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Aflatoxin B2 aptamer affinity column and preparation method and application thereof

A kind of technology of aflatoxin and aptamer, which is applied in the direction of chemical instruments and methods, biochemical equipment and methods, separation methods, etc., can solve the problem of inability to meet the requirements of rapid screening of large batches of samples, high detection cost, and relatively expensive, etc. problem, achieve the effect of reducing variance, reducing cross-reaction, and low price

Active Publication Date: 2018-07-06
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high requirements for sample purity, some pretreatment processes are required, resulting in high detection costs and long cycles, which cannot meet the requirements of rapid screening of large batches of samples.
Traditional pretreatment technologies include immunoaffinity columns, multifunctional purification columns, etc. These purification columns are more expensive and mostly disposable.

Method used

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  • Aflatoxin B2 aptamer affinity column and preparation method and application thereof
  • Aflatoxin B2 aptamer affinity column and preparation method and application thereof
  • Aflatoxin B2 aptamer affinity column and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 utilizes the aflatoxin B of amino modification 2 Aptamer Preparation Aptamer Affinity Column

[0049] 1. Preparation of cyanogen bromide-modified agarose. Sepharose 4B was used as the carrier and activated with cyanogen bromide.

[0050] 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, add cyanogen bromide to the above agarose solution at an amount of 100 mg per mL of agarose solution , 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; Buchner funnel, with cold buffer (200mM Na 2 HPO 4 ,5mM MgCl 2 , pH 8.0) was washed by suction filtration, and the hydroxyl groups on the surface reacted with cyanogen bromide to obtain cyanogen bromide-modified agarose.

[0051] 2. Amino-modified aflatoxin B 2 Preparation of aptamer (SEQ ID NO: 1).

[0052] At the 3' ...

Embodiment 2

[0065] Embodiment 2 utilizes aflatoxin B 2 Purification of Aflatoxin B from Peanut Samples by Aptamer Affinity Column 2 and its detection

[0066] In this embodiment, aflatoxin B is quantitatively added to normal peanut samples 2 Standard substance, then use the aflatoxin B prepared by embodiment 1 2 The aptamer affinity column was used for purification, and after purification, it was detected by high performance liquid chromatography-fluorescence detector, and the recovery rate was determined. details as follows:

[0067] 1. Peanut sample processing

[0068] 1) Crush the peanut sample.

[0069] 2) According to the standards of 0.5, 5, and 50 μg per gram of sample, add aflatoxin B to the crushed peanut samples 2 Standard.

[0070] 3) Weigh 5 g of the sample, add 25 mL of methanol-water (70:30, v / v), and place it on a homogenizer for 11,000 rpm high-speed homogenization for 3 min.

[0071] 4) Filter with a 0.45 μm syringe filter.

[0072] 5) Take 5 mL of the filtrate, ...

Embodiment 3

[0084] Embodiment 3 Aflatoxin B 2 Investigation on Reusability and Column Capacity of Aptamer Affinity Column

[0085] This embodiment utilizes aflatoxin B 2 The standard substance, and the aflatoxin B prepared with embodiment 1 2 Aptamer affinity column for purification. Aflatoxin B retained on an affinity column 2 After elution with methanol, its concentration was detected by high performance liquid chromatography-fluorescence detector. Repeat loading and elution after re-equilibration of the affinity column and measure the eluted aflatoxin B 2 concentration. The main purpose is to test for aflatoxin B 2 Reusability and column capacity of aptamer affinity columns.

[0086] 1. Prepare aflatoxin B with different solubility 2 Standard.

[0087] 2. Remove aflatoxin B 2 For the aptamer affinity column, the plug of the injection port is opened, the injection port is connected with the needle hole of the syringe, and the syringe is inserted into the air control operation ...

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Abstract

The invention provides an aflatoxin B2 aptamer affinity column and a preparation method thereof, wherein the affinity column uses cyanogens-bromide-modified agarose as a vector, a nucleic acid aptamercapable of recognizing aflatoxin B2 with high affinity and high specificity is covalently coupled to the vector, and the coupled aflatoxin B2 aptamer complex vector is loaded into the affinity column. The affinity column is mainly used for purification and cleaning of the aflatoxin B2 in food, feeds, milk, blood samples, traditional Chinese medicines and other various samples so as to facilitatehigh performance liquid chromatography and fluorescence detection of the aflatoxin B2 in the samples.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, in particular, relates to a kind of aflatoxin B 2 Aptamer affinity column and its preparation method and application. Background technique [0002] Aflatoxin B 2 (Aflatoxin B 2 ,AFB 2 ) is a kind of structurally similar toxic secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus, etc. of the fungal genus. It has strong carcinogenic, teratogenic and mutagenic effects, and is the most stable mycotoxin found so far. , It is not easy to destroy under general food processing conditions, so it has planted a huge hidden danger to the dietary safety of consumers. About 25% of the food in the world may be contaminated by aflatoxin in production, processing, transportation, storage and other links every year. Due to the seriousness of aflatoxin to human health, many countries and international organizations have Toxin residues in food or traditional Chinese med...

Claims

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Application Information

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IPC IPC(8): C12N15/115B01J20/281B01J20/30B01D15/38C07D493/14
CPCB01D15/3804B01J20/281C07D493/14C12N15/115C12N2310/16
Inventor 刘洪美栾云霞陆安祥付海龙王纪华
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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