Group of oligonucleotides aptamers capable of specifically recognizing aflatoxin B1

A kind of aflatoxin and oligonucleotide technology, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., to achieve the effect of simple operation, low preparation cost and stable single-stranded oligonucleotide

Active Publication Date: 2013-03-06
JIANGNAN UNIV
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Problems solved by technology

[0005] In view of the advantages of SELEX technology over traditional antigen-antibody technology, it has higher selectivity, specificity and affinity, and the characteristics of being able to distinguish substances with very similar structures. At present, SELEX technology is mainly used in macromolecules such as whole bacteria and cells. In the screening of small molecules, especially in the application of mycotoxins, there are few mycotoxin aptamers. It is reported that the only aptamers of mycotoxins screened are ochratoxin and fumonisin aptamers. If aflatoxin B can be screened 1 The aptamers will be of great significance to the detection of mycotoxins, and will also promote the development of SELEX technology in the field of small molecules, which is also the improvement of SELEX technology itself.

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  • Group of oligonucleotides aptamers capable of specifically recognizing aflatoxin B1
  • Group of oligonucleotides aptamers capable of specifically recognizing aflatoxin B1
  • Group of oligonucleotides aptamers capable of specifically recognizing aflatoxin B1

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Embodiment Construction

[0016] The following is the preparation method of nucleic acid aptamers that screen the dissociation constants of specific binding part of aptamers and AFB1 through SELEX technology and the rapid detection of part of aptamers and AFB 1 The application of the dissociation constant.

[0017] 1. Synthesize random single-stranded DNA library and primers (synthesized by IDT)

[0018] Random single-stranded DNA (ssDNA) library: 5'-AGC AGC ACA GAG GTC AGATG(N40)CCTATGCGT GCT ACC GTG AA-3', a random ssDNA library with a length of 80nt was constructed, with fixed primer sequences at both ends and 40 in the middle Random sequence of 10 bases, the library capacity is 10 14 Above; Primer I: 5’-AGCAGCACAGAG GTCAGATG-3’; Primer II: 5’-PO 4 -TTC ACG GTA GCA CGC ATA GG-3’, the random ssDNA library and the two primers are prepared into 100μmol / L stock solution with TE buffer and stored at -20℃ for later use.

[0019] 2. PCR amplification of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) ...

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Abstract

The invention discloses a group of oligonucleotides aptamers capable of specifically recognizing aflatoxin B1. A single-chain deoxyribonucleic acid (DNA) oligonucleotides aptamer capable of specifically recognizing the aflatoxin B1 is acquired by a systematic evolution of ligands by exponential enrichment (SELEX) technology; the aptamer can be transformed into a report aptamer by a fluorescein mark and the like; the report aptamer is used for detecting the aflatoxin B1; and the aptamer sequence can accurately and rapidly check the aflatoxin B1 in the food; and therefore, the oligonucleotides aptamer has the wide application prospect.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to the use of SELEX technology (exponentially enriched ligand system evolution technology) in molecular biology technology to prepare a set of aflatoxin B 1 The nucleic acid aptamer with high specificity and high affinity is used for the detection of aflatoxin B 1 The application in provides a scientific basis and theoretical basis. Background technique [0002] Aflatoxins (AF) are mainly secondary metabolites of Aspergillus flavus and Aspergillus parasiticus, and their chemical structures are similar. They are all derivatives of dihydrofuranocoumarin. The structure of aflatoxin has been determined to be AFB 1 , AFB 2 , AFM 1 Among other 18 species, their basic structures all contain difuran ring and oxaphthalone (also known as coumarin). The former is a toxic structure, and the latter may be related to its carcinogenesis. Aflatoxin is resistant to high temperatures, and usually heat...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12Q1/68
Inventor 王周平王文凤段诺吴世嘉夏雨马小媛
Owner JIANGNAN UNIV
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