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Method for detecting ochracin A based on fluorescence anisotropy of nucleic acid aptamer

A nucleic acid aptamer and ochratoxin technology, which is applied in fluorescence/phosphorescence, material excitation analysis, etc., can solve problems such as complex methods, and achieve the effects of simple detection process, easy preparation, and easy operation

Active Publication Date: 2014-02-12
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported methods for the detection of ochratoxin A using nucleic acid aptamers are often complicated and require rational design and separation, such as electrochemical sensing, affinity chromatography, etc. (Yang, X.H.; Kong, W.J.; Yang, M.H.; Zhao, M.; Ouyang, Z. Chin. J. Anal. Chem. 2013, 41, 297? 306; Hayat, A.; L. Food Control 2012, 26, 401–415; Chapuis-Hugon, F.; du Boisbaudry, A.; Madru, B.; Pichon, V. Anal. Bioanal. Chem. 2011, 400, 1199–1207.)

Method used

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  • Method for detecting ochracin A based on fluorescence anisotropy of nucleic acid aptamer
  • Method for detecting ochracin A based on fluorescence anisotropy of nucleic acid aptamer
  • Method for detecting ochracin A based on fluorescence anisotropy of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Detection of different concentrations of ochratoxin A ( figure 1 ).

[0016] The nucleic acid aptamer marked by TMR has the following sequence: 5'-GAT CGG GTG TGG GTG GCG TAA AGGGAG CAT CGG ACA-3, TMR is marked on the 10th T base, and the corresponding TMR-labeled aptamer is abbreviated as It is T10-TMR-O36.

[0017] In buffer solution (10mM Tris-HCl, 120mM NaCl, 20mM CaCl 2 , 0.1% Tween20, pH 8.5) mixed T10-TMR-O36 (27nM concentration) with different concentrations of ochratoxin A, incubated at room temperature for 40 minutes, and then measured the fluorescence of T10-TMR-O36 Anisotropy, the excitation light wavelength is 560 nm, and the emission light wavelength is 578 nm. As the concentration of ochratoxin A (Ochratoxin A) increases, the fluorescence anisotropy value r of T10-TMR-O36 gradually decreases, the detection limit is 3nM, the detection range is 3nM to 3μM, and the relative standard deviation of detection is less than 3%.

Embodiment 2

[0018] Example 2: Investigating the selectivity of TMR-labeled nucleic acid aptamer (T10-TMR-O36) in detecting ochratoxin A ( figure 2 ).

[0019] In buffer solution (10mM Tris-HCl, 120mM NaCl, 20mM CaCl 2 , 0.1% Tween20, pH8.5) T10-TMR-O36 (27nM concentration) was mixed with different analytes, incubated at room temperature for 40 minutes, and the fluorescence anisotropy change Δr of T10-TMR-O36 was measured. Only ochratoxin A (620nM) caused a significant decrease in the fluorescence anisotropy value of T10-TMR-O36, and none of the other tested substances could cause a significant change in the fluorescence anisotropy value of T10-TMR-O36. figure 2 In (a) is a buffer solution that does not contain ochratoxin A, (b) is 620nM ochratoxin A, and the tested substances from (c) to (j) are 10μM N-acetyl-L-phenylalanine (NAP ) (c), 7 μM warfarin (d), 9 μM 7-amino-4-methylcoumarin (AMC) (e), 62 μM arginine (f), 62 μM phenylalanine (g), 62 μM aspartic acid (h), 62 μM serine (i), 6...

Embodiment 3

[0020] Example 3: Detection of ochratoxin A ( image 3 ).

[0021] Red wine was diluted 50 times with buffer solution (10mM Tris-HCl, 120mM NaCl, 20mM CaCl2, 0.1%Tween20, pH8.5), and then T10-TMR-O36 (27nM concentration) was mixed with Different concentrations of ochratoxin A were mixed, incubated at room temperature for 40 minutes, and then the fluorescence anisotropy of T10-TMR-O36 was measured, the excitation light wavelength was 560 nm, and the emission light wavelength was 578 nm. As the concentration of ochratoxin A (Ochratoxin A) increased, the fluorescence anisotropy value r of T10-TMR-O36 decreased gradually.

[0022]

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Abstract

The invention provides a method for detecting ochracin A based on the fluorescence anisotropy (fluorescence polarization) of a nucleic acid aptamer marked by a fluorescent dye tetramethylrhodamine (TMR for short). According to the method, the TMR is modified to a specific position of the aptamer of the ochracin A, the fluorescence anisotropy (fluorescence polarization) value of the TMR-marked nucleic acid aptamer is obviously changed due to the combination of the ochracin A, and the fluorescence anisotropy (fluorescence polarization) value is detected, so as to realize the quantitative detection of the ochracin A. The method has the advantages of simplicity and rapidness in operation, easiness in high throughput test, good specificity, high sensitivity and low cost.

Description

technical field [0001] The invention relates to a mycotoxin detection technology, in particular to a fluorescence detection technology for detecting ochratoxin A based on nucleic acid aptamers. Background technique [0002] Ochratoxin A, the English name is Ochratoxin A, is a mycotoxin with a wide range of effects, a secondary metabolite produced by certain species of Aspergillus and Penicillium, and the toxin-producing bacteria include Ochratoxin and Penicillium verrucous and Aspergillus carbon niger, etc. Ochratoxin A can directly contaminate cereals, fodder, fruits, dried fruits and other crops and a variety of foods. People and animals can absorb them into the body through ingestion of contaminated plant foods. It enters the human body through the ingestion of animal food through the accumulation effect. Ochratoxin A has strong nephrotoxicity, immunosuppressive toxicity, neurotoxicity, carcinogenicity and teratogenicity. Highly sensitive and specific detection of ochr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 赵强吕琴
Owner SHANXI UNIV
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