as follows: figure 1 As shown, the specific process is:

[0033] (a) Prepare 10mM streptomycin solution, adjust the pH to 8.0, take 50μL, 10mg / mL epoxy-modified magnetic beads, wash with PBS buffer of pH 7.4 for 3-5 times, add the prepared streptomycin 200 μL of prime solution, and shake overnight at 37°C. Streptomycin not bound to the magnetic beads was washed with pH 7.4 PBS buffer and magnetically separated. Add 200 μL of 0.5 M ethanolamine at pH 8.0 to the streptomycin-magnetic bead complex, and combine with a shaker at 37°C for 6 hours to block the uncoupled groups on the magnetic beads and improve the specificity of aptamer screening. The ethanolamine not bound to the magnetic beads was washed with PBS buffer solution of pH 7.4 to obtain the affinity magnetic beads coupled with streptomyci...

Embodiment 2

[0035] Example 2. Construction of random ssDNA library and primers thereof

[0036] (a) Construction of a random ssDNA library with a length of 79 bases

[0037] 5'-TAGGGAATTCGTCGACGGATCC-N35-CTGCAGGTCGACGCATGCGCCG-3' where N represents any of the bases A, T, C, and G, and N35 represents a random fragment length of 35 bases.

[0038] (b) Synthesis of upstream primers

[0039] Upstream primer 1: 5'-TAGGGAATTCGTCGACGGAT-3'

[0040] Upstream primer 2: 5'-biotin-TAGGGAATTCGTCGACGGAT-3'

[0041] (c) Synthesis of downstream primers

[0042] Downstream primer 1: 5'-CGGCGCATGCGTCGACCTG-3'

[0043] Downstream primer 2: 5'-biotin-CGGCGCATGCGTCGACCTG-3'

Embodiment 3

[0044] Example 3. In vitro screening of nucleic acid aptamers

[0045] In order to screen out ssDNA aptamers with high affinity and specificity to streptomycin, a total of 8 rounds of nucleic acid aptamers were screened. In order to improve the specificity of screening, a reverse screening experiment of ethanolamine-immobilized magnetic beads was carried out every two rounds of screening. In each round of screening, the binding rate of the aptamer to the target streptomycin was as follows: figure 2 shown.

[0046] (a) Take 1 μL of ssDNA primary library, add 200 μL of binding buffer, bathe in 90°C water for 10 minutes, rapidly cool to 4°C, let stand for 15 minutes, and then stand at room temperature for 7 minutes, then add it to the affinity magnetic beads coupled with streptomycin , react at room temperature for 30min (slight shaking). Wash 5 times with binding buffer and magnetically separate to remove unbound ssDNA. Add 150 μL of elution buffer (40 mM Tris-HCl, 10 mM EDT...

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Abstract

The invention discloses a single-chain DNA aptamer specifically recognizing streptomycin and application of the DNA aptamer, belonging to the field of biochemistry and molecular biology, analytical chemistry and combinatorial chemistry. The invention provides a method for preferably selecting the streptomycin aptamer by using an improved magnetic bead SELEX technology and four single-chain DNA aptamers with high appetency with the streptomycin and sequences of A1, A3, A6 and A12, and provides a high-specificity detection recognition element with the advantages of better stability, high sensitivity, low cost, and easy preparation, modification and mark for the detection of residue of the streptomycin, especially streptomycin in foods.

Description

technical field [0001] The invention relates to the screening and application of a novel recognition element-DNA aptamer (Aptamer) specific to streptomycin, and relates to the fields of biochemistry and molecular biology, analytical chemistry and combinatorial chemistry. Background technique [0002] Aminoglycoside antibiotics, such as streptomycin, gentamicin, neomycin, dihydrostreptomycin, etc., have significant antibacterial effects on a variety of Gram-positive and Gram-negative bacteria, and can effectively inhibit The growth and reproduction of bacteria, so it is one of the commonly used veterinary drugs in my country's agriculture, animal husbandry and aquaculture. However, the main toxic and side effects of this type of antibiotics are reflected in the damage to the cranial nerves, hearing and kidneys. Therefore, many countries and institutions have stipulated clear maximum residue limits (MRIJs) for the residues of this type of drugs in food. Due to the illegal use ...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/10C12Q1/68
Inventor 周楠迪王靖元田亚平
Owner 苏州医疗器械产业发展集团有限公司
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