Method for detecting dimethachlon residue in tobacco by competition-indirect time-resolved fluoroimmunoassay system

A technique of fluorescence immunoassay and competition time, which is applied in the field of detection of net sclerotia residues in tobacco, which can solve the problems of low sensitivity and achieve the effect of high detection signal and strong anti-matrix interference ability

Inactive Publication Date: 2012-07-18
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to: for the problem that there are few researches on the detection methods of sclerotia at present, and the sensitivity of the reported gas chromatography detection method and enzyme-linked immunoassay detection method is low, the present invention provides a time-resolved detection method for sclerotia residues Fluorescence immunoassay method, and applied it to the detection of net sclerotia residues in tobacco, and developed a simple and fast pretreatment method that does not require sample purification

Method used

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  • Method for detecting dimethachlon residue in tobacco by competition-indirect time-resolved fluoroimmunoassay system
  • Method for detecting dimethachlon residue in tobacco by competition-indirect time-resolved fluoroimmunoassay system
  • Method for detecting dimethachlon residue in tobacco by competition-indirect time-resolved fluoroimmunoassay system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Preparation of Eu-N1 labeled goat anti-rabbit IgG

[0019] Weigh 4 mg of Eu-N1 (provided by Wallac, Finland), dissolve it in 350 μL of distilled water, and set aside. Dissolve 1 mg of affinity-purified goat anti-rabbit IgG (provided by Wuhan Boster Company) in 150 μL of distilled water, add 15 μL of 0.5 mol / L carbonate buffer (pH 9.8), mix well, and mix the dissolved Eu-N1 with Goat anti-rabbit IgG was mixed and allowed to stand overnight at 4°C. The next day, the reactant was put into a dialysis bag (molecular weight cut-off: 8000), dialyzed for 3 days, and the retentate was divided and stored at -20°C for later use.

Embodiment 2

[0020] Example 2. Establishment of CI-TRFIA (Competitive indirect time-resolved fluorescence immunoassay)

[0021] Pre-wash the microtiter plate once with washing buffer, add 100 μL of sclerotinia-OVA (made in the laboratory) with a final concentration of 6.9 mg / L, diluted in detection buffer (Wuxi Jiangyuan Industrial Technology and Trade Corporation), at room temperature After shaking and incubating at 700r / min for 30min, wash the plate 4 times with washing buffer (Wuxi Jiangyuan Industrial Technology and Trade Corporation); add 50μL of 2000-fold diluted sclerotinia polyclonal antibody (self-made in the laboratory) and 50μL Sclerotinia standard solutions of different concentrations were shaken and incubated at room temperature at 700r / min for 1 hour, and then the plate was washed 4 times; then 100 μL of Eu-N1-labeled goat anti-rabbit IgG diluted 1000 times was added, and incubated at room temperature with shaking at 700r / min After 1 h, wash the plate 4 times; add 100 μL of f...

Embodiment 3

[0023] Example 3. Research on the Influence of Tobacco Leaf Matrix on the Time-Resolved Fluorescence Detection System

[0024] Considering that immunological detection methods are mainly used for rapid screening of a large number of samples, the sample pretreatment methods used usually do not use complicated sample purification steps, but simply extract the samples, and then dilute, reduce or remove the samples. Matrix interference with the assay. However, sample dilution will lead to a decrease in the actual sensitivity of the detection method. In this experiment, by studying the influence of different concentrations of matrix on the CI-TRFIA analysis system (in the linear range) after the tobacco leaves were extracted with acetone, the dilution factor required for the detection method after the pretreatment of the tobacco leaf samples was determined.

[0025] Specific steps are as follows:

[0026] Weigh 3 groups of 1g tobacco leaves (8 parts for each group), put them into...

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Abstract

The invention relates to a method for detecting dimethachlon residue in tobacco by an competition-indirect time-resolved fluoroimmunoassay system, wherein the CI-TRFIA (competition-indirect time-resolved fluoroimmunoassay) system takes dimethachlon-OVA as a coating antigen, a dimethachlon polyclonal antibody as a recognition antibody and a Goat Anti-rabbit IgG (immunoglobulin G) marked with Eu-N1 as a secondary antibody to establish a standard inhibition curve corresponding to dimethachlon pesticide; and the detection of the dimethachlon residue in tobacco is realized by the following steps: after pretreatment on a tobacco sample, detecting the tobacco sample by the CI-TRFIA system and then judging the quantity of the dimethachlon residue in the tobacco corresponding to the sample.

Description

Technical field: [0001] The invention relates to a method for detecting net sclerotia residues in tobacco by a CI-TRFIA detection system. Background technique: [0002] Sclerotin (N-3,5-dichlorophenylsuccinamide) is a high-efficiency, low-toxic imine fungicide. It has the characteristics of broad-spectrum, bactericidal, systemic, penetrating, and long-lasting effects, and is widely used in the prevention and treatment of tobacco red spot and rice sheath blight. Considering the potential risks of net sclerotia residues to human health and the environment, it is imperative to establish a detection method for net sclerotia residues in tobacco. However, the residue limit of sclerotia in tobacco in my country has not yet been formulated, and the detection standard is only the gas chromatography method established by the tobacco industry standard [Gas Chromatography for Determination of Sclerotia Pesticide Residues in Tobacco and Tobacco Products. Tobacco Industry Standard of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558
Inventor 刘媛刘贤金张霄王冬兰
Owner JIANGSU ACAD OF AGRI SCI
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