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Rapid detection method of ochratoxin A by fluorescence polarization based on exonuclease I circular enzyme digestion and amplification

A technology of ochratoxin and exonuclease, which is applied in the fields of molecular biology and fluorescence detection, can solve the problems of high cost, low sensitivity, and complicated operation, and achieve the effects of high specificity, improved sensitivity, and simple and convenient operation

Inactive Publication Date: 2014-07-16
HENAN ACAD OF AGRI SCI
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Problems solved by technology

[0006] In order to solve the shortcomings of low sensitivity, complicated operation and high cost of the current OTA detection method, the present invention provides a rapid detection method for ochratoxin A fluorescence polarization based on exonuclease I cyclic enzymatic digestion amplification

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  • Rapid detection method of ochratoxin A by fluorescence polarization based on exonuclease I circular enzyme digestion and amplification
  • Rapid detection method of ochratoxin A by fluorescence polarization based on exonuclease I circular enzyme digestion and amplification
  • Rapid detection method of ochratoxin A by fluorescence polarization based on exonuclease I circular enzyme digestion and amplification

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Embodiment Construction

[0021] The present invention will be described in detail below in conjunction with specific embodiments.

[0022] (1) Ochratoxin A nucleic acid aptamer hybridizes with a single-stranded fluorescently labeled oligonucleotide probe to form a nucleic acid aptamer double-stranded complex

[0023] First, the ochratoxin A nucleic acid aptamer and the single-stranded fluorescently labeled oligonucleotide probe were pretreated: denatured at 95°C for 5 minutes, and cooled at 25°C for 30 minutes. The sequence of the ochratoxin A nucleic acid aptamer (aptamer) was 5'-CTTCGGGTGTGGGTGGCATTCCGGTAGGCTCTG-3'; the sequence of the single-stranded fluorescently labeled oligonucleotide probe (FAM-probe) was 5'-FAM-CAGAGCCTAC-3'.

[0024] (2) Cycle of strand displacement reaction and enzyme cleavage reaction

[0025]Take 50 μL of 10 mM Tris-HCl reaction buffer solution (pH 8.5), which contains 50 nM nucleic acid aptamer double-stranded complex, 0.8 U / μL exonuclease I and an appropriate concentrat...

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Abstract

The invention discloses a rapid detection method of ochratoxin A (OTA) by fluorescence polarization based on exonuclease I (ExoI) circular enzyme digestion and amplification. According to the method, hybridization between an aptamer and a single-stranded fluorescent labeled oligonucleotide probe is performed to form a double-stranded complex; a specific strand displacement reaction between the complex and OTA can be carried out and a fluorescent labeled probe and OTA-aptamer complex is released so as to trigger an enzyme digestion reaction of ExoI to the single-stranded fluorescent labeled oligonucleotide probe and the OTA-aptamer complex to release OTA; and the released OTA can trigger next strand displacement reaction and enzyme digestion reaction, followed by recycling so as to realize signal amplification. The defect that a present antibody dependent OTA rapid detection method easily causes a cross reaction, has low sensitivity, is complicated to operate and has too high costs is solved by the method provided by the invention. The method provided by the invention is simple to operate and is convenient and fast. By the method, it only takes 15 min to realize rapid detection of OTA, and sensitivity can reach 0.5nM.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and fluorescence detection, and specifically relates to a method for detecting ochratoxin A by using the change of the fluorescence polarization value of the reaction system, which is characterized in that the nucleic acid aptamer-based strand displacement reaction and the nucleic acid-based The application of the signal amplification mechanism of Dicer I, and the method has fast detection speed, high sensitivity and strong operability. Background technique [0002] Ochratoxin A (OTA) exists widely in nature and is mainly produced by Aspergillus and Cerium. Common agricultural products susceptible to OTA contamination mainly include wheat, corn, barley, oats, rye, rice and millet, etc. Food, as well as crops such as peanuts and vegetables (beans). Toxicological studies have shown that OTA has multiple toxicities such as liver and kidney toxicity, strong teratogenic, carcinogenic and mut...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N21/31
CPCG01N21/21G01N21/6402G01N33/53G01N33/533
Inventor 刘继红王红旗张军锋张玲周玲王建王静尹海燕祁玉峰
Owner HENAN ACAD OF AGRI SCI
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