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Organophosphorus pesticide multi-residue bio-barcode immunodetection kit based on hairpin self-assembly and application of organophosphorus pesticide multi-residue bio-barcode immunodetection kit

A technology of biological barcodes and organophosphorus pesticides, which is applied in biological testing, microbial measurement/inspection, biological material analysis, etc., and can solve problems such as false positives, difficulties in fast and efficient detection, and long detection cycles.

Active Publication Date: 2021-10-29
INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is no report on the application of catalytic hybridization self-assembly technology in pesticide detection.
For the existing pesticide residue confirmation detection technologies such as LC-MS, GC-MS and other technologies, but because chromatography and mass spectrometry methods require professional equipment and personnel, the sample pretreatment operation is complicated, the detection cycle is long, and the cost is high, so it is difficult to meet the fast The requirements for efficient detection, with the rise of rapid detection technologies based on modern biotechnology such as enzyme-linked immunosorbent assay technology and enzyme inhibition technology, have the characteristics of fast, simple, and low cost, but at the same time, false positives and sensitivity cannot be achieved. Meet the trace detection of pesticides and other issues

Method used

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  • Organophosphorus pesticide multi-residue bio-barcode immunodetection kit based on hairpin self-assembly and application of organophosphorus pesticide multi-residue bio-barcode immunodetection kit
  • Organophosphorus pesticide multi-residue bio-barcode immunodetection kit based on hairpin self-assembly and application of organophosphorus pesticide multi-residue bio-barcode immunodetection kit
  • Organophosphorus pesticide multi-residue bio-barcode immunodetection kit based on hairpin self-assembly and application of organophosphorus pesticide multi-residue bio-barcode immunodetection kit

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Experimental program
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preparation example Construction

[0051]In the present invention, the detecting kit comprises a black reaction plate that is coated with an organophosphorus semi-antigen-oval protein. The preparation method of the black reaction plate of the package is organic phosphorus semi-antigen-oval protein, preferably includes the steps of adding the organophosmic semi-antibiotic - oval protein to the reaction hole of the black reaction plate, 4 ° C overnight, closed after the washing, the washing plate gave a black reaction plate that was packaged by organophosphorus half-antine-oval protein. The addition amount of the packaged liquid of the organophosmic semi-antine-oval protein is 100 μl / well. The black reaction plate is preferably 96 well black enzyme panels. The organophosphorus semi-antine-oval-binding solution of a coated liquid is 0.05 mol / L carbonate buffer (CBS, pH = 9.6). The preparation method of the organophosphorus semi-antine-oval protein is to weigh the organophosphorus semi-antigen in DMF (N, N-dimethyl...

Embodiment 1

[0064] An organic phosphorus pesticide-free biological barcode immunoassay test kit based on hair clip self-assembly

[0065] 1) The black reaction plate package is packaged by organophosphorus semi-antigens, the preparation method is as follows:

[0066] In a black enzyme board, 100 μL per well is added to a certain amount of triazolophos, and the thiophosphorus, the poison is completely antigen (OVA coupling), 4 ° C, and 300 μl of 1% after washing. BSA was closed for 1 h, washing the plate, and obtained a black reaction plate that was packaged by organophosphorus semi-antibiotic-oval protein;

[0067] 2) Gold nanoparticles in which biological barcode and organophosphorus antibody are modified, the preparation method is as follows:

[0068] A. Synthesis of colloidal gold

[0069] 100ml 1mmol / L of Haucl 4 The aqueous solution is added to the round bottom flask, connect the condenser and start reflux. Heating to boiling under magnetic stirring, rapidly adding 10 ml of newly prepa...

Embodiment 2

[0092] Example 1 The detection kit prepared in the detection of triazolophosphorus phosphorus, thiophoschrazole, phosphorus and poisonous pesticides, including the following steps:

[0093] 1) Indirect competition Immunization: 50 μL of three pesticides mixed standard solutions or samples to be tested, 50 times diluted 10 times, three-kind gold probe mixture, slightly shock on the microscope, incubate 37 ° C Incubate 1h. During this process, pesticides in three pesticide standards or samples, the pesticides, respectively, compete with the coated antigen, bind to antibodies across the corresponding colloidal gold probe. Three probes specifically bind to the bottom of the microplate are preserved by the washing plate, and the remaining components are removed.

[0094] 2) Catalytic hair clamping self-assembly reaction: 100 μl of a concentration of DTT is added per well, then 2 μm hairpin structure H1, H3, H5 μL and H2, H4, H6 15 μL, slightly oscillated for 2 minutes, at 37 ° C consta...

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Abstract

The invention provides an organophosphorus pesticide multi-residue bio-barcode immunodetection kit based on hairpin self-assembly and application thereof, and belongs to the technical field of pesticide residue detection. The detection kit comprises the following components: a black reaction plate coated with organophosphorus hapten-ovalbumin, gold nanoparticles modified with a bio-barcode and an organophosphorus antibody, hairpin structure DNA H, hairpin structure DNA H marked with a fluorophore and a quenching group, dithiothreitol and an organophosphorus standard solution. According to the invention, bio-barcode immunoassay and a catalytic hairpin self-assembly technology are combined to develop a simple, convenient, rapid, high-sensitivity and trace pesticide detection method, and the method has important significance in meeting the requirements of rapid pesticide field detection and food safety supervision, aiming at organophosphorus pesticides including triazophos, parathion and chlorpyrifos, a multi-residue bio-barcode immunoassay method based on hairpin self-assembly is established, and the method has high sensitivity and accuracy.

Description

Technical field [0001] The present invention belongs to the field of pesticide residue detection, and in particular, the present invention relates to an organophosphoric pesticide-based organophosphoric acid poly residual biological barcode immunoassay test kit and its application based on hair clip self-assembly. Background technique [0002] Organic phosphorus pesticides are one of the broadest pesticides in China, in which rhizophosphorus is a high toxic pesticide, it has been comprehensively banned in 2008, and triazole phosphorty phosphorus and poisonous miutants have also prohibited in use in vegetables in 2016, but illegal use is still happens sometimes. The toxicity of organophosphorus pesticides and problems such as food safety from their residues are increasingly concerned. Therefore, developing a simple and fast, high sensitivity, pesticide trace detection method, and meets the need for rapid detection of pesticides and food safety supervision. [0003] Traditional con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/543G01N33/58C12Q1/682C12Q1/6804
CPCG01N33/5308G01N33/54346G01N33/582C12Q1/682C12Q1/6804G01N2430/10C12Q2563/107C12Q2563/185C12Q2563/155Y02A50/30
Inventor 金茂俊王源上王静邵华曹振王珊珊郑鹭飞佘永新金芬王淼
Owner INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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