Screening method and application of nucleic acid aptamers for specifically recognizing streptomycin
A nucleic acid aptamer and screening method technology, applied in the field of nucleic acid aptamer screening, can solve the problems of complex screening method steps, poor accuracy and sensitivity, poor affinity and specificity, etc., so as to easily eliminate interference from other factors , good stability and low cost
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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 were 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 temper...
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 quantitative detection method in Example 2 was used to detect the concentration of streptomycin in the samples....
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