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A new method for purification of ochratoxin a in traditional Chinese medicine by aptamer affinity column

A technology of ochratoxin and aflatoxin, which is applied in the field of preparation of aptamer affinity columns, can solve problems such as improper processing and storage, difficult detection, and limit blank, and achieves simple and easy preparation process, low detection cost, no The effect of batch-to-batch variance

Active Publication Date: 2018-12-04
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the wide variety of Chinese herbal medicines, complex matrix, improper processing and storage, OTA trace (ppb), pH sensitivity and other characteristics, the actual detection is difficult.
Therefore, except for the European Union (EU) that has made relevant OTA limit regulations on licorice and aromatic plants, other countries are still in the exploratory stage, especially the relevant organizations in my country have no limits on OTA limits for Chinese herbal medicines.

Method used

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  • A new method for purification of ochratoxin a in traditional Chinese medicine by aptamer affinity column
  • A new method for purification of ochratoxin a in traditional Chinese medicine by aptamer affinity column
  • A new method for purification of ochratoxin a in traditional Chinese medicine by aptamer affinity column

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the preparation of AAC

[0027] (1) Preparation steps:

[0028] a. Aptamer refolding: Dissolve 2.7 nmol of 5' amino-modified aptamer in 400 μL buffer (200 mM Na 2 HPO 4 5mM MgCl 2 , pH 8.0), renaturation at 75°C for 5 minutes, and left at room temperature for 30 minutes;

[0029] b. Washing: Take about 200 μL of NHS-activated sephaorse 4FF gel and wash with 1 mL of HCl (1 mM, pH 3.0) rapidly and repeatedly for 5 times;

[0030] c. Coupling: Remove excess hydrochloric acid, quickly add refolded aptamer buffer, adjust pH to 8.0, and shake for 3 hours at room temperature;

[0031] d. Blocking: After the reaction is completed, use 3mL Na 2 HPO 4 (200mM, pH 8.0) to wash, add 1mL Tris-HCl buffer (0.1M, pH 8.0) to react at room temperature for 2h to block the remaining active sites;

[0032] e. Washing: alternately wash 5 times with 2mL Tris-HCl buffer (0.1M, pH 8.0, containing 0.5M NaCl) and 2mL acetate buffer (0.1M, pH 4.0, containing 0.5M NaCl);

[003...

Embodiment 2

[0036] Embodiment 2: AAC purification UPLC-FLR detects OTA in ginger powder

[0037]1. Sample extraction: Accurately weigh 20.0g of ginger powder (accurate to 0.1g), add 60mL of acetonitrile-water (60:40, v / v) solution, ultrasonically extract for 15min, filter with quantitative filter paper, take 5mL of filtrate and add 45mL BBS 2 (pH 8.0), mix well, filter with glass fiber filter paper, and the filtrate is set aside.

[0038] 2. Purification: take AAC column and use 3mL BBS 1 For activation, pipette 3 mL (equivalent to 0.1 g sample) of the above-mentioned diluted filtrate to pass through the column, and adjust the flow rate to 1 drops / s. with 1mL BBS 1 Rinse the column until the air has completely passed through the column. Add 1 mL of pure methanol, elute at a flow rate of 1 drops / s, and collect the eluate. The eluent was blown dry with nitrogen at 45°C, and finally the volume was adjusted to 0.5 mL with methanol-water (50:50, v / v), shaken and mixed, and centrifuged at ...

Embodiment 3

[0074] Example 3: AAC purification UFLC-MS / MS detection of OTA in traditional Chinese medicine

[0075] 1. Sample extraction: Accurately weigh 2.0g of medicinal material powder (accurate to 0.1g), add 8mL of acetonitrile-water (60:40, v / v) solution, vortex-assisted extraction at 2400rpm for 2min, let stand for 30min, and centrifuge at 4000rpm 10min, take 3mL filtrate and use BBS 2 Dilute to 30mL, mix well, and adjust the pH to 7.5 with hydrochloric acid, filter with glass fiber filter paper, and use the filtrate for later use.

[0076]2. Purification: take AAC column and use 3mL BBS 1 For activation, pipette 2 mL (equivalent to 50 mg sample) of the above-mentioned diluted filtrate to pass through the column, and adjust the flow rate to 1 drops / s. with 1mL BBS 1 Rinse the column until the air has completely passed through the column. Add 1 mL of pure methanol, elute at a flow rate of 1 drops / s, and collect the eluate. Dry the eluent with nitrogen gas at 45°C, add 5 μL of i...

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Abstract

The invention relates to a preparation method of an aptamer affinity column having the advantages of simple operation, fast coupling speed and high coupling efficiency, and the aptamer affinity column is prepared from NHS activated sephrose4FF as a carrier and ochratoxin A specific aptamer as a ligand. After systematical review, the affinity column has good applicability in traditional Chinese medicines, and can achieve the purpose of rapid purification of the ochratoxin A in the traditional Chinese medicines, the established detection method is good in accurate degree and high in sensitivity, and can be used for rapid screening of the ochratoxin A in the traditional Chinese medicines. In addition, the preparation method of the aptamer affinity column is simple, easy, low in production cost, and stable and controllable in quality, can meet the rapid detection of the ochratoxin A in the traditional Chinese medicines, and has broad development prospects.

Description

technical field [0001] The invention relates to a new purification method of ochratoxin A in traditional Chinese medicine, in particular to the preparation of an aptamer affinity column (AAC), AAC purification ultra-high-efficiency liquid phase fluorescence detection and liquid mass spectrometry detection of ochratoxin A in traditional Chinese medicine , which belongs to the field of mycotoxin detection. Background technique [0002] Ochratoxin A (ochratoxin A, OTA) is a derivative of isocoumarin linked to L-phenylalanine, a secondary metabolite produced by toxin-producing strains such as Aspergillus and Penicillium, and has strong liver and kidney Toxicity, neurotoxicity and immunotoxicity, etc., so in 1993 the International Cancer Organization IARC has defined it as a 2B carcinogen. OTA has a wide range of pollution in the world, and it is widely found in various foods and crops, and the pollution level is relatively high. In recent years, some Chinese herbal medicines s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06B01D15/38B01J20/281
Inventor 杨美华杨锡辉孔维军
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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