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ScFv-ELISA kit for detecting thiamethoxam residues by one-step method and application thereof

A technology of scfv-ap and thiamethoxam, which is applied in the field of genetic engineering and ELISA detection, can solve the problems of long time-consuming, complicated pre-treatment, and many operation steps, and achieve the effect of less time-consuming and simple pre-treatment process

Pending Publication Date: 2019-09-17
苏州微测生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to aim at the shortcomings of current pesticide residue instrument analysis methods such as high cost, complicated pretreatment, poor specificity, low sensitivity, and difficulty in on-site detection in experiments; Influenced by the specificity and sensitivity of the secondary antibody, it has disadvantages such as relatively low stability, and provides a high-specificity, high-sensitivity, high-accuracy, high-precision, simple operation method, and can be used for large-scale samples quickly Detectable, one-step ELISA kit for the detection of thiamethoxam residues

Method used

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  • ScFv-ELISA kit for detecting thiamethoxam residues by one-step method and application thereof
  • ScFv-ELISA kit for detecting thiamethoxam residues by one-step method and application thereof
  • ScFv-ELISA kit for detecting thiamethoxam residues by one-step method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The preparation of embodiment 1 thiamethoxam coating antigen

[0030] (1) Weigh 8.269mg of thiamethoxam-5C (MW=413.457, 0.02mmol), 2.76mg of NHS (MW=115, 0.024mmol), 4.738mg of DCC (MW=206, 0.23mmol) and dissolve them in 200μL of anhydrous DMF Then place it on a magnetic stirrer, stir and react overnight at room temperature, centrifuge the reaction solution at 5000rmp for 15min the next day, and the supernatant is the active ester solution.

[0031] (2) Dissolve 20mg of BSA in 2mL of CB (0.05M, pH 9.6), place it on a magnetic stirrer and stir slowly, slowly add the above-mentioned active ester solution drop by drop for about 20min, then continue at room temperature The reaction was stirred for 6h.

[0032] (3) The reaction solution was dialyzed with PBS for 5-6 times, and the PBS was changed once every 6 hours. The supernatant was collected by centrifugation, and the supernatant was the synthesized thiamethoxam-coated antigen Thi-5C-BSA.

Embodiment 2

[0033] Example 2 Construction of anti-thiamethoxam avian scFv-AP genetically engineered antibody

[0034] Firstly, the specific thiamethoxam scFv gene fragment, ie nanobody (SEQ ID NO: 1), was screened by phage display technology.

[0035] Then the specific thiamethoxam single-chain antibody plasmid was extracted, and the scFv gene fragment was amplified by PCR.

[0036] The PCR reaction system is as follows:

[0037]

[0038] The reaction procedure is as follows:

[0039] Pre-denaturation at 93°C for 2 minutes;

[0040]

[0041] The PCR primer sequences are as follows (SEQ ID NO:2-3):

[0042] AP-F: 5'-CATGCCATGACTGTGGCCCAGCCGGCCTTGGACGAGTCCGG-3'

[0043] AP-R: 5'-CATGCCATGACTCGCGGCCCCCGAGGCACCTAGAGGTCAGGG-3'

[0044] Afterwards, the scFv gene was modified with restriction endonuclease SfiI to obtain cohesive ends, and then the scFv gene fragment was ligated to the vector Pecan 45 by T4 ligase, which contained the AP gene, to obtain the scFv-AP fusion plasmid. 【Wa...

Embodiment 3

[0046] Example 3 Expression of anti-thiamethoxam avian scFv-AP genetically engineered antibody

[0047] The positive monoclonal scFv-AP plasmid was extracted, transformed into Escherichia coli TOP10F' competent cells, and spread on solid medium for overnight culture after recovery. The next day, a single clone was picked and cultured in SB-carboxybenzyl medium, and IPTG (isopropylthiogalactopyranoside) was added to induce overnight expression. The next day, the cells were lysed with an ultrasonic breaker, filtered with a filter membrane and purified with a nickel column, that is, the scFv-AP genetic engineering antibody was separated and purified by affinity chromatography using the histidine tag and nickel chloride in the nickel column to obtain high-purity Anti-thiamethoxam avian scFv-AP genetically engineered antibody. After amino acid sequencing analysis, the amino acid sequence of the obtained scFv-AP genetically engineered antibody is shown in SEQ ID NO:4.

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Abstract

The invention discloses a rapid detection kit for detecting neonicotine pesticide thiamethoxam residues by a one-step method and application thereof. The kit comprises a box body and an ELISA plate and a reagent which are set in the box body, wherein each hole of the ELISA plate is coated with a thiamethoxam coating antigen, the reagent comprises a thiamethoxam avian source scFv-AP genetically engineered antibody, a thiamethoxam standard solution, a buffer solution PBS, a washing solution PBST, a developing solution, a reaction termination solution and the like. In the detection process, the thiamethoxam coating antigen to-be-tested thiamethoxam which are adsorbed in the holes of the ELISA plate and the antibody are subjected to a competitive reaction, and the results are observed through a developing reaction. The rapid detection kit has the advantages that the residual amount of the thiamethoxam in water, soil, vegetables and fruits is accurately detected by using the one-step detection method, the sample pretreatment process is simple, the time consumption is small, a large number of samples can be detected simultaneously, the sample detection cost is much lower than that of an existing detection method, and compared with a traditional ELISA kit, the time required for sample detection is greatly shortened.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and ELISA detection, in particular to an ELISA kit for one-step detection of thiamethoxam residue and application thereof. Background technique [0002] In 1991, Syngenta successfully developed the second-generation neonicotinoid insecticide Thiamethoxam (Thiamethoxam, Thi, chemical structure formula: figure 1 shown), because of its low toxicity to mammals and its broad-spectrum insecticidal activity, it can be quickly absorbed by plants and transmitted to various tissues of plants after application, and can be used to control a variety of chewing mouthparts pests in most crops and piercing-sucking mouthparts pests, and thus has been widely used around the world, but its unreasonable application has caused harm to animals, plants and the environment, making people increasingly concerned about its residues in animals, plants and the environment. Gas chromatography completes the separat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00G01N33/535G01N33/543
CPCC07K16/44G01N33/535G01N33/54326C07K2319/61C07K2317/622
Inventor 许艇何金鑫徐波杰王楷马凤飞李季韩跃国
Owner 苏州微测生物技术有限公司
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