Alternariol aptamer affinity column and its preparation method and application

A technology of aptamer and Alternaria, which is applied in the field of Alternariol aptamer affinity column and its preparation, can solve the problem that it is difficult to meet the requirements of Alternaria enrichment/purification

Active Publication Date: 2020-10-20
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] At present, the aptamer affinity columns reported at home and abroad are mainly aimed at ochratoxin, and it is difficult to meet the requirements for t

Method used

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  • Alternariol aptamer affinity column and its preparation method and application
  • Alternariol aptamer affinity column and its preparation method and application
  • Alternariol aptamer affinity column and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0076] Example 1 Preparation of aptamer affinity column using amino-modified alternariol aptamer

[0077] 1. Preparation of NHS-modified agarose

[0078] ①Wash 10mL of agarose gel with 4FF water and drain it, wash it with 30% and 70% acetone with 5 times the volume of the agarose gel in sequence in a sand core funnel, and finally wash it with 100% dioxane with 5 times the volume of the agarose gel The ring was washed 5 times, drained, and transferred to a 100 mL Erlenmeyer flask with a stopper, 10 mL of dioxane, 100 μL of allyl glycidyl ether and 300 μL of boron trifluoride diethyl ether were added, and reacted in a water-bath shaker at 140 rpm at 35°C for 45 minutes. The reacted matrix was washed successively with 30% and 70% acetone of 5 times the volume of agarose gel, and finally washed with a large amount of deionized water to obtain an activated matrix.

[0079] ② Carboxylation of thioglycolic acid: Take 10 mL of activated matrix, add 1.20 mL of thioglycolic acid, 10 mL...

Embodiment 2

[0095] Example 2 Purification of Alternaria in Lotus Seed Samples and Its Detection Using Alternaria Aptamer Affinity Column

[0096] In this example, quantitatively add alternariol standard substance to the lotus seed sample, then use the alternariol aptamer affinity column prepared in Example 1 to purify, and then detect with high performance liquid chromatography-fluorescence detector after purification. Determine recovery. details as follows:

[0097] 1. Lotus seed sample processing

[0098] 1) Pulverize the lotus seed sample.

[0099] 2) According to the standards of 0.5ng, 5ng, and 50ng per gram of sample, add alternariol standard substance to the crushed lotus seed sample.

[0100] 3) Weigh 5 g of the sample, add 25 mL of methanol-water (7:3, v / v), and place it on a multi-tube shaker at 2500 rpm for high-speed homogenization for 20 min.

[0101] 4) Take 5 mL of supernatant, add 45 mL of Binding Buffer (binding buffer), vortex, centrifuge at 10,000 rpm for 10 min, fi...

Embodiment 3

[0112] Example 3 Design and Screening of Alternariol-specific DNA Aptamers

[0113] This example aims at the problems that the small molecule target has few binding sites and nucleic acid, is difficult to separate, is not easy to fix, and will have a great impact on its chemical bonds after fixation, resulting in the failure of screening. The fixed library is used instead of the traditional method of fixing small molecules. A suitable screening method for small molecular compounds such as AOH was established, which solved the difficult problems of difficult fixation and separation of small molecular targets. The main principle is to anneal and pair the biotin-modified capture nucleic acid with the library. Through the interaction between biotin and SA, the library is connected to the SA magnetic beads and incubated with the target. The specifically bound aptamer will be eluted by competition and collected. Nucleic acid amplification to prepare single strands for the next round...

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Abstract

Provided are an alternariol aptamer affinity column and a preparation method and an application therefor. The affinity column uses agarose modified with N-hydroxysuccinimide as a carrier, and then covalently couples to the carrier a nucleic acid aptamer capable of recognising alternariol with high affinity and high specificity, the coupled product being used as a filler for packing the affinity column.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, and in particular relates to an alternariol aptamer affinity column and a preparation method and application thereof. Background technique [0002] Alternariol (Alternaria mycotoxin, AOH) is a secondary metabolite produced by the fungus Alternaria genus. It is a toxin commonly found in the environment of fruits, vegetables and field crops. Fungi of the genus can grow and reproduce in low temperature and humid environments, so Alternaria toxin contamination is common in rotten samples such as fruits, vegetables, and grains during refrigeration or long-distance transportation. Alternariol has chronic or acute toxic effects such as mutagenicity, carcinogenicity and teratogenicity on humans or livestock, and AOH has potential genotoxicity. When the concentration of AOH is ≥ 1 μM, it is more destructive to DNA in human cells . The mycotoxin properties of AOH can significantly inhibit t...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/53
CPCC12N15/115C12N2310/16G01N33/5308
Inventor 栾云霞刘洪美陆安祥郭晓军冯晓元
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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