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Deoxynivalenol aptamer affinity column as well as preparation method and application thereof

A technology of aptamers and affinity columns, applied in chemical instruments and methods, separation methods, preparation of test samples, etc., can solve problems such as poor linearity of standard curves, large coefficient of variation, matrix interference, etc., to reduce Cross-reactivity, high purification efficiency, and high-affinity effects

Inactive Publication Date: 2017-10-20
BEIJING MEIZHENG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are the following problems in GC chromatography: poor linearity of the standard curve, response drift, retention and memory effects of the last sample injection, large coefficient of variation in repeated injections during MS detection, and matrix interference.

Method used

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  • Deoxynivalenol aptamer affinity column as well as preparation method and application thereof
  • Deoxynivalenol aptamer affinity column as well as preparation method and application thereof
  • Deoxynivalenol aptamer affinity column as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of affinity column using amino-modified deoxynivalenol aptamer

Embodiment approach

[0041] A preferred embodiment of the present invention to prepare deoxynivalenol aptamer affinity column is as follows:

[0042] 1. Vector activation

[0043] Select N-hydroxysuccinimide (NHS) modified agarose carrier sepharose4B for activation

[0044] 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

[0045] 2. 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 deoxynivalenol aptamer, and couple at room temperature for 8 hours

[0046] 3. Wash the coupled deoxynivalenol aptamer-agarose carrier with 20mM, pH7.4 phosphate buffer PBS for 3 times

[0047] 4. closed

[0048] Add 100mmol / L Tris.ClPH8.0 to the coupled deoxynivalenol aptamer-agarose carrier, and react at room temperature for 2 hours

[0049] 5. Wash the blocked deoxynivalenol aptamer-a...

Embodiment 2

[0060] Example 2: Preparation of an affinity column using biotin-modified deoxynivalenol aptamer

[0061] A preferred embodiment of the present invention to prepare deoxynivalenol aptamer affinity column is as follows:

[0062] 1. Take 1ml streptavidin (SA)-modified agarose gel sepharose4B, wash 3 times with 10ml ultrapure water

[0063] 2. Wash the (SA) modified sepharose4B three times with coupling buffer (0.02M PBS, 0.8M NaCl, pH7.4). Add 20mol / L of biotin-modified deoxynivalenol aptamer, and couple at room temperature for 4 hours

[0064] 3. Wash the coupled deoxynivalenol aptamer-agarose carrier with 20mM, pH7.4 phosphate buffer PBS for 3 times

[0065] 4. Column packing

[0066] Put the deoxynivalenol-agarose carrier into the chromatographic column according to the needs, and the deoxynivalenol affinity purification columns of different capacities can be prepared according to the needs

[0067] 1) Take the empty cylinder, add the lower sieve plate, add 1ml PBS, let i...

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Abstract

The invention relates to a deoxynivalenol aptamer affinity column as well as a preparation method and an application thereof. The affinity purification column adopts a chemical modified solid-phase carrier, a nucleic acid aptamer of deoxynivalenol and the carrier are subjected to covalent coupling, and then the affinity column is filled. The aptamer affinity column is mainly used for purifying deoxynivalenol in food samples, feed samples, milk samples, blood samples and other various samples, so that following HPLC (high performance liquid chromatography) detection and fluorescence detection of deoxynivalenol in the samples can be facilitated.

Description

technical field [0001] The invention relates to an aptamer affinity column for deoxynivalenol, a preparation method and application thereof. It belongs to the field of food safety testing. Background technique [0002] Deoxynivalenol (DON), also known as vomitoxin (VT), is mainly one of the secondary metabolites produced by certain Fusarium fungi—a trichothecene toxin. DON is the most common type of trichothecene toxins. Studies have shown that although DON has no obvious carcinogenicity and mutagenicity, it has a wide range of toxicity, especially has a significant impact on immune function. According to the dose of DON and Depending on the duration of exposure, it can cause immunosuppression or immunostimulation. Animals can be poisoned by eating feed containing mycotoxins. Pigs are most sensitive to the emetic effect of DON, and the minimum amount of oral vomiting is 0.1-0.2 mg / kg. Bacteria etc. DON is one of the mycotoxins that contaminate feed and grain all over the...

Claims

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Application Information

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IPC IPC(8): B01J20/281B01J20/30B01D15/38G01N1/34
CPCB01J20/281B01D15/3804B01J2220/86G01N1/34G01N1/405
Inventor 张彦明
Owner BEIJING MEIZHENG BIOLOGICAL TECH
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