Kit for detecting picoxystrobin as well as preparation method and application thereof

A picoxystrobin and kit technology, applied in the field of immunochemistry, can solve the problems of inability to adapt to the simple and fast market demand, genotoxicity of teratogenic cells, accumulation of picoxystrobin, etc., to stimulate animal immune response, purity and high yield and enhanced immunogenicity

Pending Publication Date: 2022-07-22
SOUTH CHINA AGRI UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Experiments have shown that picoxystrobin has good stability and low mobility in soil, but long-term high-frequency application can easily lead to accumulation of picoxystrobin in soil
In 2017, the European Commission issued a decision to gradually abolish the registration and use of picoxystrobin, mainly because of the risk of mutagenicity and cytogenetic toxicity of its metabolite IN-H8612, and the risk of its metabolite IN-QDY68 on aquatic organisms and Mammals that feed on earthworms are at high risk
[0003] At present, the main detection methods for picoxystrobin are chromatographic detection methods such as LC, GC and GC-MS. These methods are expensive and require professionals to operate. Although the accuracy is high, the detection is cumbersome and costly, and it is difficult to carry out on-site Detection cannot meet today's simple and rapid market demand; although the enzyme-linked immunoassay method has high sensitivity and high throughput, it also requires professional operation, and the detection time is long, which also cannot meet the needs of rapid on-site screening; The colloidal gold method has the advantages of high throughput, simple operation, short detection time and no need for professional and technical personnel to operate. However, due to the difficulty in quantitative analysis, it cannot meet the needs of increasingly heavy emergencies and emergency monitoring work

Method used

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  • Kit for detecting picoxystrobin as well as preparation method and application thereof
  • Kit for detecting picoxystrobin as well as preparation method and application thereof
  • Kit for detecting picoxystrobin as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 Preparation method of picoxystrobin hapten

[0050] The synthetic circuit diagram of picoxystrobin hapten is shown in figure 1 , the specific preparation method is as follows:

[0051] 1. (1) Take 1.0 g (2.72 mmol) of picoxystrobin whose structural formula is shown in formula I, add 8 mL of methanol in a 50 mL round-bottomed flask, stir and dissolve at room temperature, and then add 4 mL of lithium hydroxide (1 mol / L) Reaction at room temperature (20℃~30℃) for 72h;

[0052] (2) After heating and evaporating the solvent of step 1 to dryness, the obtained residue was directly dissolved in 10 mL of pure water, extracted with ethyl acetate to remove impurities twice, adjusted to pH 3 to 5 with hydrochloric acid, and a large amount of white precipitates were produced, filtered, and dried. After drying, 0.53 g of the product was obtained, which was the picoxystrobin hapten with the structural formula shown in formula II.

[0053] 2. (1) Take 1.0 g (2.72 mmol)...

Embodiment 2

[0060] The synthesis of embodiment 2 picoxystrobin artificial antigen

[0061] 1. The synthetic method of picoxystrobin artificial antigen whose carrier protein is bovine serum albumin

[0062] (1) Take 5 mg of the picoxystrobin hapten in Example 1, dissolve it in 0.25 mL of dimethylformamide (DMF), stir well, add 5 mg of carbodiimide hydrochloride (EDC) and 4 mg of N -Hydroxysuccinimide (NHS), stirring at room temperature (20℃~30℃) for 5h, the picoxystrobin hapten activated ester can be obtained;

[0063] (2) Weigh 24 mg of bovine serum albumin (BSA), fully dissolve it in 2.5 mL of PBS solution with a concentration of 0.01 mol / L to form a carrier protein solution, mix the picoxystrobin prepared in step 1 with stirring The hapten-activated ester is slowly added dropwise to the carrier protein solution, and stirred at room temperature (20°C to 30°C) for 16 to 24 hours;

[0064] (3) The solution prepared in step (2) was dialyzed with 0.01 mol / L PBS at room temperature (20°C to...

Embodiment 3

[0070] Example 3 Preparation of picoxystrobin monoclonal antibody

[0071] BALB / C mice were immunized with the picoxystrobin artificial antigen-KLH prepared in Example 2 as the immunogen and emulsified with an equal volume of Freund's adjuvant. The immunization dose of each mouse was 50-100 μg, the immunization interval was 2 weeks, and 7 days after the second booster immunization, the tail vein blood of the mice was collected to detect the serum titer. If the antibody titer does not meet the requirements, booster immunization is required. After the antibody titer no longer increases, subcutaneous booster immunization is performed with 100 μg of whole antigen, and mouse spleen cells are fused with SP20 cells 3 days later. The fused cells were selected in HAT medium and cultured after 5 days with complete medium replaced with HAT medium. The cell supernatant was detected by ELISA, and the cells in the wells with strong positive results were cloned and cultured by the limiting ...

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Abstract

The invention discloses a picoxystrobin detection kit and a preparation method and application thereof.The picoxystrobin hapten prepared through the method retains the characteristic structure of picoxystrobin to the maximum extent, the immunogenicity of the picoxystrobin hapten is obviously enhanced, the sensitivity of the obtained picoxystrobin monoclonal antibody is high, and the picoxystrobin monoclonal antibody can be used for detecting picoxystrobin through the ELISA detection technology. The lowest detection limit of picoxystrobin is 0.5 [mu] g / L; the preparation method of the picoxystrobin hapten is simple in reaction operation, reaction conditions are easy to control, and the prepared picoxystrobin hapten is high in purity and yield. The picoxystrobin fluorescent quantitative immunochromatographic test strip prepared by the invention has the detection sensitivity up to 1 microgram / L, is high in precision, can be stably stored for more than one year at room temperature, is beneficial to storage and transportation, and can realize rapid quantitative detection of picoxystrobin.

Description

technical field [0001] The invention relates to the technical field of immunochemistry, in particular, to a kit for detecting picoxystrobin and a preparation method and application thereof. Background technique [0002] Picoxystrobin (Picoxystrobin), the chemical name is (E)-3-methoxy-2-{2-[6-(trifluoromethyl)-2-pyridyloxymethyl]phenyl} acrylate, methyl ester, Molecular formula: C 18 H 16 F 3 NO 4 , relative molecular weight: 367.32. Picoxystrobin is a kind of methoxyacrylate fungicide. It was first developed by Syngenta in 2001. After DuPont acquired the product in 2006, it began to be widely used in the world. Pyraclostrobin was officially registered by the Chinese Ministry of Agriculture in 2012. This is a systemic fungicide, mainly used to control crop diseases, such as leaf blight, leaf rust, glume blight, brown spot, powdery mildew, net spot, white rot and downy mildew, etc. Therefore, picoxystrobin is widely used and has been reported in agricultural products s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/558
CPCG01N33/577G01N33/558
Inventor 徐振林梁科许晶冰邓浩王炳志孙远明
Owner SOUTH CHINA AGRI UNIV
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