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Deoxynivalenol and its derivative aptamer affinity column and its preparation method and application

A technology of vomitoxin and its derivatives, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of unsatisfactory vomitoxin and its derivatives, and achieve the goal of improving pretreatment efficiency, reducing cost and time, and saving costs Effect

Active Publication Date: 2021-02-26
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, the aptamer affinity column reported at home and abroad can only target a certain toxin, which cannot meet the requirement of one-step purification and enrichment of deoxynivalenol and its derivatives in samples in practical applications. toxin requirements

Method used

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  • Deoxynivalenol and its derivative aptamer affinity column and its preparation method and application
  • Deoxynivalenol and its derivative aptamer affinity column and its preparation method and application
  • Deoxynivalenol and its derivative aptamer affinity column and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Example 1 Preparation of aptamer affinity column using amino-modified deoxynivalenol and its derivative aptamers

[0058] 1. Preparation of N-hydroxysuccinimide modified agarose. Sepharose 4B was used as the carrier and activated with N-hydroxysuccinimide.

[0059] Mix agarose with an equal volume of water in a fume hood, and add it to a reactor equipped with a pH electrode and a magnetic stirrer, and add 100 mg of N-hydroxysuccinimide per mL of agarose solution to the above agarose solution For N-hydroxysuccinimide, adjust the pH value to 11 with NaOH, control the pH value of the whole reaction at 11, control the temperature at about 20°C, and complete the reaction in 10 minutes; after the reaction, add an equal volume of ice chips to the above reaction quickly solution, and quickly poured into the Buchner funnel, with 10 times the volume of the agarose solution of cold buffer (200mM Na 2 HPO 4 , 5 mM MgCl 2 , pH 8.0) was washed by suction, and the hydroxyl groups ...

Embodiment 2

[0074] Example 2 Purification and detection of deoxynivalenol and its derivatives in wheat samples by using aptamer affinity column for deoxynivalenol and its derivatives

[0075] In this example, quantitative addition of deoxynivalenol and its derivatives standard substance to normal wheat samples, followed by purification with the aptamer affinity column of deoxynivalenol and its derivatives prepared in Example 1, followed by purification by high performance liquid chromatography- Fluorescence detector detection, determination of recovery. details as follows:

[0076] 1. Wheat sample processing

[0077] 1) Pulverize the wheat sample.

[0078] 2) According to the standards of 0.5, 5, and 50 μg per gram of sample, respectively, add deoxynivalenol and its derivative standard to the pulverized wheat sample.

[0079] 3) Weigh 5g of the sample, add 25mL of methanol-water (7:3, v / v), and place it on a homogenizer for 11000rpm high-speed homogenization for 3min.

[0080] 4) Filt...

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Abstract

The invention provides vomitoxin and its derivative aptamer affinity column as well as its preparation method and application. The affinity column uses N-hydroxysuccinimide-modified agarose as a carrier, and then it will be able to identify deoxynivalenol and deoxynivalenol-3-in the sample with high affinity and high specificity. The nucleic acid aptamers of glucoside, 3‑acetyl deoxynivalenol and 15‑acetyl deoxynivalenol are covalently coupled to the carrier, and the coupled aptamer composite carrier is filled with an affinity column. The affinity column is mainly used for the purification and purification of deoxynivalenol and its derivatives in food, feed, milk, food crops, traditional Chinese medicine and other samples, so as to facilitate the high-performance liquid chromatography and purification of such toxins in the samples in the later stage. Quick check. One pass through the column can purify and enrich 4 kinds of toxins, the number of repeated use can reach 15 times, and the recovery rate is above 80%, which reduces the cost and improves the pretreatment efficiency of samples.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, and in particular relates to vomitoxin and its derivative aptamer affinity column and its preparation method and application. Background technique [0002] DON and its derivatives belong to a kind of mycotoxin, also known as vomitoxin, which is a kind of trichothecene toxin, which is mainly produced by certain Fusarium fungi. Vesonder et al. isolated such compounds from feed and corn that caused American sows to refuse to eat and vomit in 1973, and named them vomitoxin. Trichothecene toxoids have a double bond between C-9 and C-10 in the chemical structure, an oxygen ring at C-12 and C-13, and other positions also contain hydroxyl and acetyl groups group. DON is composed of a 12,13-epoxy group, an α,β-unsaturated ketone group and 3 -OH functional groups, and its chemical name is 3,7,l5-trihydroxyl-12,l3-epoxy mono Teprosporine-9-en-8-one (3,7,15-trihydroxy-12,13-epoxytrichothec-9...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115
Inventor 栾云霞刘洪美陆安祥付海龙郭晓军冯晓元
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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