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Enzyme-linked immunosorbent assay kit for detecting iprodione and its application

A technology of immunological reagents and ibacteria urease, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problem of fewer types of substrates, and achieve the effects of enhanced immunogenicity, high specificity, and simple pretreatment methods

Active Publication Date: 2017-09-12
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the detection methods of iprodione are liquid chromatography and gas chromatography, which can detect fewer types of substrates, and there are few related patents and literature reports on the detection method of iprodione in tobacco

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting iprodione and its application
  • Enzyme-linked immunosorbent assay kit for detecting iprodione and its application
  • Enzyme-linked immunosorbent assay kit for detecting iprodione and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 Preparation of kit components

[0024] 1. Preparation of iprodione hapten

[0025] Synthesis of compound a: get 5.0 g 2,6-dichloro-4-nitrophenol and 3.2 g tert-butyl 4-bromobutyrate in acetone, then add 2.6 g of anhydrous potassium carbonate as a catalyst at 60 After reaction at ℃ for 8 h, evaporate the solvent to dryness, add water and ethyl acetate to extract, and dry with anhydrous sodium sulfate, concentrate, pass through a 200-300 mesh silica gel column, and chromatographically separate and purify to obtain 5.23 g of compound a. The rate is 87%. 1H NMR (CDCl3, 300MHZ) δ: 7.945 ( 1H, d, J=0.000), 4.144 ( 2H, t, J=7.500), 7.945 ( 1H, d, J=0.000), 1.973 ( 2H, tt, J= 7.500, J=7.367), 2.359 (2H, t, J=7.367), 1.414 ( 3H), 1.414 ( 3H), 1.414 ( 3H,s).

[0026] Synthesis of compound b: add 3.1 g of zinc powder and 1 mL of glacial acetic acid to 20 mL of water, react at 90 °C for 30 min, then add 5.2 g of compound a in ethanol solution 80 mL, react at 60 °C f...

Embodiment 2

[0054] Example 2 Formation of an enzyme-linked immunosorbent assay kit for detecting iprodione

[0055] An enzyme-linked immunosorbent assay kit for detecting iprodione was set up to include the following components:

[0056] (1) ELISA plate coated with iprodione-conjugated antigen;

[0057] (2) 6 bottles of iprodione standard solution, the concentrations are 0 µg / L, 4 µg / L, 12 µg / L, 36 µg / L, 108 µg / L, 324 µg / L;

[0058] (3) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0059] (4) Iprodione-specific antibody;

[0060] (5) The substrate chromogenic solution is composed of liquid A and liquid B, liquid A is carbamide peroxide, and liquid B is tetramethylbenzidine;

[0061] (6) The stop solution is 2 mol / L sulfuric acid;

[0062] (7) The washing solution has a pH value of 7.4, contains 0.5%~1.0% Tween-20, 0.01‰~0.03‰ sodium azide preservative, and 0.1~0.3 mol / L phosphate buffer solution, the percentages are by weight volume percentage;

[0063] (8) Th...

Embodiment 3

[0064] Example 3 Detection of iprodione in tobacco leaves

[0065] 1. Sample pretreatment

[0066]Weigh 1.0±0.05 g of tobacco leaf sample into a 50 mL polystyrene centrifuge tube, add 10 mL of tobacco leaf extract (weigh 100 mL of methanol, add 100 mL of deionized water, and mix thoroughly); Crush; filter the crushed sample with a filter membrane; pipette 200 mL of the filtrate and add 800 mL of complex solution, mix well; take 50 mL for analysis.

[0067] 2. Detection with kit

[0068] Add 50 μL of standard / sample to the corresponding microwells, then add 50 μL of antibody working solution / well, shake gently to mix, cover the plate with a cover film and place it in a dark environment at 25°C for 30 min. Carefully uncover the cover plate membrane, shake off the liquid in the wells, wash fully with 250 μL / well of washing working solution for 4-5 times with an interval of 10 s each time, and pat dry with absorbent paper. Add 100 μL / well of the enzyme-labeled secondary antibod...

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PUM

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Abstract

The invention provides an enzyme linked immunosorbent assay kit for detecting iprodione and application thereof. The enzyme linked immunosorbent assay kit comprises an elisa plate coated with a coating antigen, an iprodione standard substance solution, an enzyme-labeled secondary antibody, an iprodione specific antibody, a substrate color developing solution, a stop solution, a scrubbing solution and a redissolved solution. The coating antigen is an iprodione coupling antigen, and the enzyme-labeled secondary antibody is an enzyme-labeled goat-anti-mouse antibody. The invention further discloses a method using the enzyme linked immunosorbent assay kit for detecting iprodione. The enzyme linked immunosorbent assay kit can be used for detecting the content of iprodione in tobacco leaf samples, and is easy to operate, low in cost, high in sensitivity, capable of achieving on-site monitoring and suitable for screening a great number of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting iprodione and its application, which can qualitatively and quantitatively detect the residual amount of iprodione in tobacco leaves. Background technique [0002] Iprodione, also known as diphenhydramine, has the molecular formula C 13 h 13 Cl 2 N 3 o 3 , belonging to dicarboximides, is a broad-spectrum contact-type protective fungicide, widely used in tobacco, fruit trees, vegetable disease control and fruit storage and preservation. Iprodione can be absorbed systemically through roots, and can effectively control fungi that are resistant to benzimidazole systemic fungicides. Its main control targets are diseases caused by Botrytis, Alternaria, and Sclerotinia, such as Botrytis cinerea, early blight, black spot, and Sclerotinia. Iprodione pesticide is a testing item with national limit requirements...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/535
CPCG01N33/535
Inventor 陈黎范子彦杨昌松冯静潘立宁胡斌唐纲岭刘惠民鲁亚辉
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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