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Construction method of immunosensor for measuring DNA (Deoxyribose Nucleic Acid) mark of melamine

An immunosensor and DNA labeling technology, applied in the field of immuno-PCR, can solve the problems of limited sensitivity, limited detection content and harmfulness.

Active Publication Date: 2012-12-19
JIANGNAN UNIV
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Problems solved by technology

Melamine is harmful to the body and cannot be used in food processing or food additives
[0003] Antigen-antibody reaction-based enzyme-linked immunosorbent ELISA method and immunochromatography technology use horseradish peroxidase (HRP) and colloidal gold as signal marker molecules respectively. Due to the limitation of their sensitivity, the detection content is limited

Method used

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  • Construction method of immunosensor for measuring DNA (Deoxyribose Nucleic Acid) mark of melamine
  • Construction method of immunosensor for measuring DNA (Deoxyribose Nucleic Acid) mark of melamine
  • Construction method of immunosensor for measuring DNA (Deoxyribose Nucleic Acid) mark of melamine

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Embodiment 1

[0027] (1) Preparation of DNA-melamine antibody conjugate

[0028]2.5 mg of diisofunctional group coupling agent 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (Sulfo-SMCC) was dissolved in 200 μL ultrapure Water, take 2 μL Sulfo-SMCC solution and dilute to 200 μL with pH 7.4 100 mM PBS buffer containing 150 mM NaCl, then take 200 μL 6 mg / mL melamine antibody and mix with the above-mentioned diluted 200 μL Sulfo-SMCC to make the final volume 400 μL, Shake the reaction at room temperature for 30 minutes, and ultrafilter the reactant with an ultrafiltration tube with a molecular weight cut-off of 3000 to remove excess coupling agent; dissolve the ultrafiltration retentate with 100mM PBS buffer containing 5mM EDTA and restore its original volume to 400μL ;Take 200 μL of the above dissolving solution for the next reaction, add 200 μL of 4nmol 89bp ssDNA to 200 μL of the dissolving solution, shake and react at room temperature for 30 minutes,...

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Abstract

The invention discloses a construction method of an immunosensor for measuring the DNA (Deoxyribose Nucleic Acid) mark of melamine, belonging to the field of immune PCRs (Polymerase Chain Reactions). The method comprises the following steps of: preparing a DNA-melamine antibody conjugate; coating a PCR tube with a melamine coating antigen; and constructing a melamine immunosensor. The invention provides a DNA-melamine antibody conjugate which is subjected to immunological recognition and fluorescent quantitation PCR amplification of DNA, and an amplified fluorescence signal is used for detecting melamine. The method can be used for detecting melamine at ultrahigh sensitivity, and has the advantages of low detection limit, high detection sensitivity and high specificity; and an effective method is provided for the detection of a trace quantity of melamine medicaments.

Description

technical field [0001] The invention relates to a method for constructing a DNA-marked immunosensor for measuring melamine, which belongs to the field of immuno-PCR. Background technique [0002] Melamine (Melamine, MEL), commonly known as melamine and protein essence, is a three-sulfone nitrogen-containing heterocyclic organic compound, which is used as a chemical raw material and is widely used in wood, plastics, coatings, papermaking, textiles, leather, electrical appliances, and medicine. and other industries. Melamine is harmful to the body and cannot be used in food processing or food additives. However, due to the high nitrogen content of melamine, the food industry often uses the Kjeldahl method to determine the total nitrogen content to estimate the protein content. Therefore, melamine is often used by unscrupulous traders as food and feed additives to increase protein in food testing. content index. On October 8, 2008, the Ministry of Health, the Ministry of Ind...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 胥传来尹红红徐丽广王利兵匡华
Owner JIANGNAN UNIV
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