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A nucleotide aptamer probe kit for rapid detection of doxycycline residues and its application

A nucleic acid aptamer and doxycycline technology, which is applied in the field of biotechnology and food antibiotic detection, can solve the problems of cumbersome pretreatment and inability to realize rapid on-site detection, and achieve good stability, easy modification, and high sensitivity.

Active Publication Date: 2018-08-24
CHONGQING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aiming at the shortcomings of traditional large-scale instruments that cannot realize on-site rapid detection, cumbersome pretreatment, and the need for antibody preparation in immunoassay methods, the present invention establishes a fluorescence detection method based on magnetic bead separation-aptamer recognition, which can be used for doxycycline in food Rapid, highly sensitive detection of residues

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of Nucleotide Aptamer Probe Kit for Rapid Detection of Doxycycline Residues

[0025] ① Preparation of aminated nano-magnetic beads

[0026] Add 13 g of 1,6-hexanediamine, 4.0 g of anhydrous sodium acetate (CH3COONa) and 2.0 g of ferric chloride hexahydrate (FeCl3 6H2O) into 60 mL of ethylene glycol, and stir at 50 ° C to obtain a colloidal solution. The solution was transferred to a 100mL reaction kettle lined with polytetrafluoroethylene, and reacted at 198°C for 6h. Cool at room temperature, discard the liquid in the upper layer, wash out the solid material in the lower layer with deionized water, and collect it by magnetic separation. Wash twice with deionized water and ethanol respectively, and dry at 50°C for 5-10h.

[0027] ②Avidin-coupled aminated nano-magnetic beads

[0028]The nanomaterials were quantitatively weighed and suspended in PBS (10 mM), ultrasonically dispersed for 15 min, and glutaraldehyde solution was added to make the fin...

Embodiment 2

[0039] Example 2 Application of Nucleotide Aptamer Probe Kit for Rapid Detection of Doxycycline Residues

[0040] ①Establishment of standard curve

[0041] Apply the method in the summary of the invention to measure doxycycline standard substance, and establish a standard curve. The serial standard concentrations of doxycycline are respectively configured as 0, 0, 2.5, 5, 10, 20, 40, and 80 μg / L, with different standard concentrations as the abscissa, and the inhibition rate B / B 0 As the ordinate, draw a standard curve and establish a regression equation. The standard curve is a typical S-type, and the curve regression equation is y=-43.76x+94.95, R 2 =0.99, its IC 50 It was 10.7μg / L, the lowest detection line was 1.79μg / L, and the detection range of the standard curve of the detection method was 1.8~80μg / L.

[0042] ②Tissue sample matrix effect test

[0043] Doxycycline standard working solution was serially diluted with three sample matrices including PBS, pig liver sam...

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Abstract

Provided is a nucleotide aptamer probe kit used for quick detection of Doxycycline residue and application thereof. The Doxycycline nucleic acid aptamer nucleic acid probe adopts a nano-magnetic sphere as a core, the outer surface of the probe is bonded with a Doxycycline nucleic acid aptamer chain; wherein, the Doxycycline nucleic acid aptamer chain (Doxycycline nucleic acid aptamer artificial sequence) is 5'- GTA CGG AAT TCGCTA GCC GGG CGG CAG GCC ACG GCT GGG GTT GGT CCC ACT GCG GAT CCG AGC TCC ACG TG-3'. The kit aptamer probe is a novel recognition element of Doxycycline, and has the advantages of being good in stability, high in sensitivity, low in cost, easy to prepare and modify and high in specificity of marks.

Description

technical field [0001] The invention relates to a screening method for DNA aptamers specifically recognized by doxycycline and its application, in particular to the fields of biotechnology and food antibiotic detection. Background technique [0002] Doxycycline (Doxycycline,) is a tetracycline antibiotic (Tetracyclines), which is usually used to treat animal infectious diseases, and is also used as a feed additive to prevent diseases and promote animal growth. In actual work, due to arbitrarily increasing the dosage and not observing the drug withdrawal period, the drug remains in animal tissues. Tetracyclines remain in animal tissues for a long time, leading to the spread of drug-resistant bacteria among humans and seriously damaging human health. Many countries and international organizations control drug residues by establishing effective detection methods for veterinary drug residues in animal foods. For example, my country and the European Union have clearly stipulate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115
CPCG01N33/54326G01N33/54346G01N33/9446G01N2446/20
Inventor 乐涛张磊孙琦张志浩
Owner CHONGQING NORMAL UNIVERSITY