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Pesticide and veterinary drug multi-residue detection method based on microarray detection chip

A microarray chip and detection chip technology, applied in the direction of biological testing, material inspection products, etc., can solve the problems of expensive instruments, false positives, complicated and difficult operations, etc., to ensure accuracy and sensitivity, reduce preparation costs, and operate The effect of simple process

Active Publication Date: 2015-06-10
内蒙古敖敦食品股份有限公司
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AI Technical Summary

Problems solved by technology

(3) Chromatography-mass spectrometry (GC-MS, HPLC-MS): It can achieve qualitative and quantitative detection purposes at the same time, especially suitable for the detection of pesticide metabolites, degradation products and multi-residue detection, etc., but this method requires expensive instruments And the operation is complicated and difficult, not suitable for frequent detection
(4) Supercritical Fluid Chromatography (SFC): It not only has the characteristics of fast, efficient, and sensitive gas spectroscopy, but also has the characteristics of liquid chromatography that can detect thermally unstable and macromolecular compounds, but requires expensive instruments and complicated operations Difficulty, etc.
(5) Capillary electrophoresis (CE): It is suitable for the separation and analysis of some ionized samples that are difficult to separate by traditional chromatography. It has 10-1000 times higher analytical capacity than HPLC. It requires expensive instruments and is complicated and difficult to operate. It is not suitable for for regular testing
However, due to the influence of antibodies such as dependence on foreign imports, at present, the ELISA finished kits in the Chinese market rely on imports from abroad.
(3) Enzyme inhibition method is the most mature and widely used rapid pesticide residue detection technology. This method can only detect organophosphorus and carbamate pesticides, and the inhibition rate of different pesticides of the same kind varies greatly. , so using a uniform inhibition rate to determine whether pesticide residues exceed the standard will inevitably lead to false positive or false negative missed detection, which can be said to be the current "rapid detection method adopted as a last resort", which is only applicable to the initial inspection at the grassroots level and plays a warning role , when the phenomenon of exceeding the standard is found, it must be retested and confirmed by standard methods, negative reactions should also be sampled in proportion, and retested and confirmed by reliable methods
[0013] Existing research at home and abroad is mainly focused on establishing a specific immunoassay method for a single pesticide and veterinary drug, but in fact there are often many kinds of pesticide and veterinary drug residues in agricultural products, Immunoassay methods and corresponding detection kits for single agricultural and veterinary drug components have been difficult to meet the needs of actual detection

Method used

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  • Pesticide and veterinary drug multi-residue detection method based on microarray detection chip
  • Pesticide and veterinary drug multi-residue detection method based on microarray detection chip
  • Pesticide and veterinary drug multi-residue detection method based on microarray detection chip

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Experimental program
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Effect test

Embodiment 1

[0066] Part I: Synthesis of Pefloxacin (PEFloxacion, PEF) Immunization Antigen and Detection Antigen

[0067] (1) Accurately weigh 45 mg of N-hydroxysuccinimide (NHS), 210 mg of carbodiimide (EDC), 36 mg of pefloxacin (PEF), and add 5 mL of N,N-dimethyl Formamide (DMF). And incubate at room temperature for 24 h with shading and stirring, and this solution is called A.

[0068] (2) Accurately weigh 105 mg bovine serum albumin (BSA), add 0.01 mol / L (pH 7.4) phosphate buffer saline (PBS) 15 mL, this solution is called B.

[0069] (3) Add the incubated solution A to solution B dropwise, stir while adding, and dialyze with 0.01 mol / L (pH value 7.4) PBS magnetic stirring for 6 days, and change the solution several times during the period to Remove unreacted small molecule species. Centrifuge at 12,000 r / min for 30 min, collect the supernatant to obtain the PEF-BSA conjugate, and store the obtained PEF-BSA conjugate in a -20°C refrigerator for future use.

[0070] After replaci...

