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VHH-ELISA kit for one-step detection of fipronil residues and application thereof

A kit, fipronil technology, applied in the field of genetic engineering and ELISA detection, can solve the problems of complex pretreatment, high cost, poor specificity, etc.

Pending Publication Date: 2019-10-25
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

[0006] The purpose of the present invention is to provide a method with high specificity, high sensitivity, high accuracy, and high precision for the shortcomings of the current pesticide residue instrument analysis method, such as high cost, complicated pretreatment, poor specificity, low sensitivity, and difficulty in on-site detection in experiments. , The operation method is simple, and it can be used for rapid detection of large batches of samples, and it is an ELISA kit for one-step detection of fipronil residues

Method used

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  • VHH-ELISA kit for one-step detection of fipronil residues and application thereof
  • VHH-ELISA kit for one-step detection of fipronil residues and application thereof
  • VHH-ELISA kit for one-step detection of fipronil residues and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of fipronil-coated antigen

[0029] A conjugated complex was prepared with hapten and bovine serum albumin as the coating antigen. The preparation method is as follows:

[0030](1) Dissolve 8.8mg of fipronil-6C (MW=440.15, 0.02mmol), 2.65mg of NHS (MW=115, 0.024mmol), 4.8mg of DCC (MW=206, 0.023mmol) in 200μL of anhydrous DMF , stirred overnight at room temperature. Centrifuge the reaction solution (5000rpm, 10min), discard the precipitate, and the supernatant is the active ester.

[0031] (2) Dissolve 20mg of BSA (MW=67000) in 2mL of carbonate buffer solution (0.05mol / mL, pH 9.5), add 150μL of active ester solution drop by drop under stirring, slowly add in about 20-30min Finish. Then the stirring reaction was continued at room temperature for 4-6 h.

[0032] (3) The reaction solution was put into a dialysis bag and dialyzed with PBS (0.01 mol / L, pH7.4). The liquid was changed every 6-8 hours, and the liquid was changed 5-6 times in total. ...

Embodiment 2

[0033] Example 2 Construction of anti-fipronil VHH-AP genetically engineered antibody

[0034] Firstly, the specific fipronil VHH gene fragment, ie nanobody (SEQID NO: 1), was screened by phage display technology.

[0035] Then the specific fipronil nanobody plasmid was extracted, and the VHH gene fragment was amplified by PCR.

[0036] Wherein the PCR reaction system is as follows:

[0037]

[0038] The reaction procedure is as follows:

[0039]

[0040] The PCR primer sequences are as follows (SEQ ID NO:2-3):

[0041] AP-F:

[0042] 5'-CATGCCATGACTGTGGCCCAGCCGGCCCAGKTGCAGCTCGTGGAGTCNGGNGG-3'

[0043] AP-R5:

[0044] 5'-CATGCCATGACTCGCGGCCCCCGAGGCCTGCTTGCATACTTCATTCGTTCCTG-3'

[0045] Wherein, K represents the base G / T, and N represents the base A / T / G / C.

[0046] Then modify the VHH gene with restriction enzyme SfiI to obtain sticky ends, and then connect the VHH gene fragment to the vector Pecan 45 by T4 ligase. The vector Pecan 45 contains the AP (alkaline phos...

Embodiment 3

[0048] Example 3 Expression of anti-fipronil VHH-AP genetically engineered antibody

[0049] The positive monoclonal VHH-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 VHH-AP plasmid was separated and purified by affinity chromatography using the histidine tag and nickel chloride in the nickel column to obtain high-purity fluorine Chlornil genetically engineered antibody. After amino acid sequencing analysis, the amino acid sequence of the obtained anti-fipronil VHH-AP genetically engineered antibody is shown in SEQ ID NO:4.

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Abstract

The invention discloses a VHH-ELISA kit for one-step detection of fipronil residues and an application thereof. The kit comprises a box body, and an enzyme labeling plate and a reagent arranged in thebox body, wherein each hole of the enzyme labeling plate is enveloped with a fipronil envelope antigen, and the reagent comprises fipronil-resistant VHH-AP genetically engineered antibodies, a fipronil standard solution, a buffer solution PBS, a washing solution PBST, a color developing solution, a reaction termination solution and the like. During the detection process, the envelope antigens adsorbed to the hole walls of the enzyme labeling plate and fipronil to be detected compete with each other for reaction with the antibodies, and a result is observed through a color developing reaction.The VHH-ELISA kit has the advantages of being capable of precisely and sensitively detecting fipronil residues in chicken and eggs, the pretreatment process of the sample is simple and requires shorttime, a large number of samples can be simultaneously detected, and the sample detection cost is far lower than that of the traditional instrument detection method. Compared to the traditional ELISAkit, the time required for sample detection is greatly shortened by means of the VHH-ELISA kit.

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 fipronil residue and its application. Background technique [0002] Fipronil (Fipronil, chemical structure such as figure 1 Shown) also known as fipronil, fipronil, fipronil, is a widely used N-phenylpyrazole insecticide with trifluoromethylsulfonyl group, which is harmful to grain, cotton It has a good control effect on pests of major crops such as oil, fruit and vegetables, and tea. During the use of pesticides, many pests develop resistance to organophosphate pesticides, carbamate pesticides and pyrethroid insecticides. For such resistant insects, fipronil is also effective. Fipronil is highly toxic to aquatic products, bees, silkworms, rodents, and birds, and moderately toxic to mammals. Fipronil can damage human β3GABAA receptor subunits and cause human neurodevelopmental disorders, such as autism , A...

Claims

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Application Information

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IPC IPC(8): G01N33/541G01N33/535
CPCG01N33/541G01N33/535
Inventor 许艇王楷徐波杰周航李季韩跃国
Owner 苏州微测生物技术有限公司