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Enzyme-linked immunoassay kit for detecting dimethomorph, and applications thereof

A technology of dimethomorph and dimethomorph couple, applied in the field of enzyme-linked immunoassay, can solve problems such as high detection technology requirements, unreasonable use of pesticides, damage to consumers' health, etc., and achieve simple sample pretreatment process, The effect of simple structure and high accuracy

Inactive Publication Date: 2018-06-26
ZHENJIANG YITE BIOTECH DEV CO LTD
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

At present, some national and regional food safety regulations have stipulated the maximum residue limit of dimethomorph. The MRL value of dimethomorph in some crops varies greatly, and the MRL value of some crops is relatively low. The requirements are relatively high, and there is no standard analysis method and MRL value regulation for dimethomorph residues in my country, which makes there is no unified and reliable basis for judging important information such as whether the pesticide is used unreasonably and whether the residue in agricultural products damages the health of consumers. and method

Method used

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  • Enzyme-linked immunoassay kit for detecting dimethomorph, and applications thereof
  • Enzyme-linked immunoassay kit for detecting dimethomorph, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 Preparation of kit components

[0035] 1. Preparation of dimethomorph hapten

[0036] 1) Dissolve 20 mg of dimethomorph, 10 ul, 1,3-propylenediamine, and a catalytic amount of 4-dimethylaminopyridine in 2 ml of N, N'-dimethylformamide to obtain liquid I;

[0037] 2) Dissolve 20mg of N,N'-dicyclohexylcarbodiimide in 0.5ml of DMF to obtain liquid II;

[0038] 3) Slowly add solution II to solution I dropwise at 0°C, and continue to react for 20 h after returning to room temperature;

[0039] 4) Evaporation of the solvent, column chromatography (eluent: dichloromethane / methanol, volume ratio 20:1), to obtain the dimethomorph hapten, such as figure 1 .

[0040] 2. Antigen preparation

[0041] Immunogen preparation—Dimethomorph hapten was coupled with bovine serum albumin (BSA) to obtain immunogen.

[0042] 1) Dissolve 5.3 mg of dimethomorph hapten in 0.5 ml of DMF to obtain liquid I;

[0043] 2) Dissolve 30mg of BSA in 2.0ml, pH7.0, 0.1mol / L phosphate buff...

Embodiment 2

[0064] Example 2 The formation of the enzyme-linked immunosorbent assay kit for detecting dimethomorph

[0065] An enzyme-linked immunosorbent assay kit for detecting dimethomorph was set up to include the following components:

[0066] (1) a microtiter plate coated with dimethomorph-coupled antigen;

[0067] (2) 6 bottles of dimethomorph standard solution, the concentrations are 0ug / L, 1ug / L, 3ug / L, 9ug / L, 27ug / L, 81ug / L.

[0068] (3) concentrated enzyme conjugate;

[0069] (4) enzyme conjugate dilution;

[0070] (5) Substrate chromogenic solution is made up of substrate liquid A liquid and substrate liquid B liquid, and substrate liquid A liquid is carbamide peroxide, and substrate liquid B liquid is tetramethylbenzidine;

[0071] (6) The stop solution is 2mol / L sulfuric acid.

Embodiment 3

[0072] The detection of dimethomorph in the sample of embodiment 3

[0073] 1. Sample pretreatment

[0074] Homogenize the vegetable sample with a homogenizer; weigh 5.0g±0.05g of the homogenized vegetable sample into a 50ml polystyrene centrifuge tube, add 25ml of 50% methanol, shake vigorously with an oscillator for 5min, over 3000r, at room temperature ( Centrifuge at 20-25℃ / 68-77℉) for 5min; take 500ul supernatant to a 2ml polystyrene centrifuge tube, add 500ul10% sodium chloride aqueous solution and shake for 1min with a shaker, mix well; take 20ul for analyze.

[0075] 2. Detection with kit

[0076] Add ochratoxin A standard solution / sample 20 ul to the microwells of the microtiter plate coated with dimethomorph-coupled antigen, and then add 100 ul of enzyme conjugate working solution (concentrate the enzyme conjugate with enzyme Dilute the diluent according to the volume of 1:20), seal the plate with a cover film, and react in the dark at 25°C for 10 minutes, pour ...

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Abstract

The present invention provides an enzyme-linked immunoassay kit for detecting dimethomorph. The enzyme-linked immunoassay kit contains an enzyme labeled plate coated with a dimethomorph-conjugated antigen, a dimethomorph standard substance solution, a concentrated enzyme conjugate, an enzyme conjugate working solution, a substrate chromogenic solution and a terminating solution. The invention further discloses a method for detecting dimethomorph in a sample by using the enzyme-linked immunoassay kit, wherein the method comprises: pre-treating a sample, detecting by using the kit, and analyzingthe detection result. According to the present invention, the enzyme-linked immunoassay kit can be used for detecting the residual dimethomorph in vegetable samples (raw materials, matching materialsand concentrated materials), has advantages of simple operation, low cost, high sensitivity and on-site monitoring, and is suitable for screening of a large number of samples.

Description

technical field [0001] The invention relates to an ELISA detection technology, in particular to an ELISA kit for detecting dimethomorph, which is suitable for the determination of dimethomorph in vegetables. technical background [0002] Dimethomorph is a fungicide specific to oocidal fungi, and has a remarkable control effect on downy mildew and Phytophthora fungal diseases. It is one of the fungicides widely used in my country. At present, the pesticide preparations with dimethomorph as the main ingredient are mainly registered in my country for cucumbers and grapes, which are used to prevent and control downy mildew; there are fewer preparations registered for crops such as peppers, melons, potatoes, cabbage, tomatoes, and tobacco. Used to control blight and downy mildew in these crops and blackleg in tobacco. Dimethomorph is a low toxicity pesticide. But it is widely used. The level of pesticide residues is concerned. At present, some national and regional food safety...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/531G01N33/543G01N21/31G01N21/78G01N1/34G01N1/38
CPCG01N33/558G01N1/34G01N1/38G01N21/31G01N21/78G01N33/531G01N33/54306G01N2021/3185
Inventor 洪霞刘静立雯馨
Owner ZHENJIANG YITE BIOTECH DEV CO LTD
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