Embodiment 2

[0138] The first part of melamine (Melamine) immune antigen and detection antigen synthesis

[0139] The immune antigen melamine-BSA and the detection antigen melamine-OVA were synthesized by combining polybasic anhydrides and mixed anhydrides.

[0140] (1) Synthesis of melamine glutaric anhydride semialdehyde by polybasic anhydride method (where pyridine is used as solvent and catalyst). Weigh 125 mg of melamine (about 1 mmol), add it into 4 mL of pyridine solution containing 114 mg (about 1 mmol) of glutaric anhydride, and stir the reaction with a magnetic stirrer for 22 h at room temperature. After the reaction was complete, the pyridine was blown dry with nitrogen.

[0141] (2) Use the above product to react with isobutyl chloroformate to obtain mixed anhydrides. Dissolve melamine-glutaric anhydride semialdehyde in 40 mL solvent (DMF and 1,4-dioxane 1:1 mixture), add 262 μL (about 1 mmol) n-tributylamine, stir in ice for 10 min, add chloroformic acid 144 μL (about 1 m...

Embodiment 3

[0148] The first part of the synthesis of methamidophos immune antigen and detection antigen

[0149] Weigh 100 mg of methamidophos and dissolve in 10 mL of 0.1 mol / L HCl, add pre-cooled 1 % NaNO 2 Continue to stir for 30 min until the starch potassium iodide test paper turns blue; take another 10 mg BSA and dissolve it in 2 mL of borate buffer solution with pH 8.7, take 2 mL of the above diazotization product and add it dropwise to the protein solution, and stir in an ice bath After 2 hours, place overnight at 4°C and put the reaction solution into a dialysis bag. Dialyze with PBS at 4°C for 3 days, freeze-dry the dialysate to obtain a white powder, store at -20°C for use. The coated antigen methamidophos-OVA was prepared in the same way.

[0150] The second part is the preparation of monoclonal antibodies to agricultural and veterinary drugs, and the third part is the preparation of microarrays of monoclonal antibodies to agricultural and veterinary drugs.

[0151] U...

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Abstract

The invention prepares an immune antigen by coupling small molecular agricultural and veterinary drug haptens with bovine serum albumin, and prepares corresponding agricultural and veterinary drug monoclonal antibodies by immunizing mice with the immune antigen, and uses a biochip spotter for capturing monoclonal antibody probes The samples were printed on the agarose-modified glass slide solid phase carrier to prepare a multi-repeated "capture monoclonal antibody probe" microarray. The hapten of agricultural and veterinary drugs was coupled with ovalbumin to prepare a "hapten-OVA" conjugate, which was then labeled with fluorescent molecule Cy3. Mix the sample solution to be tested with the "labeled detection antigen" at a certain concentration, and then incubate with the chip. Competitive binding, eluted under certain conditions. The results were detected with a biochip scanner. The method can simultaneously detect a variety of agricultural and veterinary drug residues in agricultural and sideline product samples, and is suitable for high-throughput, rapid and accurate detection of drug residues in planting and breeding production.

Description

technical field [0001] The invention relates to a method for preparing a multi-residue detection microarray chip of agricultural and veterinary drugs in the field of food safety. Background technique [0002] Agricultural and veterinary drugs are effective means to prevent and control crop diseases and insect pests and aquaculture diseases in countries around the world. The use of a large number of chemical pesticides and veterinary drugs, especially the highly toxic and high-residue pesticides and veterinary drugs, has become the main "killer" of food safety and endangering people's health. [0003] At present, the main chemical pesticides used are mainly divided into three categories: insecticides, herbicides and fungicides according to their different functions. According to different chemical structures, they can be divided into organochlorines, organophosphorus, pyrethroids, carbamates, and inorganic pesticides. The common characteristics of these chemical pesticides...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
Inventor 李同祥孙会刚黄天姿候进慧
Owner 内蒙古敖敦食品股份有限公司
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