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Method for detecting nitrofuran metabolites in animal-derived food through liquid-phase chip

A liquid phase chip detection and nitrofuran technology, which is applied in the fields of immunization and food public health detection, can solve the problems of high cost, time-consuming, single detection of veterinary drug residues, etc., and achieves simple operation, economical benefits, and high-throughput detection. Effect

Inactive Publication Date: 2014-06-25
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It aims to solve the problems of single, time-consuming, and high cost of veterinary drug residue detection, and lays the foundation for the rapid detection technology research of multi-residue veterinary drugs; the establishment of this method can meet the requirements of accurate and rapid detection of AHD, SEM, AOZ and AMOZ in animal-derived food At the same time, it provides a new detection method for the daily monitoring of veterinary drug residues in food of animal origin in my country

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0026] The biotinylation of embodiment 1AHD, SEM, AOZ and AMOZ monoclonal antibody

[0027]Weigh 1 mg of AHD, SEM, AOZ and AMOZ monoclonal antibodies, respectively, and dissolve in 45 μL of 10 mM Sulfo-NHS-Biotin (BNHS) (Sigma Company) solution. Stir the reaction for 4-5 hours at room temperature to fully biotinylate the antibody. Then pass through the YM-10 column with 0.1 mol / L PBS at pH 7.4 to replace the buffer and remove unreacted small molecule BNHS. AHD, SEM, AOZ and AMOZ biotinylated antibodies were aliquoted in small quantities and stored at -20°C.

Embodiment 2

[0028] The preparation of embodiment 2 coupled microspheres

[0029] Place the microspheres stored at 4°C at room temperature for 20-30 minutes, fully vortex the microspheres to make them into a suspended state, pipette 100 μL of the microsphere solution into two 1.5 ml centrifuge tubes, and centrifuge at 13000 r / min for 4 minutes to remove the protective solution , After washing with triple distilled water, resuspend the microspheres in 400 μL of 0.1M pH6.2 phosphate buffer. Add 10 μL, 50 mg / mL N-hydroxy-sulfosuccinimide (Sulfo-NHS) (Pierce Company) and 10 μL, 50 mg / ml 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (Pierce Company) respectively , after mixing, act at 37°C for 20 minutes to obtain coated microspheres. Centrifuge to remove the supernatant, resuspend the coated microspheres with 900 μL, 0.05M MES, centrifuge to remove the supernatant, and wash the microspheres twice. The microspheres were resuspended with 400 μL, 0.05 M MES to obtain the re...

Embodiment 3

[0030] Example 3 Pretreatment of samples to be detected

[0031] Accurately weigh 1±0.01g tissue homogenate into a 50mL centrifuge tube, add 5mL of distilled water and mix well, add 0.5mL of 1mol / L HCl solution, vortex for 1min, then add 100μL of 0.01mol / L p-nitrobenzaldehyde (2 Methyl sulfoxide solution), vortex for 1 min, sonicate for 20 min, and incubate at 55°C for 3 h in a constant temperature shaker. After incubation, add 5mL0.1mol / LNa2HPO4, vortex for 1min, add 0.4mL1mol / LNaOH, vortex for 1min, then add 6mL ethyl acetate, vortex for 1min, centrifuge at 5000r / min for 15min at room temperature, absorb 3.0mL ethyl acetate Put the supernatant into a 15mL centrifuge tube, dry it with nitrogen at 45°C, redissolve it in 1mL of n-hexane and mix it with 1mL of sample diluent, centrifuge at 5000r / min for 15min at room temperature, take the bottom aqueous phase, and measure the pH value (should be neutral), get the sample to be tested, and store it at 4°C until use.

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Abstract

The invention provides a method for detecting nitrofuran metabolites in animal-derived food through a liquid-phase chip. The method comprises the steps of coupling four microspheres with different numbers with artificial antigens for nitrofuran metabolites such as AHD, SEM, AOZ and AMOZ to obtain coupled microspheres; performing biotin reaction on four AHD, SEM, AOZ and AMOZ monoclonal antibodies and a BNHS (biotin-hydroxysuccinimide) solution to obtain a biotin antibody; performing reaction on the nitrofuran metabolites, the coupled microspheres, the biotin antibody and streptavidin-phycoerythrin (SA-PE) to obtain liquid to be detected; finally detecting a mean fluorescence intensity (MFI) value of the reaction liquid, and performing data analysis. The method disclosed by the invention can be used for simultaneously detecting the AHD, SEM, AOZ and AMOZ in the animal-derived food according to the specificity; furthermore, the whole process is simple to operate and is economical, practical, quick and accurate, and high-flux detection can be realized.

Description

technical field [0001] The invention belongs to the technical field of immunity and food public health detection, and in particular relates to a liquid phase chip detection method for nitrofuran metabolites in animal source food. Background technique [0002] Nitrofurantoin, nitrofurazone, furazolidone, and furaltadone are a class of broad-spectrum antibiotics that have been used as fungicides, insect repellants and animal growth promoters in many countries. Studies have shown that nitrofurantoin, nitrofurazone, furazolidone, and furaltadone are extremely unstable in animals, and are metabolized into corresponding metabolites 1-aminohydantoin (1-aminohydanto, AHD), semicarbazide (semicarbazide) after a few hours. , SEM), 3-amino-2-oxazolidinone (3-amino-2-oxazolidinone, AOZ) and 5-morpholinomethyl-3-amino-2-oxazolidinone (3-Amino- 5-morpholinomethyl-2-oxazolidinone, AMOZ), and most of the metabolites are stably combined with animal tissue proteins and exist in animals for a...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N21/64
CPCG01N33/545G01N33/5375G01N33/542
Inventor 刘迎春王权陈永军蒋蔚张华景张梦肖燕
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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