Aptamer AFB1-01 of aflatoxin B1 and application of aptamer AFB1-01

An aflatoxin and nucleic acid aptamer technology, applied in the field of nucleic acid, can solve the problems of limited application scope and rapid development of immunological detection methods, short life of reagents, difficult to store, difficult antibody preparation, etc., and achieves simple and rapid in vitro screening and detection. Easy fixed-point modification mark, promising effect

Inactive Publication Date: 2014-04-02
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But AFB 1 It is a highly toxic small molecule, the preparation of its antibody is relatively difficult, the life of the reagent is short, it is difficult to store, and the difference between batches is also large. These bottlenecks limit the scope of application and rapid development of immunological detection methods (3. Jin Hwan Do;Dong-Kug Choi.Aflatoxins: Detection,Toxicity,and Biosynthesis.Biotechnology and Bioprocess Engineering2007,12,585-593;4.Liu Z.;Gao J..Advances in research on detection methods for aflatoxins.Journal of Anhui Agricultural University2004 , 31, 223-226)

Method used

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  • Aptamer AFB1-01 of aflatoxin B1 and application of aptamer AFB1-01
  • Aptamer AFB1-01 of aflatoxin B1 and application of aptamer AFB1-01
  • Aptamer AFB1-01 of aflatoxin B1 and application of aptamer AFB1-01

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Experimental program
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Effect test

Embodiment 1

[0020] Example 1 Target AFB 1 Synthesis and characterization of -beads

[0021] Using agarose microbeads as the solid phase carrier of target molecules is beneficial to the separation of unbound or weakly bound and non-specific binding sequences, and is suitable for flow cytometry detection. will AFB 1 The synthetic route coupled to agarose microbeads is as follows:

[0022]

[0023] Take 7.6mg AFB 1 Dissolve in 4mL pyridine, add 35mg carboxymethylhydroxylamine hydrochloride, reflux at 80°C overnight, concentrate in vacuo and silica gel column chromatography (chloroform / methanol=10:1), isolate AFB 1 -oxime was dissolved in 3 mL of anhydrous dichloromethane, 5.4 mg of NHS, 9.6 mg of dicyclohexylcarbodiimide and 5 mg of dimethylaminopyridine were added, stirred overnight with a magnetic rotor, filtered and concentrated by evaporation to obtain AFB 1 -oxime activated ester (11.Chu, F.S.; Hsia, M.T.; Sun, P.S.: Preparation and characterization of aflatox-n B1-1-(O-carboxyme...

Embodiment 2

[0024] Embodiment 2 Aflatoxin B 1 Screening of specific aptamers

[0025] (1) Design and synthesis of random oligonucleotide library

[0026] Design and synthesize a random oligonucleotide library with a fixed region of 18 nucleotides at both ends and a random region of 45 nucleotides in the middle as follows: 5'-ATA CCA GCT TAT TCA ATT-N45-AGA TAG TAA GTG CAA TCT -3', storage capacity in 10 15 . The primer sequences used were forward primer (FP): 5'-ATA CCA GCT TAT TCA ATT-3', reverse primer (RP): 5'-AGA TTG CAC TTA CTA TCT-3', fluorescent labeling primer (FFP ): 5'-FAM-ATA CCA GCTTAT TCA ATT-3', biotin-labeled primer (BRP): 5'-Bio-AGA TTG CAC TTA CTA TCT-3' (12. Shangguan, D.; Li, Y. ;Tang,Z.;Cao,Z.C.;Chen,H.W.;Mallikaracchy,P.;Sefah,K.;Yang,C.J.;Tan,W.Aptamers evolved from live cells as effective molecular probes for cancer study.Proc Natl Acad Sci USA2006 , 103, 11838-11843).

[0027] (2) Screening of nucleic acid aptamers

[0028] AFB 1 -beads are used as target m...

Embodiment 3

[0030] Affinity Characterization of Example 3 Nucleic Aptamers

[0031] Label the nucleic acid aptamer molecule with carboxyfluorescein (FAM) at the 5' end, prepare a 0-7000nM gradient solution in 200μL of binding buffer, heat denature, and mix with 0.4μL AFB 1 -beads were incubated at room temperature for 30min. After washing with the binding buffer, the microbeads were suspended in the binding buffer, and the fluorescence intensity on the surface of the microbeads was measured by flow cytometry. Plot fluorescence intensity versus aptamer concentration using the formula Y=Bmax X / (K d +X) carrying out the fitting of the binding curve to determine the binding and dissociation constant K of the nucleic acid aptamer d (see Figure 5 ).

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Abstract

The invention discloses an aptamer AFB1-01 of aflatoxin B1 and an application of the AFB1-01, and relates to a nucleic acid. The aptamer AFB1-01 of the aflatoxin B1 is prepared by a systematic evolution of ligands by exponential enrichment (SELEX) method based on agarose beads separation-flow cytometry analysis. The aptamer AFB1-01 of the aflatoxin B1 contains a G base, has a stem-and-loop structure, is high in affinity, good in stability, free of toxicity, and easy to synthetize and mark, and can be used as a potential detection reagent of the aflatoxin B1 in a sample.

Description

technical field [0001] The present invention relates to a nucleic acid, in particular to aflatoxin B 1 aptamer AFB 1 -01 and its application. Background technique [0002] In the biological contamination of grain, oil and food, mycotoxin contamination is one of the most important factors, and aflatoxins (AF) are the most toxic and carcinogenic natural pollutants found so far. Aflatoxins are a group of secondary metabolites with similar chemical structures produced by Aspergillus flavus and Aspergillus parasiticus, which have the basic structure of difuran ring and oxinone (coumarin). species, mainly including B 1 , B 2 , G 1 , G 2 , M 1 and M 2 Wait. Among them, AFB 1 It has the strongest toxicity, the largest amount, and the highest stability. Its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. It mainly acts on the liver and other organs, and can induce primary liver cancer, gastric cancer and lung cancer. In 1993, it was designated ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N21/64
Inventor 杨朝勇朱玲邹远刘如迪朱志庄峙厦
Owner XIAMEN UNIV
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