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Vomitoxin aptamer affinity column and preparation method and application thereof

A nucleic acid aptamer and vomitoxin technology, which is applied in the field of affinity columns, can solve the problem of no relevant research reports on vomitoxin, and achieve the effect of reducing the cost of pretreatment

Pending Publication Date: 2018-03-16
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, there have been many products and applications of OTA aptamer affinity columns for ochratoxin, but there have been no relevant research reports on vomitoxin

Method used

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  • Vomitoxin aptamer affinity column and preparation method and application thereof
  • Vomitoxin aptamer affinity column and preparation method and application thereof
  • Vomitoxin aptamer affinity column and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] This embodiment provides a deoxynivalenol aptamer affinity column, such as figure 1 Shown: a CNBr-activated agarose gel 4 comprising a column tube 2 and a surface-coupled 3' amino-modified DON aptamer filled at the bottom of the column tube;

[0028] The mass ratio of the 3' amino-modified deoxynivalenol aptamer b to the CNBr-activated agarose gel a is 1:1000. The aptamer is specifically: 5'-GCCCGGATCGAGTTGATTTCAAGCGCATGAAGGCTA-CCCCCCC-NH 2 -3'; in actual application, the nucleic acid aptamer b can capture vomitoxin c;

[0029] The ratio of the filling volume of the CNBr-activated agarose gel to the volume of the column tube is 0.5:1;

[0030] Both ends of the column tube are respectively covered with an upper cap 1 and a lower cap 6; the agarose gel is fixed on the bottom of the column tube through the upper sieve plate 3 and the lower sieve plate 5 .

Embodiment 2

[0032] This example provides the preparation method of the deoxynivalenol aptamer affinity column described in Example 1, specifically:

[0033] a. Aptamer renaturation: Dissolve 1OD of 3’ amino-modified DON aptamer in 200 μL buffer (200 mM Na2HPO4 5 mM MgCl2, pH 8.0), refold at 95°C for 5 min, and place at room temperature for 30 min;

[0034] b. Washing: Take about 60mg of CNBr-activated agarose and activate it with 2mL HCl (1mM, pH3.0) for 1h, wash with 1mL HCl (1mM, pH3.0) repeatedly for 6 times, wash twice with 1ml distilled water and 1ml buffer ( 200mM Na2HPO4 5mM MgCl2, pH8.0) washed twice;

[0035] c. Coupling: Remove the excess hydrochloric acid, quickly add the refolded aptamer buffer, and shake the reaction at room temperature (30 degrees) on a shaker overnight;

[0036] d. Blocking: After the reaction is completed, wash with 3mL Na2HPO4 (200mM, pH 8.0), add 1mL Tris-HCl buffer (0.1M, pH 8.0), and shake the reaction at 28 degrees for 2h to block the remaining activ...

Embodiment 3

[0040] This example provides the application method of the deoxynivalenol aptamer affinity column described in Example 1, specifically:

[0041] (1) Pretreatment of wheat samples: pulverize the wheat samples; add standard vomitoxin in the pulverized wheat samples according to the standards of 0.2, 0.5, and 1.0 μg per gram of sample respectively; weigh 5 grams of samples, add 25 mL of methanol- Water (70:30, v / v), placed on a homogenizer at 11,000rpm for high-speed homogenization for 3min; filtered through a 0.45μm syringe filter; took 5mL of the filtrate, blown to nearly dry with nitrogen at 40°C, added 0.5mL of methanol- Reconstitute with water (70:30, v / v), and dilute to 5mL with Binding buffer; take the above reconstituted solution for sample purification and detection;

[0042] (2) Affinity column pretreatment: take out the vomitoxin aptamer affinity column, open the injection port plug (ie upper cap), connect the injection port to the syringe barrel, and connect the syrin...

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Abstract

The invention relates to a vomitoxin aptamer which particularly is 5'-GCCCGGATCGAGTTGATTTCAAGCGCATGAAGGCTA-CCCCCCC-NH2-3'. The invention relates to a vomitoxin aptamer affinity column which comprisesa column pipe and a CNBr (cyanogen bromide) activated sepharose, the bottom of the column pipe is filled with the CNBr activated sepharose, and a 3'-terminal amino-modified vomitoxin aptamer is coupled on the surface of the CNBr activated sepharose. The invention further relates to a preparation method of the vomitoxin aptamer affinity column. The affinity column takes high-affinity and specificity vomitoxin aptamer as a recognition element, the vomitoxin aptamer is connected on a cyanogen bromide activated sepharose solid-phase vector, washed, closed and packed to prepare the affinity column.Agricultural products and products prepared from the agricultural products are treated by the aptamer affinity column and used for subsequent detection, vomitoxin can be effectively enriched and purified, and the affinity column has the advantages that the affinity column is high in selectivity and good in stability, false positive of detection is reduced, and sensitivity is improved.

Description

technical field [0001] The invention relates to the field of pretreatment for the detection of deoxynivalenol in agricultural products and products thereof, in particular to the application of a highly selective, stable and easy-to-synthesize nucleic acid aptamer to solid-phase extraction using the deoxynivalenol aptamer as a recognition material Technology, prepared as an affinity column for the enrichment, purification and purification of deoxynivalenol in samples. Background technique [0002] Deoxynivalenol (DON), also known as vomitoxin, is a metabolite of strains such as Fusarium graminearum, Fusarium yellow, Cephalosporium, Urudenella, Trichoderma and other strains of the Fusarium genus, the main pollution Wheat, corn, barley, etc. are highly toxic. The survey of DON pollution in wheat, corn and rice in my country in 2003 showed that DON pollution in wheat and corn was more serious than that in rice. In 2005, my country promulgated the limit standard of DON in grain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/34G01N1/40G01N30/08B01D15/38C12N15/115
CPCB01D15/3819G01N1/34G01N1/40G01N30/08C12N15/115
Inventor 栾云霞刘洪美陆安祥付海龙王纪华
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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