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Specific nucleic acid aptamer for recognizing streptomycin and application of nucleic acid aptamer to streptomycin detection

A nucleic acid aptamer and specific recognition technology, which is applied in the field of biotechnology detection, can solve the problems of cumbersome detection process of streptomycin, poor accuracy and sensitivity, and cumbersome detection procedures, and achieve flexible methods, easy preservation, and low cost. Effect

Active Publication Date: 2015-07-01
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 nucleic acid aptamer that specifically recognizes streptomycin, and apply it to qualitative or quantitative detection of streptomycin, so as to solve the problem of cumbersome detection process of existing streptomycin and the accuracy of detection. poor problem
[0006] The second object of the present invention is to provide a method for qualitative and quantitative detection of streptomycin, to solve the problem of detecting streptomycin in the prior art, either the detection process is cumbersome, time-consuming and laborious, the cost is high, or the accuracy and sensitivity are relatively low. bad question

Method used

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  • Specific nucleic acid aptamer for recognizing streptomycin and application of nucleic acid aptamer to streptomycin detection
  • Specific nucleic acid aptamer for recognizing streptomycin and application of nucleic acid aptamer to streptomycin detection
  • Specific nucleic acid aptamer for recognizing streptomycin and application of nucleic acid aptamer to streptomycin detection

Examples

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Embodiment 1

[0036] Example 1 Acquisition of a nucleic acid aptamer that specifically recognizes streptomycin

[0037] (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

[0066] Example 2 Qualitative and quantitative detection of streptomycin using aptamers

[0067] (1) Colloidal gold preparation: 20 mL of HAuCl with a mass ratio concentration of 0.01% 4 Heat to boil, and add 500 μL of sodium citrate with a mass ratio concentration of 1% 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; the colloidal gold can be stored in a mass ratio concentration of 0.05% sodium azide, spare;

[0068] (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 1 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 temperature Incubate for 1 h; finally...

Embodiment 3

[0073] Quantitative detection of streptomycin residues in embodiment 3 milk and honey

[0074]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:

[0075] 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;

[0076] Honey sample processing: honey is diluted five times with double distilled water, and used as a test sample;

[0077] 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 quantitative detection method in Example 2 was used to detect the concentration of streptomycin in the samples. ...

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Abstract

The invention discloses a specific nucleic acid aptamer for recognizing streptomycin. The nucleotide sequence of the aptamer is shown as SEQ ID NO: 1. Meanwhile, the invention further discloses a method for qualitatively and quantitatively determining streptomycin by using the nucleic acid aptamer and application of the nucleic acid aptamer to food inspection. According to the invention, streptomycin is taken as a target substance, and a high-affinity and high-specificity nucleic acid aptamer for recognizing streptomycin is screened by an SELEX technology through affinity chromatography and is combined with colloidal gold colorimetry, so that a novel streptomycin detection method based on the aptamer is built. The method can be used for qualitative or quantitative detection of streptomycin simply, quickly, accurately, economically and specifically, and can be widely applied to the fields of food, sanitation, import and export inspection and quarantine, and the like.

Description

technical field [0001] The invention relates to the field of biotechnology detection, in particular to a nucleic acid aptamer specifically recognizing streptomycin and its application. 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 to detect whether strep...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/53
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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