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Method and kit for detecting organophosphorus and carbamate pesticides

A carbamate and organophosphorus technology is applied in the field of detecting organophosphorus and carbamate pesticides, and achieves the effects of high sensitivity detection lower limit, saving detection cost and low cost.

Inactive Publication Date: 2017-10-31
赵芳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both ELISA and immunochromatographic test strips can only detect one or two (at most five or six) pesticide residues at a time, which cannot meet the actual needs of hundreds of pesticides currently in use.

Method used

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  • Method and kit for detecting organophosphorus and carbamate pesticides
  • Method and kit for detecting organophosphorus and carbamate pesticides
  • Method and kit for detecting organophosphorus and carbamate pesticides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Processing method of samples to be tested

[0023] 1. Meat: 1) Take 0.5g of meat in a 10ml beaker, add 5ml of acetone to soak for 5min, vortex and mix once every 1min, then add 0.2g of calcium carbonate; 2) Take 0.5ml of the above liquid in a 5ml beaker , dry the acetone with nitrogen; add 0.3ml 0.1MTris-HCl (pH8.0) buffer solution to dissolve; 3) add 0.1ml 0.3% calcium hypochlorite, let stand for 10min; 4) add 0.3ml 10% sodium nitrite, Shake well and prepare for inspection.

[0024] 2. Milk: 1) Raw milk and pasteurized milk do not need to be processed. 2) Fermented milk: Adjust the pH to pH 8.0 with twice the mass of 0.1M Tris-HCl (pH 8.0) for inspection. 3) Milk powder: Take 1g of milk powder, add 9ml of deionized water, dissolve, and prepare for inspection.

[0025] 3. Honey: dilute 10 times with 0.1M Tris-HCl (pH8.0) for inspection.

[0026] 4. Urine: Dilute 2 times with 0.1M Tris-HCl (pH8.0) for inspection.

[0027] 5. Fruits and vegetables: Take 1g ...

Embodiment 2

[0030] The preparation of embodiment 2 monoclonal antibody

[0031] 1. Preparation of Primary Monoclonal Antibody

[0032] a. Immunize Balb / c mice with active cholinesterase that is highly sensitive to organophosphorus and carbamate pesticides (such as acetylcholinesterase / butyrylcholinesterase derived from animals, plants, and genetic recombination), Immunization dose: 100μg / monkey, immunized once every 2 weeks, and immunized four times in a row;

[0033] b. Splenocytes of immunized mice were fused with SP2 / 0 cells, and then monoclonal antibodies were screened out with corresponding active cholinesterase-coated microwell plates;

[0034] c. Preparation of aging cholinesterase: add excess methyl parathion to active cholinesterase; place at 37°C for 1 hour; remove small molecules by ultrafiltration; resuspend cholinesterase with PBS, which is aging Cholinesterase.

[0035] d. Screening of the first monoclonal antibody: Dilute aged cholinesterase to 1-10 μg / ml with carbonate ...

Embodiment 3

[0045] Embodiment 3 detects the immunochromatographic method of active cholinesterase

[0046] 1. Preparation of the first gold-labeled monoclonal antibody: take 10ml of gold nanoparticles with a particle size of 40nm, add 40μg of the first monoclonal antibody; react at room temperature for 15 minutes; add BSA to a final concentration of 1% (W / V), and continue the reaction at room temperature 15min; centrifuge, resuspend the particles with 1ml reconstitution solution (10mM Tris-HCl (pH8.0), 0.5%BSA), and set aside.

[0047] 2. Preparation of chromatographic membrane: Spray the third monoclonal antibody (detection area) with a concentration of 2mg / ml and 0.02mg / ml goat antibody on Millipore's 135 nitrocellulose membrane in an amount of 1.0μl / cm Mouse IgG (quality control area), dried at 37°C for 2 hours, set aside.

[0048] 3. Preparation of the sample pad: Soak Ahlstrom 8964 glass fiber in 20mM Tris-HCl (pH8.0), 1%BSA, 0.5%Tween 20 for 10min; dry at 37°C for later use.

[00...

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Abstract

The invention discloses a method for detecting organophosphorus and carbamate pesticides. The detection method is achieved through detecting cholinesterase through a double-antibody sandwich. The invention further discloses a kit for detecting organophosphorus and carbamate pesticides. The residue conditions of the organophosphorus and carbamate pesticides in a detected sample are judged through detecting the comformational change of the cholinesterase, so that the kit and the method have the advantages of being wide in detection range, high in sensitivity, simple, feasible and low in cost.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method and a kit for detecting organophosphorus and carbamate pesticides. Background technique [0002] Organophosphorus and carbamate pesticides have been widely used in my country's agricultural production practice due to their broad insecticidal spectrum and high efficacy. Due to its large usage and wide range of applications, it has received widespread attention in terms of food safety and environmental protection. In particular, some highly toxic and highly toxic pesticides, such as methamidophos, methyl parathion, parathion and other pesticides, have been banned from being used on vegetables and fruits due to their acute toxicity and residual toxicity. However, due to the lack of scientific knowledge of pesticides used by farmers, the problem of indiscriminate use and abuse of highly toxic and highly toxic pesticides often occurs. Even for those pesticides with relat...

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 赵芳史跃光其他发明人请求不公开姓名
Owner 赵芳
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