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A method for functionalized magnetic bead separation-enzyme-linked aptamer detection of oxytetracycline in food

An enzyme-linked aptamer and magnetic bead separation technology, applied in the direction of color/spectral characteristic measurement, etc., can solve the problem that the electrochemical signal of the sample matrix is ​​easy to cause interference, cannot meet the needs of oxytetracycline detection, and the aptamer immobilization step consumes a lot of time. Time and other issues, to achieve the effect of reducing detection cost, improving specificity, and low detection cost

Active Publication Date: 2017-09-22
XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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Problems solved by technology

For example, Liu Bin (Jinan University master's thesis) established an aptamer-based biotin-avidin enzyme-linked detection method for oxytetracycline, but the detection limit (1.5g / L) could not meet the needs of oxytetracycline detection ( Limit value: 100mg / kg)
Kim et al. (Anal. Chim. Acta, 2009, 634: 250-254) established an analytical method for the detection of oxytetracycline with an aptamer electrochemical sensor, but the aptamer immobilization step is relatively time-consuming, and the sample matrix has a great influence on the electrochemistry. Chemical signals are prone to interference
In 2010, he also used gold nano-modified aptamers as probes to colorimetrically detect oxytetracycline (Biosens. Bioelectron 26: 1644-1649), but could not be accurately quantified
In 2014, Kim et al. (Biosens. Bioelectron 51: 426–430) established an indirect enzyme-linked aptamer assay (ic-ELAA) method for oxytetracycline in milk based on the ELISA principle, with a detection limit of 12.3 ng / mL. However, the method needs to coat OTC-BSA on the microtiter plate, resulting in a long detection time (>10h)

Method used

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  • A method for functionalized magnetic bead separation-enzyme-linked aptamer detection of oxytetracycline in food
  • A method for functionalized magnetic bead separation-enzyme-linked aptamer detection of oxytetracycline in food
  • A method for functionalized magnetic bead separation-enzyme-linked aptamer detection of oxytetracycline in food

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Determination of milk samples

[0042] 1. Preparation of nucleic acid aptamer-magnetic nanoparticle complex probe

[0043] The preparation of the nucleic acid aptamer-magnetic nanoparticle complex probe was prepared by the method reported in the literature (Wu SJ, Duan N, Wang ZP, et al. Analyst, 2011, 136, 2306-2314).

[0044] 1. Preparation of aminated nano-magnetic beads: Add 6.5 g of 1,6-hexanediamine, 2.0 g of anhydrous sodium acetate (CH 3 COONa) and 1.0 g ferric chloride hexahydrate (FeCl 3 ·6H 2 O), stirred at 50°C to obtain a colloidal solution, and transferred the solution to a 50mL reactor lined with polytetrafluoroethylene, and reacted at 198°C for 6h. Cool to room temperature, discard the upper liquid, wash out the lower solid material with deionized water, and collect it by magnetic separation. Wash twice with deionized water and ethanol respectively, and dry at 50°C for 5-10h. Scanning electron microscope images of aminated nano-magneti...

Embodiment 2

[0056] Embodiment 2: Determination of animal tissue samples

[0057] Method is the same as embodiment one, difference is the pretreatment method of sample:

[0058] Weigh 1.0 g of chicken sample into a 50 mL centrifuge tube, add 4 mL of extraction solution, vortex mix for 10 min, and centrifuge at 15000 r / min for 10 min. Take 200uL of the supernatant and dilute to 1 mL with 10mmol / L PBS buffer for analysis. Substitute into the standard curve to calculate the content of oxytetracycline in the sample.

Embodiment 3

[0059] Embodiment 3: honey sample determination

[0060] Method is the same as embodiment one, difference is the pretreatment method of sample:

[0061] Accurately weigh 1.0g honey sample into a 50mL screw cap centrifuge tube, add 10mL PBS buffer (10 mmol / L, pH 7.4), vortex for 5min, sonicate for 5min, and take 100μL of the supernatant for analysis. Calculate the content of the analyte in the sample according to the standard curve.

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Abstract

The invention discloses a method for functionalized magnetic bead separation-enzyme-linked aptamer to detect oxytetracycline in food. OTC directly competes with the OTC aptamer on the surface of the magnetic beads, and the substrate is chromogenic to detect the change in absorbance to quantitatively analyze the content of oxytetracycline in the sample. Compared with the traditional ELISA and enzyme-linked aptamer (ELAA), the present invention fully combines the rapid separation and enrichment effect of magnetic nanomaterials, the high specificity of nucleic acid aptamers and the high-throughput screening characteristics of ELISA, The detection time can be greatly shortened, the detection sensitivity and specificity can be improved, and the detection cost can be reduced.

Description

technical field [0001] The invention relates to a detection method for oxytetracycline in food, and belongs to the technical field of food safety analysis. Background technique [0002] Oxytetracycline (OTC), also known as 5-hydroxytetracycline, oxytetracycline, oxytetracycline, oxytetracycline, etc., is a broad-spectrum antibacterial drug, often used as a preventive agent for cattle, pigs, poultry, fish and other animal diseases and treatment of infections. However, due to abuse and non-compliance with the withdrawal period regulations, its residues in animal products not only bring serious harm to human health, but also pose a serious threat to the environment. The Food and Agriculture Organization of the World, the World Health Organization, the European Union and our government have made regulations on the residue limits of oxytetracycline in food. [0003] At present, the detection methods of oxytetracycline mainly include high performance liquid chromatography (HPLC)...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/33
Inventor 卢春霞唐宗贵刘长彬孙凤霞康立超陈霞王国红
Owner XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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