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Test kit and method for Ochratoxin A (OTA) detection

An ochratoxin and kit technology, which is applied in the field of kits for detecting ochratoxin A, can solve the problems of prolonging the time-consuming of detection and increasing the cost of detection, and achieves high signal amplification efficiency, fast detection speed and high speed. Effect

Active Publication Date: 2019-07-09
SHANGHAI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods often require the participation of nucleic acid tool enzymes, which not only increases the detection cost, but also prolongs the detection time.

Method used

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  • Test kit and method for Ochratoxin A (OTA) detection
  • Test kit and method for Ochratoxin A (OTA) detection
  • Test kit and method for Ochratoxin A (OTA) detection

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preparation example Construction

[0072] In the present invention, the preparation method of the suspension of streptavidin-functionalized magnetic particles described in step (3) preferably includes the following steps:

[0073] ① Mixing streptavidin functionalized magnetic particles with phosphate buffer solution, magnetically separating and discarding the solution, and obtaining streptavidin functionalized magnetic particles after washing;

[0074] ② Resuspending the washed streptavidin-functionalized magnetic particles in phosphate buffer to obtain a resuspension of streptavidin-functionalized magnetic particles.

[0075] The concentration of the phosphate buffer solution is preferably 9-11 mmol / L, more preferably 10 mmol / L; the pH value is preferably 7.4-7.6, more preferably 7.5.

[0076] In the present invention, the experimental conditions for the determination in step (6) are preferably: the excitation wavelength is 490 nm, and the emission wavelength scanning range is 510-600 nm.

[0077] In the pres...

Embodiment 1

[0085] The verification of the specific recognition and binding of ochratoxin A, the steps are as follows:

[0086] (a) Take 5 μL stock solutions of 20 μM DNA probe AP and SP (fluorescence-labeled) chains in microtubes, mix well, place in a 95°C water bath for 5 minutes, and cool to room temperature naturally.

[0087] (b) Take 5 μL of commercially available streptavidin functionalized magnetic particles and place them in a microtube, add 95 μL of 10 mMPBS (pH 7.5), mix thoroughly and wash for magnetic separation, discard the solution; repeat the above washing process three times, Resuspend the magnetic particles in 100 μL of 10 mM PBS (pH 7.5); then, add 5 μL of the AP / SP double-strand solution obtained in step (a) to the resuspension, mix well and react at 25°C for 20 minutes; the reaction is complete Finally, the magnetic particles were washed three times with 10 mM PBS (pH 7.5), the solution was discarded, and the obtained AP / SP double-stranded modified magnetic particles ...

Embodiment 2

[0094] The quantitative detection of ochratoxin A, its steps are as follows:

[0095] (a) Take 5 μL stock solutions of 20 μM DNA probe AP and SP chains and place them in microtubes, mix them evenly, place them in a water bath at 95°C for 5 minutes, and let them cool down to room temperature naturally.

[0096] (b) Take 10 μL stock solutions of DNA probes b1 and b2 chains with a concentration of 1 μM and put them in microtubes, mix them evenly, place them in a water bath at 95°C for 5 minutes, and let them cool down to room temperature naturally.

[0097] (c) Put 5 μL of commercially available streptavidin functionalized magnetic particles into a microtube, add 95 μL of 10 mMPBS (pH 7.5), mix thoroughly and wash for magnetic separation, discard the solution; repeat the above washing process three times, Resuspend the magnetic particles in 100 μL of 10 mM PBS (pH 7.5); then, add 5 μL of the AP / SP double-strand solution obtained in step (a) to the resuspension, mix well and react...

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Abstract

The invention belongs to the technical field of biological detection, and provides a test kit and method for Ochratoxin A (OTA) detection. The test kit comprises an AP strand solution, an SP strand solution, a b1 strand solution, a b2 strand solution, a c strand solution, and functional magnetic streptavidin particles. A nucleic acid signal amplification strategy mediated by cascade DNA strand replacement reaction is utilized to achieve sensitive OTA detection. Compared with the prior art, the test kit for the OTA detection provided by the invention has the advantages that the operation only lasts for 20 minutes, and the minimum detection line is 0.056ng / mL. The test kit for the OTA detection provided by the invention has the advantages that participation of a nucleic acid tool enzyme is not required, and high signal amplification efficiency, a high signal amplification speed, a high detection speed, high detection sensitivity and the like can be achieved.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a kit and method for detecting ochratoxin A. Background technique [0002] Ochratoxin (Ochratoxin) is a class of secondary metabolites produced by certain species of Aspergillus and Penicillium whose basic structure is isocoumarin benzoate, including 7 structural analogs. Among them, ochratoxin A (Ochratoxin A, OTA) widely exists in cereals, coffee, wine and other crops and related products, has teratogenic, mutagenic and carcinogenic effects, and is the most toxic and the greatest threat to human health. Ochratoxin-like. Therefore, the detection of ochratoxin A is of great significance in food safety and other fields. The commonly used detection methods for ochratoxin A mainly include chromatography (such as high performance liquid chromatography, gas chromatography-mass spectrometry, etc.), thin-layer chromatography, and enzyme-linked immunosorbent assay. Althou...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428G01N2021/6432
Inventor 曹亚王莹韩兵赵婧
Owner SHANGHAI UNIV
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