A group of oligonucleotide aptamers for identifying clenbuterol hydrochloride, salbutamol and ractopamine with high specificity

A technology of salbutamol oligonucleotide and clenbuterol hydrochloride, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, material inspection products, etc., can solve the problem of time-consuming and cumbersome, expensive and non-immunogenicity And other issues

Active Publication Date: 2016-08-24
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since clenbuterol hydrochloride, salbutamol, and ractopamine are all small molecular haptens, which are not immunogenic, they need to be combined with BSA and other macromolecular carrier proteins to prepare complete antigens before stimulating animals to secrete antibodies. Therefore, antibody p

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  • A group of oligonucleotide aptamers for identifying clenbuterol hydrochloride, salbutamol and ractopamine with high specificity
  • A group of oligonucleotide aptamers for identifying clenbuterol hydrochloride, salbutamol and ractopamine with high specificity
  • A group of oligonucleotide aptamers for identifying clenbuterol hydrochloride, salbutamol and ractopamine with high specificity

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Embodiment 1

[0013] Embodiment 1: Clenbuterol hydrochloride, salbutamol, the SELEX screening of ractopamine specific binding oligonucleotide aptamer 1, in vitro chemical synthesis of initial random oligonucleotide (ssDNA) library and primers (by U.S. Integrated DNA Technologies completed by the company), the sequence is as follows:

[0014] 5'-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3' (40N represents 40 random nucleotides);

[0015] Upstream primer:: 5′-AGCAGCACAGAGGTCAGATG-3′

[0016] 5′ phosphorylation downstream primer: 5′-P-TTCACGGTAGCACGCATAGG-3′

[0017] Biotinylated library complementary short strand P1: 5′-Bio-AGCACGCATAGG-3′

[0018] The random ssDNA library and primers were made into 100 μM stock solution with TE buffer and stored at -20°C for later use.

[0019] 2. Fixation and incubation of random ssDNA library

[0020] The first round of screening reaction system was 600 μL, 1nmol ssDNA library and short chain P1 were added to BB buffer (50mM Tris-HCl, 5mM KCl, 100m...

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Abstract

The invention provides a group of oligonucleotide aptamers Apt-1, Apt-2 and Apt-3 which are capable of simultaneously identifying clenbuterol hydrochloride and salbutamol, an oligonucleotide aptamer CLB-2 which is capable of identifying the clenbuterol hydrochloride with high specificity, an oligonucleotide aptamer SAL-5 which is capable of identifying the salbutamol with high specificity and two oligonucleotide aptamers RAC-5 and RAC-6 which are capable of identifying ractopamine with high specificity. Through an SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology based on Fe3O4 magnetic nanoparticle separation, a random oligonucleotide library is immobilized on avidin-enveloped magnetic nanoparticles by virtue of a complementary chain of a biotinylation marker, and the oligonucleotide aptamers, which are high in specific affinity, are finally obtained by conducting screening by 16 turns. The aptamers are broad in application prospect; and the aptamers, by virtue of marker function genes or fluorescent dyes, are applicable to detection of the clenbuterol hydrochloride, the salbutamol and the ractopamine in food; therefore, a new choice is provided for existing detection methods which depend on antibodies.

Description

technical field [0001] The invention relates to the field of food safety biotechnology, in particular to screening a group of oligonucleotide aptamers that recognize clenbuterol hydrochloride and a group of aptamers that recognize albuterol by using SELEX technology (exponential enrichment ligand system evolution technology). The oligonucleotide aptamer and a group of oligonucleotide aptamers recognizing ractopamine provide scientific and theoretical basis for the application of oligonucleotide aptamer-based detection of clenbuterol in food. Background technique [0002] Clenbuterol, as a synthetic β-adrenergic receptor activator, can reduce animal fat content, increase lean meat percentage, promote animal growth, and reduce feed consumption, so it is used in animal husbandry. However, clenbuterol metabolizes slowly in animals, and accumulates in the human body through meat products, causing poisoning symptoms such as dizziness, fatigue, heart palpitations, and numb limbs, w...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/53
CPCC12N15/115C12N2310/16G01N33/53
Inventor 王周平巩文慧段诺吴世嘉夏雨马小媛
Owner JIANGNAN UNIV
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