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Biosensing probe kit for detecting sulfadiazine on basis of nucleotide aptamer specificity, and application thereof

A technology of nucleic acid aptamer and sulfadiazine, which is applied in the field of biology and food safety analysis, can solve the problems of high specificity of aptamers, failure of detection methods, change of ion concentration in reaction system, etc., and achieve broad application prospects and rapid detection , the effect of high sensitivity

Active Publication Date: 2017-09-01
CHONGQING NORMAL UNIVERSITY
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the aptamer has high specificity and SDZ has no cross-reaction
And this aptamer-based detection method is mainly aimed at the detection of relatively simple biological matrices, while animal-derived foods with complex matrix components such as chicken and eggs are rich in salt ions (Na + or K + ), which may significantly change the ion concentration of the reaction system, so that the nucleic acid aptamer cannot be folded into the correct configuration, which will directly cause the nucleic acid aptamer to fail to bind to the target, resulting in the failure of the detection method
The above-mentioned chemiluminescent aptamer analysis method is greatly limited when it is applied to the detection of animal-derived food clinical samples

Method used

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  • Biosensing probe kit for detecting sulfadiazine on basis of nucleotide aptamer specificity, and application thereof
  • Biosensing probe kit for detecting sulfadiazine on basis of nucleotide aptamer specificity, and application thereof
  • Biosensing probe kit for detecting sulfadiazine on basis of nucleotide aptamer specificity, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1 prepares the nucleic acid aptamer of SDZ

[0058] (1) SDZ ​​and sulfamethoxine coupled with agarose magnetic beads

[0059] Take 10mg of SDZ, first dissolve in 2mL of DMF, and then add 2mL of coupling buffer after it is completely dissolved, then add 30mg of NHS (N-hydroxysulfosuccinimide) and 26mg of EDC to the solution in sequence [1-Ethyl-(3-dimethylaminopropyl) carbodiimide, then completely dissolve NHS and EDC; place the solution in a shaker, react at 4°C for 15 minutes, and activate the amino group in SDZ; 2mL Amino Sepharose magnetic beads were washed 3 times with coupling buffer, mixed with the above-mentioned activated SDZ solution, and placed in a shaker at room temperature to react for 2 hours; after the reaction was completed, the reactant was washed 5 times with an acid-base alternating buffer solution, Wash with PBS for 5 times, and finally store in PBS at 4°C; the same method is used for the coupling reaction of sulfamethoxine and amino-seph...

Embodiment 2

[0076] The determination test of embodiment 2 equilibrium dissociation constant

[0077] The nucleic acid aptamers obtained by synthesis and sequencing were measured by fluorescence method for their equilibrium dissociation constant Kd. The synthesized 5'-FAM-labeled aptamer was configured into different concentration gradient solutions (0.05, 0.1, 0.2, 0.4, 0.8, 0.16, 0.32 and 0.64 μmol / L) with binding buffer, and 10 μL of SDZ-modified agarose beads were taken, Mix 200 μL of 5'-FAM-labeled aptamers with different concentration gradients and 10 μL of SDZ-modified agarose beads, incubate at room temperature for 30 minutes, centrifuge to discard unbound DNA; then wash the agarose magnetic beads 3 times with binding buffer , use 200 μL of binding buffer each time; then elute the DNA bound to SDZ with 100 μL of 2mol / L NaOH solution at 95°C, and elute twice to obtain a total of 200 μL of eluent; dilute the same eluate with different eluents multiple, then placed in a 96-well black...

Embodiment 3

[0078] Example 3 Aptamer specificity test

[0079] Take standard stock solutions of SDZ, sulfamethoxine, sulfamethoxine, sulfadimethoxine, sulfamethoxine, sulfaquinoxaline, sulfamethoxazine and sulfamethoxazole, and dilute to The final concentration is 5 μmol / L. Dissolve 5 μg of nucleic acid aptamer in 100 μL of binding buffer, denature at 95°C, place it on ice for 10 minutes, and then place it at room temperature for 5 minutes. Take 100 μL of the prepared SDZ and other standard solutions into the pretreated aptamer solution, and incubate at 37°C for 30 minutes. Then add the above mixed solution to the SDZ-agarose magnetic bead conjugate, and incubate at 37°C for 30min. The supernatant after incubation was taken out, and the concentration of ssDNA in the supernatant was measured by NanoDrop. For the reference group, a drug-free binding buffer was used to incubate with the pretreated nucleic acid aptamer (the operation was the same as above), and then the incubation solution...

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Abstract

The invention provides a biotin-labeled sulfadiazine nucleotide aptamer. The biotin-labeled sulfadiazine nucleic acid aptamer is characterized by comprising a probe, wherein the nucleotide sequence of the probe is S-SDZ No.1. The kit and the method provided by the invention have the advantage of speediness, stability and simpleness in detection, and the prepared nucleotide aptamer is rapid in SDZ residue detection, high in sensitivity, good in repeatability and high in specificity, and has a wide application prospect in rapid detection of food safety.

Description

technical field [0001] The invention belongs to the field of biology and food safety analysis, and in particular relates to a preparation method and application of a biosensing probe for sulfadiazine residues and a detection kit thereof. [0002] technical background [0003] The phenomenon of abuse and illegal use of veterinary drugs is intensifying, and its residues not only directly affect human health (carcinogenic, teratogenic, sensitization, neurotoxicity), but also induce drug resistance in some bacterial species, causing serious harm to the human ecological environment. Sulfadiazine (SDZ) is a synthetic broad-spectrum antibacterial drug that can inhibit most Gram-positive and Gram-negative bacteria. Since this type of drug has a good effect on the prevention and treatment of animal diseases, it is widely used in animal husbandry production. The unreasonable use of SDZ antibiotics leads to its residues in animal-derived foods, and enters the human body through the foo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/535
CPCC12N15/115C12N2310/16G01N33/535G01N33/9446
Inventor 乐涛孙琦张磊张志浩
Owner CHONGQING NORMAL UNIVERSITY
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