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A screening method and application of a nucleic acid aptamer that specifically recognizes streptomycin

A nucleic acid aptamer, streptomycin technology, applied in the field of biotechnology detection, can solve the problems of poor affinity and specificity, complicated screening method steps, poor accuracy and sensitivity, etc., achieves good stability, and is easy to eliminate other problems. The effect of factor interference and low cost

Active Publication Date: 2017-10-20
HEBEI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] One of the purposes of the present invention is to provide a screening method for nucleic acid aptamers that specifically recognize streptomycin, to solve the complex steps of the existing common aptamer screening methods, and the screened aptamers have high false positives and high affinity. Problems with poor harmony and specificity
[0006] The second object of the present invention is to use the aptamer that specifically recognizes streptomycin obtained by the screening method in the detection of streptomycin to solve the problem of detecting the presence of streptomycin in the prior art or the detection process is cumbersome and time-consuming. , high cost, or relatively poor accuracy and sensitivity

Method used

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  • A screening method and application of a nucleic acid aptamer that specifically recognizes streptomycin
  • A screening method and application of a nucleic acid aptamer that specifically recognizes streptomycin
  • A screening method and application of a nucleic acid aptamer that specifically recognizes streptomycin

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1 Acquisition of a nucleic acid aptamer that specifically recognizes streptomycin

[0049] (1) Coupling of epoxy-based gel and streptomycin: Take 3 ml of epoxy-based activated gel and wash it five times with double distilled water, then filter and dry; add the dried epoxy-based activated gel to 10 ml of 0.1mol / L Na containing 20 mg / ml streptomycin 2 CO 3 -NaHCO 3 In the coupling buffer (pH value 10.5), incubate at 37°C for 16 h; after the reaction, wash the incubated product three times with 0.1 mol / L PBS solution, then add 1 mol / L ethanolamine for blocking, and incubate at room temperature for 1 h ; The blocked gel conjugate was sequentially washed with 5 times deionized water, 0.1 mol / L HAc-NaAc buffer solution (pH 4.0) containing 0.5 mol / L NaCl, deionized water, 0.1 mol / L containing 0.5 mol / L NaCl boric acid-sodium tetraborate buffer solution (pH value 8.0) and deionized water were fully washed, dried by suction filtration, and unreacted ligands were remov...

Embodiment 2

[0078] Example 2 Qualitative and quantitative detection of streptomycin using the aptamers screened in Example 1

[0079] (1) Preparation of colloidal gold: 20 mL of HAuCl with a mass ratio concentration of 0.015% 4 Heat to boil, and add 500 μL of sodium citrate with a mass ratio concentration of 0.95% while stirring. After the color changes, the solution continues to boil for 10 minutes, remove the heat source and stir until room temperature to obtain colloidal gold; colloidal gold can be stored in a mass ratio concentration of 0.05% sodium azide, spare;

[0080] (2) Qualitative detection: Add 100 nmol / L nucleic acid aptamer that specifically recognizes streptomycin to 50 µL colloidal gold, and incubate at room temperature for 0.8 h to prepare aptamer-labeled colloidal gold; the final concentrations are respectively Streptomycin at 25 nmol / L, 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L, 800 nmol / L, and 1600 nmol / L was added to aptamer-labeled colloidal gold at room tempera...

Embodiment 3

[0085] Quantitative detection of streptomycin residues in embodiment 3 milk and honey

[0086] Streptomycin-free honey and milk samples were purchased from local supermarkets, and streptomycin was quantitatively detected by standard recovery experiments. The specific operations were as follows:

[0087] Milk sample processing: Add 3 mL ethyl acetate and 3 mL water to 1 mL milk sample, vortex for 15 min; then centrifuge at 8000 rpm / min at 4°C for 15 min; the mixture is divided into three layers, and the aqueous layer is collected as the next One-step testing of samples;

[0088] Honey sample processing: honey is diluted five times with double distilled water, and used as a test sample;

[0089] A series of standard concentrations of streptomycin: 25nmol / L, 50nmol / L, 100nmol / L, 200nmol / L, 400nmol / L were added to the treated milk and honey samples respectively, and the streptomycin in the sample was detected by the quantitative detection method in Example 2. The concentration o...

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Abstract

The invention discloses a screening method of nucleic acid aptamers for specifically recognizing streptomycin. The screening method comprises the following steps: (A) coupling epoxy group gel with streptomycin; (B) preparing a negative electrode pole and a positive electrode pole; (C) constructing an ssDNA random library, and screening and determining aptamers with relatively high affinity to streptomycin; and (D) authenticating the aptamer with the highest affinity for specifically recognizing streptomycin by virtue of a fluorescence method. According to the screening method, the screened nucleic acid aptamer is utilized for qualitatively and quantitatively determining streptomycin in food. The screening method is high in condition preciseness, other interference factors are easily eliminated, the screened nucleic acid aptamer has very high affinity and high specific recognizability, and furthermore, a false positive phenomenon can be reduced; the screening method is a novel method for simply, rapidly, scientifically and accurately screening the nucleic acid aptamers; by combining the screened nucleic acid aptamer with a colloidal gold colorimetric method, streptomycin can be simply, rapidly and accurately detected; and the screening method can be widely applied to various fields of foods, sanitation, import and export inspection and the like.

Description

technical field [0001] The invention relates to the field of biotechnology detection, in particular to a screening method and application of a nucleic acid aptamer that specifically recognizes streptomycin. Background technique [0002] Streptomycin is a broad-spectrum aminoglycoside antibiotic that can inhibit Gram-negative bacilli and some Gram-positive bacilli, and is widely used in medicine, agriculture and animal husbandry. However, in actual use, due to unreasonable or illegal use, it causes a large amount of residues in animal-derived foods such as meat, liver, kidney, milk and honey, which seriously endangers human health and can cause people to have a A series of allergic reactions, nephrotoxicity, ototoxicity and other diseases can even lead to deafness. Therefore, many countries and regions such as the FDA, the European Union, and my country have made strict regulations on the maximum residue limit of streptomycin. It can be seen that it is of great significance ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/53
Inventor 刘中成樊武舫张艳芬解瑶赵丽君赵丽健王向欢
Owner HEBEI UNIVERSITY
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