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Oligonucleotides aptamer special for distinguishing zearalenone

A technology of zearalenone oligonucleotide and zearalenone, which is applied in the direction of recombinant DNA technology, microbial determination/inspection, DNA/RNA fragment, etc., can solve the problem that there is no zearalenone oligonucleotide The preparation method and application of acid aptamers can achieve the effect of simple preparation method, low cost and good stability

Active Publication Date: 2013-04-03
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 made into 100 μM stock solution with TE buffer and stored at -20°C for later use.

[0024] 2. Conditions for PCR amplification and Lambda exonuclease digestion to prepare single-strand sub-libraries

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

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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 with high specificity and high affinity binding to zearalenone prepared by SELEX technology (exponentially enriched ligand system evolution technology), which are used for rapid separation Enrichment or analysis and detection of zearalenone laid the foundation. Background technique [0002] Zearalenone (zearalenone, ZEN, also known as F-2 toxin) is produced by Fusarium graminearum, Fusarium pink, Fusarium oxysporum, Fusarium three-line, Fusarium moniliforme, Fusarium yellow and Fusarium nivalum, 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 effect, can cause hyperestrogenism in animals, lead to infertility or abortio...

Claims

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Application Information

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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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