Set of sulfamethazine specifically-bound aptamers and screening method and applications thereof

A technology of sulfamethazine and nucleic acid aptamer, which is applied in the field of nucleic acid aptamer that specifically binds to sulfamethazine and its screening, can solve the problems of limited use of antibodies, difficulty in reproducible production and the like, and achieves simple and convenient methods, Stable properties and small molecular weight

Inactive Publication Date: 2016-01-20
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the preparation of antibodies is inseparable from the use of experimental animals, and it is difficult to produce repeatedly. In addition, it requires strict storage conditions, which limits the use of antibodies

Method used

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  • Set of sulfamethazine specifically-bound aptamers and screening method and applications thereof
  • Set of sulfamethazine specifically-bound aptamers and screening method and applications thereof
  • Set of sulfamethazine specifically-bound aptamers and screening method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Screening of nucleic acid aptamers specifically binding to sulfamethazine

[0059] In this embodiment, SELEX technology is used for in vitro screening of nucleic acid aptamers, and sulfamethazine (SM 2 ) and Sulfamethazine (Sulfamerazine, SMR) are coupled with magnetic beads (Magneticbeads, MBs) to obtain SM 2 -MBs and SMR-MBs; and with SM 2 -MBs are positive screening targets, SMR-MBs are reverse screening targets, and a magnetic bead-assisted-exponential amplification ligand phylogenetic evolution screening system (Mag-SELEX) for sulfamethazine nucleic acid aptamer screening was established , the nucleic acid aptamer specifically binding to sulfamethazine was obtained from a random oligonucleotide library synthesized in vitro through 9 rounds of screening. Specific steps are as follows:

[0060] 1. Coupling of sulfamethazine, sulfamethazine and carboxyl magnetic beads

[0061] (1) Take 100 μL of carboxylated magnetic beads (about 0.6 μmol) into a 2 mL c...

Embodiment 2

[0087] Embodiment 2, the dissociation constant of nucleic acid aptamer is measured by ultrafiltration

[0088] The reaction equilibrium process was simulated by ultrafiltration, and the dissociation constant (Kd) of each nucleic acid aptamer SA04, SA06, SA07 and SA41 obtained in Example 1 was determined. Specific steps are as follows:

[0089] 1. Dilute the aptamers SA04, SA06, SA07 and SA41 with binding buffer to concentrations of 3 μM, 1.6 μM, 0.8 μM, 0.4 μM, 0.2 μM, 0.1 μM and 0.05 μM, respectively.

[0090] 2. Denature at 95°C for 6 minutes, then quickly place on ice for 10 minutes, and then let stand at room temperature for 5 minutes.

[0091] 3. Take 100 μL of the above-mentioned nucleic acid aptamers at each concentration and add to an equal volume of 20 μM sulfamethazine solution (the solvent is binding buffer), and incubate at 25° C. for 30 minutes.

[0092] 4. Add the entire amount of the reaction solution to Ultra-0.5 Centrifugalfilter (10KD), and centrifuge at 12...

Embodiment 3

[0098] Embodiment 3, competition method determines the specificity of nucleic acid aptamer

[0099] 1. Take sulfamethazine (SM 2 ), sulfamethazine (SMR) and sulfamethazine (SDZ) standard stock solutions were diluted with binding buffer to a final concentration of 4 μM.

[0100] 2. Dissolve 5 μg of nucleic acid aptamers SA07 and SA41 in 100 μL of drug-free binding buffer, denature at 95°C for 6 minutes, quickly place on ice for 10 minutes, and then let stand at room temperature for 5 minutes.

[0101] 3. Add 100 μL of sulfamethazine, sulfamethazine and sulfamethazine prepared in step 1 to the nucleic acid aptamer solution prepared in step 2 to obtain incubation solutions respectively, and incubate at 25°C for 30 minutes. The incubation solution contains unbound nucleic acid aptamers, unbound drugs and drug-nucleic acid aptamer complexes.

[0102] 4. Add the full amount of incubation solution to 7×10 7 Sulfamethazine-coated magnetic beads were incubated at 25°C for 30 min. F...

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Abstract

The invention discloses a set of sulfamethazine specifically-bound aptamers and a screening method and applications thereof. The aptamer for identifying sulfamethazine, disclosed by the invention, is a single-stranded DNA molecule shown in any one of formulas (1)-(4): (1) a single-stranded DNA molecule shown in a sequence 3 in a sequence table; (2) a single-stranded DNA molecule shown in a sequence 1 in the sequence table; (3) a single-stranded DNA molecule shown in a sequence 2 in the sequence table; and (4) a single-stranded DNA molecule shown in a sequence 4 in the sequence table. Experiments show that a directly competitive chemiluminescence analysis method established based on biotinylated aptamers and used for detecting sulfamethazine provides a basis for the practical application of the aptamers of sulfamethazine, and provides technical support for more efficient and extensive monitoring of antimicrobial drug residues.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a group of nucleic acid aptamers specifically binding to sulfamethazine, a screening method and application thereof. Background technique [0002] Sulfamethazine (Sulfamethazine, SM 2 ) is a broad-spectrum antibacterial agent, which has inhibitory effect on most Gram-positive bacteria and Gram-negative bacteria. widely used in the industry. At present, the residue of sulfamethazine in animal food is relatively serious, and it is the focus of supervision on the safety of animal products. my country, the European Union and other countries and institutions have stipulated that the maximum residue limit of the total amount of sulfa drugs and individual drugs such as sulfamethazine in animal foods is 100 μg / kg. [0003] At present, the research on the rapid residue detection of the drug is mainly based on the immunoassay method established by the antibody as the binding ele...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/53
Inventor 王战辉沈建忠张素霞温凯倪恒佳刘梅轩江海洋史为民
Owner CHINA AGRI UNIV
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