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Aptamer afb1-20 of aflatoxin b1 and its application

An aflatoxin and nucleic acid aptamer technology, applied in the field of nucleic acids, can solve the problems of limited application scope and rapid development of immunological detection methods, short reagent life, difficult preservation, and difficulty in antibody preparation. Modification marks, promising effects

Inactive Publication Date: 2016-04-27
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 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.JinHwanDo ; Dong-KugChoi. Aflatoxins: Detection, Toxicity, and Biosynthesis. Biotechnology and Bioprocess Engineering 2007, 12, 585-593; 4. Liu Z.; Gao J.. Advances in research on detection methods for aflatoxins. Journal of Anhui Agricultural University 2004, 31, 223-226)

Method used

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  • Aptamer afb1-20 of aflatoxin b1 and its application
  • Aptamer afb1-20 of aflatoxin b1 and its application
  • Aptamer afb1-20 of aflatoxin b1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Target AFB 1 Synthesis and characterization of -beads

[0048] 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:

[0049]

[0050] 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 and dissolved in 3 mL of anhydrous dichloromethane, added 5.4 mg of NHS, 9.6 mg of dicyclohexylcarbodiimide and 5 mg of dimethylaminopyridine, stirred overnight with a magnetic rotor, filtered and concentrated by evaporation to obtain AFB 1 -oxime activated esters (11. Chu, F.S.; Hsia, M.T.; Sun, P.S.: Preparation and characterization of aflatox-nB1-1-(O-carboxymethyl...

Embodiment 2

[0051] Embodiment 2 Aflatoxin B 1 Screening of specific aptamers

[0052] (1) Design and synthesis of random oligonucleotide library

[0053]Design and synthesize a random oligonucleotide library with fixed regions at both ends of 18 nucleotides and a random region of 45 nucleotides in the middle as follows: 5'-ATACCAGCTTATTCAATT-N45-AGATAGTAAGTGCAATCT-3', with a library capacity of 10 15 . The primer sequences used were forward primer (FP): 5'-ATACCAGCTTATTCAATT-3', reverse primer (RP): 5'-AGATTGCACTTACTATCT-3', fluorescent labeling primer (FFP): 5'-FAM-ATACCAGCTTATTCAATT-3 ', biotin-labeled primer (BRP): 5'-Bio-AGATTGCACTTACTATCT-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 living cells as effective molecular probes for cancers study. Proc Natl Acad Sci USA 2006, 103, 11838-11843).

[0054] (2) Screening of nucleic acid aptamers

[0055] AFB 1 -beads are used as target molecules, ...

Embodiment 3

[0057] Example 3 Fluorescence Polarization and Fluorescence Spectrum Research on Nucleic Acid Aptamer and Aflatoxin B 1 interaction between

[0058] Binding buffer (20mM Tris-HCl, pH8.0, 100mMNaCl, 5mMKCl, 2mMMgCl 2 , 1mM CaCl 2 ) Prepare a 2 μM unlabeled nucleic acid aptamer solution, conduct heat denaturation treatment, 95°C for 5 minutes, ice bath for 5 minutes, and place at room temperature for 30 minutes. Then add aflatoxin B to it 1 Make the final concentration 0.2μM, and incubate at room temperature for 30min. The blank group is aflatoxin B alone under the same conditions 1 Solution, the control group is random sequence Random. Determine the fluorescence anisotropy value of each group of solution systems (Ex: 365nm; Em: 442nm); scan the fluorescence spectrum of each group of solution systems (Ex: 365nm; Em: 380-650nm) (see Figure 5 and 6 ).

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Abstract

The invention discloses an aptamer AFB1-20 of an aflatoxin B1 and application of the aptamer AFB1-20 and relates to nucleic acid. The invention also relates to a derivative of the aptamer including a truncated aptamer as well as a preparation method and application of the derivative. The aptamer AFB1-20 of the aflatoxin B1 is selectively prepared through an affinity SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method based on agarose microbead separation-flow cytometry analysis. The aptamer AFB1-20 of the aflatoxin B1 and the derivative of the aptamer AFB1-20 are rich in G-base, have a stem-loop structure, high affinity, good stability and no toxicity, are easy to synthetize and label, and can be used as a potential separation, enrichment and detection reagent for 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 -20 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 Patents(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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