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Ochratoxin aptamer affinity column, preparation method and uses thereof

An ochratoxin and aptamer technology, applied in chemical instruments and methods, separation methods, preparation of samples for testing, etc., can solve the problems of less purification and purification methods, and achieve reduced cross-reactivity, good specificity, Reduce the effect of mutation

Inactive Publication Date: 2017-07-28
BEIJING MEIZHENG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] There are many detection methods based on nucleic acid aptamers, but there are few purification and purification methods

Method used

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  • Ochratoxin aptamer affinity column, preparation method and uses thereof
  • Ochratoxin aptamer affinity column, preparation method and uses thereof
  • Ochratoxin aptamer affinity column, preparation method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Preparation of affinity column using amino-modified ochratoxin A aptamer

Embodiment approach

[0043] A preferred embodiment of the present invention preparing ochratoxin A aptamer affinity column is as follows:

[0044] 1. Select N-hydroxysuccinimide (NHS) modified agarose carrier sepharose4B for activation

[0045] 2. Take 1g of N-hydroxysuccinimide-modified agarose, add 50ml of 1mM HCl, after swelling for 30min, wash the gel with 100ml of 1mM HCl for 6 times, and then wash with 100ml of ultrapure water

[0046] 3. The activated agarose gel sepharose4B with coupling buffer (0.1M NaHCO 3 , 0.8M NaCl, pH8.2) and washed 3 times. Add 20mol / L amino-modified ochratoxin A aptamer, and couple at room temperature for 8 hours

[0047] 4. Wash the coupled ochratoxin A aptamer-agarose carrier 3 times with 20mM, pH7.4 phosphate buffer PBS

[0048] 5. closed

[0049] Add 100mmol / L Tris.Cl pH8.0 to the coupled ochratoxin A aptamer-agarose carrier, and react at room temperature for 2 hours

[0050] 6. Wash the blocked ochratoxin A aptamer-agarose carrier 3 times with 20mM, pH7.4...

Embodiment 2

[0061] Example 2: Preparation of affinity column using biotin-modified ochratoxin A aptamer

[0062] A preferred embodiment of the present invention preparing ochratoxin A aptamer affinity column is as follows:

[0063] 1. Take 1ml streptavidin (SA)-modified agarose gel sepharose4B, wash 3 times with 10ml ultrapure water

[0064] 2. Wash the (SA) modified sepharose4B three times with coupling buffer (0.02M PBS, 0.8M NaCl, pH7.4). Add 20mol / L biotin-modified ochratoxin A aptamer, and couple at room temperature for 4 hours

[0065] 3. Wash the coupled ochratoxin A aptamer-agarose carrier 3 times with 20mM, pH7.4 phosphate buffer PBS

[0066] 4. Column packing

[0067] The ochratoxin A-agarose carrier is loaded into the chromatography column according to the need, and the ochratoxin A affinity purification column of different capacity can be prepared according to the need

[0068] 1) Take the empty cylinder, add the lower sieve plate, add 1ml PBS, let it drain naturally

[0...

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PUM

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Abstract

The invention relates to an ochratoxin A aptamer affinity column and a preparation method and application thereof. The affinity purification column utilizes a chemically modified solid phase carrier, and then a nucleic acid aptamer of ochratoxin A is covalently coupled to the carrier. Then pack the affinity column. The aptamer affinity column is mainly used for the purification of ochratoxin A in food, feed, milk, blood samples and other various samples, so as to facilitate the high performance liquid chromatography (HPLC) of ochratoxin A in the sample in the later stage detection and fluorescence detection.

Description

technical field [0001] The invention relates to an aptamer affinity column for ochratoxin, a preparation method and application thereof. It belongs to the field of food safety testing. Background technique [0002] Ochratoxin A (Ochratoxin A), referred to as OTA, is a toxic metabolite of some species of Aspergillus and Penicillium, and is one of the mycotoxins. There are many kinds of fungi that produce ochratoxin A in nature, but Penicillium verrucosum, Aspergillus ochraceus and A. common bacteria. Ochratoxin can cause severe renal lesions, acute liver dysfunction, fatty degeneration, hyaline degeneration and localized necrosis, as well as teratogenicity, mutagenicity, immunosuppression and carcinogenicity. In 1993, the International Center for Cancer Research listed ochratoxin A as a possible human carcinogen. Ochratoxin is widely found in cereals, soybeans, mung beans, peanuts, spices, dried fruits, animal kidneys, blood, coffee, milk, liquor, beer, wine, chocolate, d...

Claims

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Application Information

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IPC IPC(8): B01D15/38B01D15/20G01N1/34G01N1/40
CPCB01D15/3804B01D15/206G01N1/34G01N1/40
Inventor 张彦明
Owner BEIJING MEIZHENG BIOLOGICAL TECH
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