Oligonucleotides aptamer special for distinguishing zearalenone

A technology of zearalenone oligonucleotides and zearalenone, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problem of zearalenone oligonucleotides The preparation method and application of acid aptamers have achieved the effect of simple preparation method, low cost and good stability

Active Publication Date: 2014-05-28
JIANGNAN UNIV
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Problems solved by technology

[0005] So far, oligonucleotide aptamers for small molecules of mycotoxins such as ochratoxin and fumonisin B1 have been screened by SELEX technology, and a series of new detection methods based on oligonucleotide aptamers have been established; There is no research report on the zearalenone oligonucleotide aptamer and its preparation method and application

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  • Oligonucleotides aptamer special for distinguishing zearalenone
  • Oligonucleotides aptamer special for distinguishing zearalenone
  • Oligonucleotides aptamer special for distinguishing zearalenone

Examples

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Embodiment 1

[0017] Example 1: Competitive SELEX screening of zearalenone-specific binding oligonucleotide aptamers

[0018] 1. In vitro chemical synthesis of initial random single-stranded DNA (ssDNA) library and primers (completed by Integrated DNA Technologies, USA), the sequence is as follows:

[0019] 5'-AGCAGCACAGAGGTCAGATG(40N)CCTATGCGTGCTACCGTGAA-3' (40N represents 40 random nucleotides);

[0020] Upstream primer: 5'-AGCAGCACAGAGGTCAGATG-3'

[0021] Downstream primer: 5'-TTCACGGTAGCACGCATAGG-3'

[0022] 5' phosphorylation downstream primer: 5'-P-TTCACGGTAGCACGCATAGG-3'

[0023] The random ssDNA library and primers were prepared with TE buffer as a 100 μM stock solution and stored at -20°C for future use.

[0024] 2. PCR amplification conditions and Lambda exonuclease digestion conditions to prepare single-stranded secondary library

[0025] The synthetic random single-stranded library (ssDNA) was diluted as a PCR template to amplify the phosphorylated double-stranded DNA (dsDNA...

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Abstract

The invention discloses a group of oligonucleotides aptamers special for distinguishing zearalenone, and belongs to the field of food safety testing. An SELEX (systematic evolution of ligands by exponential enrichment) technology is combined with a magnetic bead with zearalenone coupled on the surface, and after 14 rounds of repeated incubation, cleaning, dissociation, amplification, gamma-exonuclease digestion are carried out to prepare a single-stranded secondary library, each oligonucleotides aptamer capable of being combined with the specificity of the zearalenone is screened out from a random single-stranded DNA library, after clone sequencing and 12 typical sequences are synthesized, the affinity is analyzed, and a specificity test is carried out to three aptamers with best affinity. The group of oligonucleotides aptamers (a nucleic acid sequence thereof selected from 1-3 in a sequence list) provides a new choice for developing a method which is for replacing the existing methods for detecting the zearalenone by depending on an antibody.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a group of oligonucleotide aptamers that bind to zearalenone with specificity and high affinity prepared by SELEX technology (exponential enrichment ligand system evolution technology), which is used for rapid separation Enrichment or analytical detection of zearalenone lays the foundation. Background technique [0002] Zearalenone (Zealenone, ZEN, also known as F-2 toxin) is produced by Fusarium graminearum, Fusarium pink, Fusarium oxysporum, Fusarium trilinea, Fusarium moniliformes, Fusarium yellow and Fusarium nivalis, etc. The secondary metabolite produced by the strain is an estrogenic mycotoxin. It mainly exists in corn and corn products, and also has a certain degree of distribution in wheat, barley, sorghum, and rice. Zearalenone has strong reproductive toxicity and teratogenic effects, which can cause hyperestrogenism in animals, leading to infertility or abort...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12Q1/68
Inventor 王周平陈秀娟段诺吴世嘉
Owner JIANGNAN UNIV
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