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Method for detecting botulinum in food through ATP bioluminescence reaction

A botulinum toxin and bioluminescence technology, which is applied in the direction of chemiluminescence/bioluminescence, and analysis by making materials undergo chemical reactions, can solve the problems of low test results, improve detection specificity, high sensitivity, and avoid interference Effect

Inactive Publication Date: 2019-06-25
张 丽英
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for the ATP bioluminescence method, the amount of ATP released in microorganisms will directly affect the test results. If the ATP release is not complete, the test results will be low.
And ATP is an easily degradable energy substance. If the reagent is released, it will destroy ATP, which will also lead to low test results.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] (1) Mix 50 g of the bacon sample to be tested with 950 g of 1×PBS buffer, stir to obtain a suspension, centrifuge to remove insoluble matter, add 200 μL of digestion solution to the obtained initial supernatant, The composition of the digestive juice is: 0.1 mol / L Triton X-100, 0.05 mol / L disodium edetate, 0.02 mol / L proteinase K, 0.5 mol / L sodium lauryl sulfate, mixed well and put in 40 degrees Carry out the digestion reaction in a water bath for 15 minutes, then centrifuge at 3000 g for 1 minute, and discard the precipitate to obtain the digestion supernatant;

[0027] (2) Take 100 mL of the digested supernatant obtained in step (1) for suction filtration. The filter membrane has a pore size of 0.25 μm, and then immerse the filter membrane in a beaker containing 50 mL of ATP release solution. The ATP The composition of the release solution is: 0.04 mol / L Triton X-100, 0.05 mol / L trimethylaminomethane, 0.03 mol / L guanidine hydrochloride, 2 mmol / L N-2-hydroxyethylpipera...

Embodiment 2

[0031](1) Mix 50 g of the canned lunch meat sample to be tested with 950 g of 1×PBS buffer, grind to obtain a suspension, centrifuge to remove insoluble matter, and add 200 μL of digestion solution to the obtained initial supernatant , the composition of the digestive juice was: 0.2 mol / L Triton X-100, 0.06 mol / L disodium edetate, 0.03 mol / L proteinase K, 0.7 mol / L sodium lauryl sulfate, mixed well and put into 40 The digestion reaction was carried out in a water bath for 18 min, then centrifuged at 3000 g for 1.5 min, and the digested supernatant was obtained after discarding the precipitate;

[0032] (2) Take 100 mL of the digested supernatant obtained in step (1) for suction filtration. The filter membrane has a pore size of 0.30 μm, and then immerse the filter membrane in a beaker containing 50 mL of ATP release solution. The ATP The composition of the release solution is: 0.05 mol / L Triton X-100, 0.06 mol / L trimethylaminomethane, 0.04 mol / L guanidine hydrochloride, 3 mmol...

Embodiment 3

[0036] (1) Mix 50 g of canned meat broth to be tested with 950 g of 1×PBS buffer solution, stir to obtain a mixed solution, centrifuge to remove insoluble matter, add 200 μL of digestion solution to the obtained initial supernatant, The composition of the digestive juice is: 0.3 mol / L Triton X-100, 0.07 mol / L disodium edetate, 0.04 mol / L proteinase K, 0.9 mol / L sodium lauryl sulfate, mix well and put in 40 degrees Carry out the digestion reaction in a water bath for 20 minutes, then centrifuge at 3000 g for 2 minutes, and discard the precipitate to obtain the digestion supernatant;

[0037] (2) Take 100 mL of the digested supernatant obtained in step (1) for suction filtration. The filter membrane has a pore size of 0.35 μm, and then immerse the filter membrane in a beaker containing 50 mL of ATP release solution. The ATP The composition of the release solution is: 0.06 mol / L Triton X-100, 0.07 mol / L trimethylaminomethane, 0.05 mol / L guanidine hydrochloride, 4 mmol / L N-2-hydro...

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Abstract

The invention discloses a method for detecting botulinum in food by using an ATP bioluminescence reaction. According to the method, a sample to be detected is mixed with PBS, and an obtained mixture is centrifuged, so that an initial supernatant can be obtained; a digestive solution is added to the initial supernatant, so that a water-bath heating reaction can be carried out; an obtained mixture is centrifuged again, so that a digestion supernatant is obtained and filtered; and a filter membrane is immersed in an ATP release solution, and an obtained mixture is subjected to oscillation extraction, so that an ATP extract can be obtained; a prepared luminescent reaction solution and the ATP extract are mixed in a luminous reaction tube; the luminous reaction tube is arranged in an ATP luminescence detector for detecting illumination intensity; and therefore, the rapid detection of botulinum in food can be realized. The method for detecting botulinum in food through the ATP bioluminescence reaction of the invention is simple, rapid and highly sensitive, can accurately detect whether food contains botulinum and the relative content of the botulinum, and greatly promote the improvementof food safety detection technologies.

Description

technical field [0001] The invention relates to the technical field of biological detection methods, in particular to a method for detecting botulinum toxin in food by using ATP bioluminescent reaction. Background technique [0002] Clostridium botulinum, also known as sausage, is a Gram-positive anaerobic clostridium, with large spores at the sub-extreme, peripheral flagella, and movement. The spores of this bacterium are extremely resistant in vitro, and can be eliminated by dry heat at 180°C for 15 minutes, damp heat at 100°C for 5 hours, and autoclaving at 120°C for 20 minutes. 5% phenol, 20% formaldehyde, 24 hours to kill it. Botulism is a bacterium that grows in an oxygen-deficient environment and is one of the most virulent bacteria. Botulism is mainly transmitted through food, and it is more common in cured meat, bacon, pork and poorly made canned food. In some areas, it has been caused by eating fermented soybeans, bean paste, stinky tofu and stale fish, pork, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76
Inventor 张丽英张华军
Owner 张 丽英
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