Aflatoxin B1 and B2 aptamer affinity column and preparation method and use thereof

An aflatoxin and aptamer technology, which is applied in the field of aflatoxin B1 and B2 aptamer affinity columns and their preparation, can solve the requirement that the rapid screening of large-scale samples cannot be met, the detection cost is high, and the price is relatively expensive. and other problems, to achieve the effects of low price, reduced cross-reaction, and easy operation

Active Publication Date: 2018-06-15
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 B1 and B2 aptamer affinity column and preparation method and use thereof
  • Aflatoxin B1 and B2 aptamer affinity column and preparation method and use thereof
  • Aflatoxin B1 and B2 aptamer affinity column and preparation method and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 utilizes the aflatoxin B of amino modification 1 and B 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 1 and B 2 Preparation of aptamer (SEQ ID NO: 1).

...

Embodiment 2

[0065] Embodiment 2 utilizes aflatoxin B 1 and B 2 Purification of Aflatoxin B from Peanut Samples by Aptamer Affinity Column 1 and B 2 and its detection

[0066] In this embodiment, aflatoxin B is quantitatively added to normal peanut samples 1 and B 2 Standard substance, then use the aflatoxin B prepared by embodiment 1 1 and B 2 The aptamer affinity column was used for purification, and after purification, it was detected by high performance liquid chromatography-online photochemical derivatization-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, respectively, add aflatoxin B to the crushed peanut samples 1 and B 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 m...

Embodiment 3

[0084] Embodiment 3 Aflatoxin B 1 and B 2 Investigation of the Specificity of Aptamer Affinity Columns

[0085] This embodiment utilizes aflatoxin B 1 , Aflatoxin B 2 , Aflatoxin G 1 and aflatoxin G 2 The standard substance, and the aflatoxin B prepared with embodiment 1 1 and B 2 Composite ligand affinity column for purification. Aflatoxin B retained on an affinity column 1 , Aflatoxin B 2 , Aflatoxin G 1 and aflatoxin G 2 After elution with methanol, its concentration was detected by high performance liquid chromatography. The main purpose is to test for aflatoxin B 1 and B 2 Specificity of complex ligand affinity columns.

[0086] 1. Prepare aflatoxin B with a concentration of 5ng / mL 1 , Aflatoxin B 2 , Aflatoxin G 1 and aflatoxin G 2 Standard solution.

[0087] 2. Remove aflatoxin B 1 and 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...

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Abstract

The invention provides an aflatoxin B1 and B2 aptamer affinity column and a preparation method thereof. The affinity column uses a cyanogen bromide-modified agarose as a carrier, then aptamers for high affinity and high specificity recognition of aflatoxin B1 and B2 and the carrier are covalently coupled, and the coupled aflatoxin B1 and B2 aptamer complex carrier fills the affinity column. The affinity column is mainly used for purification of aflatoxin B1 and B2 in multiple samples such as foods, feed, milk, blood samples and traditional Chinese medicines and is conducive to later high-performance liquid chromatography and fluorescence detection of aflatoxin B1 and B2 in the sample.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, in particular, relates to a kind of aflatoxin B 1 and B 2 Aptamer affinity column and its preparation method and application. Background technique [0002] Aflatoxin B (Aflatoxin B, AFB) is a kind of structurally similar toxic secondary metabolites produced by fungal species such as Aspergillus flavus and Aspergillus parasiticus, and the common ones are aflatoxin B 1 and aflatoxin B 2 , has strong carcinogenic, teratogenic and mutagenic effects. Among them, aflatoxin B 1 The toxicity of potassium cyanide is 10 times that of potassium cyanide and 68 times that of arsenic. It is classified as a class I carcinogen by the International Agency for Research on Cancer (IARC). Aflatoxin B is the most stable mycotoxin found so far. It is not easy to be destroyed under general food processing conditions, so it poses a huge hidden danger to consumers' dietary safety. About 25% of the foo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
CPCG01N33/53
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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