Aptamer nucleic acid probe kit for detecting doxycycline residue as well as preparation method and application thereof

A doxycycline and nucleic acid probe technology, which is applied in the fields of biotechnology and food antibiotic detection, can solve the problems of cumbersome pretreatment, inability to realize rapid on-site detection, etc., and achieves the effects of good stability, easy modification and easy preparation.

Inactive Publication Date: 2015-02-18
CHONGQING ACADEMY OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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, th...

Method used

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  • Aptamer nucleic acid probe kit for detecting doxycycline residue as well as preparation method and application thereof
  • Aptamer nucleic acid probe kit for detecting doxycycline residue as well as preparation method and application thereof
  • Aptamer nucleic acid probe kit for detecting doxycycline residue as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of Aptamer Nucleic Acid Probe Kit for Detection of Doxycycline Residues

[0028] 1. Preparation of aminated nano-magnetic beads: Add 13g of 1,6-hexanediamine, 4.0g of anhydrous sodium acetate (CH3COONa) and 2.0g of ferric chloride hexahydrate (FeCl3 6H2O) in 60mL of ethylene glycol , stirred at 50°C to obtain a colloidal solution, the solution was transferred to a 100mL reaction kettle with a polytetrafluoroethylene liner, 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.

[0029] 2. Avidin-coupling aminated nano-magnetic beads: Quantitatively weigh the nano-materials and suspend them in PBS (10 mM), ultrasonically disperse for 15 min, and add glutaraldehyde solution to make the final concentration 5% (v / v). Then ...

Embodiment 2

[0035] Example 2 Application of the Aptamer Nucleic Acid Probe Kit for Detecting Doxycycline Residues

[0036] Establishment of standard curve

[0037] Apply the method in the summary of the invention to measure doxycycline standard substance, and establish a standard curve. The series standard concentrations of doxycycline are respectively configured as 0, 0, 2.5, 5, 10, 20, 40, and 80 μg / L, with different standard concentration as the abscissa and inhibition rate B / B0 as the ordinate, draw the standard Curve, to establish a regression equation. The standard curve is a typical S-type, the curve regression equation is y=-43.76x+94.95, R2=0.99, and 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.

[0038] Tissue sample matrix effect test

[0039] Doxycycline standard working solution was serially diluted with three sample matrices including PBS, pig liver sample treatment...

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Abstract

The invention provides an aptamer nucleic acid probe kit for detecting doxycycline residue as well as a preparation method and application thereof. A nanometer magnetic sphere is an inner core and the outer surface of the nanometer magnetic sphere is bonded with a doxycycline nucleic acid aptamer chain, wherein the doxycycline nucleic acid aptamer chain is 5'-GCATGCCTTAAGCGATCG-N35-CCATATTATAAGGCATGC-3', N represents any one of A, T, C and G, N35 represents that random fragment length comprises 35 basic groups, and the probe is suitable for detection of doxycycline residue in animal body.

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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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 乐涛何红秋王利锋
Owner CHONGQING ACADEMY OF SCI & TECH
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