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Vomitoxin fluorescence immunochromatography test strip as well as preparation method and application thereof

A technique of fluorescence immunochromatography and vomitoxin, which is applied to the analysis of materials, biological tests, material inspection products, etc. It can solve the problems that positive results cannot be saved, sample background interference, and results are no longer accurate and reliable.

Inactive Publication Date: 2020-05-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, colloidal gold immunochromatographic test strips have the following defects: (1) general test strips are qualitatively analyzed by visual observation, and accurate quantitative detection cannot be achieved; (2) different material matrix effects are obvious, and the sample background Large interference, easy to produce false positive results; (3) The positive results of the test cannot be saved, usually beyond the judgment time, the results are no longer accurate and reliable

Method used

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  • Vomitoxin fluorescence immunochromatography test strip as well as preparation method and application thereof
  • Vomitoxin fluorescence immunochromatography test strip as well as preparation method and application thereof
  • Vomitoxin fluorescence immunochromatography test strip as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Preparation of Deoxynivalenol Hapten, Complete Antigen, Monoclonal Antibody, Goat Anti-mouse Secondary Antibody

[0073] 1. Preparation of vomitoxin hapten

[0074] The synthetic route of vomitoxin hapten of the present invention is as follows Figure 4 As shown, it specifically includes the following steps:

[0075] (1) Weigh 23.2 mg of vomitoxin in advance, add 10 mL of ethylene glycol dimethyl ether, dissolve it by ultrasonic, and set aside;

[0076] (2) Weigh 0.6g of sodium hydride (because sodium hydride contains kerosene, use 20mL of petroleum ether to stir magnetically for 30s, let it stand for 2min, pour off the petroleum ether; repeat the cleaning 2 to 3 times; after pouring off the petroleum ether for the last time, Blow dry with nitrogen);

[0077] (3) Add dissolved vomitoxin in sodium hydride, stir for 1 hour, add 0.5 g of succinic anhydride, stir for 3 hours, after reacting for 1 hour, slowly add 4 mL of methyl 3-bromopropionate dropwise, react...

Embodiment 2

[0108] Embodiment 2 Preparation method of vomitoxin fluorescent immunochromatography test strip

[0109] 1. Preparation of fluorescent probes for DON monoclonal antibody labeled with aggregation-induced luminescence fluorescent microspheres

[0110] a. Take 10 μL of aggregation-induced luminescent fluorescent microsphere solution and 1 mL of MES buffer solution into a centrifuge tube, mix well, centrifuge, and set aside;

[0111] The carboxylated aggregation-induced luminescent fluorescent microspheres have an excitation wavelength of 200 nm and an emission wavelength of 610 nm.

[0112] b. Add 15 μL 0.5 mg / mL EDC and 20 μL 0.5 mg / mL NHS to each tube, use a constant temperature culture shaker to shake, 200 rpm, 25 ° C for 15 min; after the catalysis is completed, 14000 rpm, 25 ° C for 15 min, discard the supernatant solution, redissolve in 1 mL of 0.05M BB buffer solution (pH=8.0);

[0113] c. Add 1 μL of deoxynivalenol monoclonal antibody, place on a constant temperature cu...

Embodiment 3

[0142] Example 3 Using the above-mentioned fluorescent immunochromatographic test strips to detect vomitoxin residues

[0143] 1. Sample pretreatment

[0144] Grind the sample (feed) to be tested into powder; weigh 1.00±0.05g of the crushed sample into a 10mL centrifuge tube; add 6mL of 70% methanol-water solution, vortex for 3min, and centrifuge at 4000rpm for 5min; take 50μL of the supernatant, Add 450 μL of sample diluent, mix well, and test.

[0145] 2. Test with test strips

[0146] Open the test paper cylinder, take out the test paper strip prepared in Example 2, mark it and place it on the table (note: after taking out the micropore and the test paper strip, cover the cylinder cover immediately to prevent moisture); absorb 100 μL of the sample to be tested , drop the test strip into the micropore of the fluorescent probe, fully immerse the sample pad in the sample, and react for 5 minutes; take out the test strip, and interpret the result in a dry fluorescence analyze...

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Abstract

The invention discloses a vomitoxin fluorescence immunochromatography test strip as well as a preparation method and application thereof. The test strip comprises a bottom plate, and a reaction film is attached to the bottom plate; one end of the reaction film covers a water absorption pad, and the other end sequentially covers a combination pad and a sample pad; a detection line and a quality control line are arranged on a non-covering surface of the reaction film along the transverse direction; a fluorescent probe of a vomitoxin monoclonal antibody marked by aggregation-induced emission fluorescent microspheres is sprayed on the combination pad; the detection line is coated with a vomitoxin complete antigen, and the quality control line is coated with a goat anti-mouse secondary antibody. According to the invention, the marking steps are greatly simplified, the marking efficiency is improved, the detection time is shortened, the physical adsorption stability is better than that of colloidal gold, and the detection accuracy and precision are high; the inter-batch difference is small, and the cost is low; in addition, the detection method is simple to operate, the whole detection process only needs 8 minutes, the detection limit is 100ppb, the quantitative detection range is 100ppb-5000ppb, and the sensitivity and specificity are high.

Description

technical field [0001] The invention belongs to the field of immune detection, in particular to the technical field of rapid determination and detection of vomitoxin in feed and grain products. More specifically, it relates to a vomitoxin fluorescent immunochromatographic test strip and its preparation method and application. Background technique [0002] Deoxynivalenol (DON for short) is a common type B trichothecene toxin, which is one of the trichothecene olefins, and it is usually produced by cereals (such as wheat, corn, etc.) and barley, etc.) in the mold, Fusarizin production. Large doses of vomitoxin can cause vomiting symptoms in humans, pigs, ruminants, mice, and poultry, so it is also commonly known as "vomitoxin", which can cause weight loss and diarrhea in humans and animals, disrupt the homeostasis of the intestinal system, And affect the endocrine system and immune system, and can reach the blood-brain barrier through blood circulation, thereby causing poten...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/577G01N33/558G01N33/53
CPCG01N33/5308G01N33/558G01N33/577G01N33/582G01N33/585
Inventor 杨金易王序曾道平苏晓娜谭庶卢迪莎吴颖许小炫
Owner SOUTH CHINA AGRI UNIV
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