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Kit for detecting ochratoxin A and detection method thereof

An ochratoxin and kit technology, which is applied in the field of photo-induced chemiluminescence immunoassay, can solve the problems of complex operation, unsuitable for large-scale sample detection, expensive equipment and other problems, and achieves high sensitivity, short detection time, and simple operation. Effect

Inactive Publication Date: 2010-05-12
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chromatography can be qualitative and quantitative, but its equipment is expensive, the operation is complicated and it is not suitable for the detection of large batches of samples

Method used

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  • Kit for detecting ochratoxin A and detection method thereof
  • Kit for detecting ochratoxin A and detection method thereof
  • Kit for detecting ochratoxin A and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 Preparation of kit and detection of corn samples

[0015] Preparation of luminescent particles coated with OTA artificial antigen:

[0016] Add 1 mg of luminescent particles to a centrifuge tube, add 12.5 μL of Tween-20 with a mass concentration of 1%, 0.05 mg of OTA-BSA artificial antigen, 10 μL of sodium borohydride, and use pH 6.0, 0.1M 2-(N-morpholine) The volume of ethanesulfonic acid (MES) buffer was added to 200 μL, and the reaction was shaken at 37° C. in the dark for 48 hours. Add 10 μL pH 5.0, 0.3M carboxymethoxylamine hemihydrochloride (CMO) solution to block unbound sites, incubate at 37°C in the dark for 1 hour, and then centrifuge to separate the luminescent particles linked to OTA-BSA, dilute and set aside .

[0017] Preparation of reagents:

[0018] Preparation of standard OTA reagent: Standard OTA: 0ng / mL, 0.1ng / mL, 0.5ng / mL, 1ng / mL, 10ng / mL, 100ng / mL, obtained by dilution from pure OTA, the diluent is methanol: water volume The ratio is 3...

Embodiment 2

[0035] Example 2 Determination of barley samples

[0036] The reagents provided by the kit are the same as in Example 1, and are used to detect barley samples.

[0037] The specific detection steps are as follows:

[0038] First process the barley sample: crush the barley sample to 20 mesh, take 5 grams of the sample and put it in a test tube, add 12.5 mL of extract (methanol:water=7:3). Stopper and vibrate for 3 minutes, filter, and use Xinhua No. 1 paper as the filter paper. Take 1mL of the filtrate and dilute it with 1mL of distilled or deionized water for later use.

[0039] Take 20 μL of OTA-BSA-coated luminescent particles and add them to a white opaque microwell plate; add 20 μL of OTA standard or processed samples to their respective microwells; add 20 μL of rabbit anti-OTA antibody; continue to add 20 μL of biotinylated goat antibody Rabbit antibody, incubate at 37°C for 15 minutes; add 175 μL photosensitive particles coated with streptavidin in the dark, incubate ...

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Abstract

The invention belongs to the technical field of light initiated chemiluminescence immunoassay, and discloses a kit for detecting ochratoxin A and a detection method thereof, which are used for detecting content of the ochratoxin A (OTA) in grain, feeds and products thereof. The base of the kit is homogeneous label immunoreaction. Luminescent particles coated with OTA-BSA are added into a white opaque microtiter plate; an OTA standard sample or treated sample is added into micropores respectively, and then rabbit anti-OTA antibody and biotinylated goat anti-rabbit antibody are added into the micropores in sequence for label immunoreactions; and photosensitive particles coated with streptavidin are added into obscure corners for reaction to detect optical signals, under the excitation of light, photoluminescent particles generate fluorescent light through the generation and transmission of singlet ionized oxygen, the detection is performed by a light initiated chemiluminescence detector, the intensity of the optical signals is reciprocal to the concentration of the OTA, and the content of the OTA in the sample is calculated by referring to a standard curve. The kit has simple structure, simple and convenient operation, short detection time and high sensitivity.

Description

technical field [0001] A kit for detecting ochratoxin A (OTA) and a detection method thereof belong to the technical field of light-induced chemiluminescence immunoassay (LICLIA), and are used for detecting OTA content in grains, feed and their products. Background technique [0002] Ochratoxin A (OTA) is a toxin produced by the fungus Aspergillus ochraceous and several Penicillium fungi. OTA has been proven to cause damage to the kidneys of animals and humans, and is also a carcinogen. Most of the poisoning caused by mycotoxins is caused by mold-contaminated grains, oil crops, and fermented foods, and mycotoxin poisoning is often manifested in obvious places The clinical manifestations are more complex, including acute poisoning, chronic poisoning, carcinogenicity, teratogenicity and mutagenicity. OTA can be isolated from most grains, including barley, wheat, oats, corn, coffee beans, etc. Poultry fed with these grains will also be contaminated. Therefore, in order to prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/52G01N21/64
Inventor 黄飚张珏张艺陈蕴金坚
Owner JIANGSU INST OF NUCLEAR MEDICINE
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