Homogeneous-phase rapid detection method of ochratoxin A based on nucleic acid chimeric dye fluorescence quenching

A technology of ochratoxin and fluorescence quenching, which is applied in the field of fluorescence detection, can solve the problems of long detection time, complex scheme, and locked operation, and achieve the effects of high specificity, simple and convenient operation, and cost reduction

Active Publication Date: 2014-05-21
河南黄淮检测科技有限公司
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Problems solved by technology

[0005] In order to solve the shortcomings of the current OTA detection method, such as complex scheme, locked operation, high cost, and long detection time, the present invention provides a homogeneous rapid detection method for ochratoxin A based on fluorescence quenching of nucleic acid chimeric dyes

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  • Homogeneous-phase rapid detection method of ochratoxin A based on nucleic acid chimeric dye fluorescence quenching
  • Homogeneous-phase rapid detection method of ochratoxin A based on nucleic acid chimeric dye fluorescence quenching

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Embodiment Construction

[0020] The present invention will be described in detail below in conjunction with specific embodiments.

[0021] (1) Formation of fluorescent complexes between ochratoxin A nucleic acid aptamers and nucleic acid chimeric dyes

[0022] First, the ochratoxin A nucleic acid aptamer was pretreated: denatured at 95°C for 5 minutes, and cooled at 4°C for 5 minutes. Then take 50 μL of 10 mM Tris-HCl reaction buffer (pH 8.5), wherein the concentration of ochratoxin A nucleic acid aptamer is 50 nM, the dilution ratio of GeneFinder nucleic acid chimeric dye is 8:10000, and react at room temperature for 30 min to make nucleic acid chimeric The dye is fully combined with the ochratoxin A nucleic acid aptamer to form a fluorescent complex of the ochratoxin A nucleic acid aptamer and the nucleic acid chimeric dye; the ochratoxin A nucleic acid aptamer is a single-stranded DNA probe with a sequence of 5 '-CTTCGGGTGTGGGTGGCATTCCGGTAGGCTCTG-3'.

[0023] Screening and preparation of an ochra...

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Abstract

The invention discloses a homogeneous-phase rapid detection method of ochratoxin A based on nucleic acid chimeric dye fluorescence quenching. The homogeneous-phase rapid detection method comprises the following steps: (1) forming an ochratoxin A nucleic acid aptamer and a nucleic acid chimeric dye fluorescence composite; and (2) specifically recognizing ochratoxin A by the ochratoxin A nucleic acid aptamer. According to the invention, the OTA (ochratoxin A) detection method is constructed by taking the ochratoxin A nucleic acid aptamer as a molecule recognition element and taking the nucleic acid chimeric dye as report molecule; the cost for detecting OTA fungaltoxin is reduced greatly; the detection method is good in reproducibility and high in specificity, is simple and convenient to operate and can detect a great quantity of samples in parallel. The homogeneous-phase rapid detection method provided by the invention can detect OTA only for 5-10 minutes after the ochratoxin A nucleic acid aptamer and the nucleic acid chimeric dye fluorescence composite are formed in advance.

Description

technical field [0001] The invention relates to the detection of ochratoxin A by fluorescence quenching of nucleic acid chimeric dyes, and is characterized in the application of ochratoxin A nucleic acid aptamers and nucleic acid chimeric dyes, and the method has fast detection speed and high detection throughput The invention has strong operability and belongs to the technical field of fluorescence detection. Background technique [0002] Ochratoxin A (OTA) is mainly produced by Aspergillus and Cerium, and widely exists in nature, among which grains (wheat, corn, barley, oats, rye, rice and millet, etc.), peanuts, vegetables ( Beans) and other crops are mostly toxic to the liver and kidney, as well as strong teratogenic, carcinogenic and mutagenic effects, and have great potential harm to animal and human health. In view of this, all countries in the world attach great importance to the detection and control of OTA, and have formulated OTA-related limit standards in grain ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 张玲王红旗刘继红张军锋周玲王静尹海燕王建
Owner 河南黄淮检测科技有限公司
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