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A quantitative detection method for Bacillus thuringiensis subsp. israeli toxin protein

A quantitative detection method, the technology of Bacillus thuringiensis, applied in the field of quantitative detection of toxin protein, can solve the problems of low Bti toxin protein content and inability to accurately quantify Bti toxin protein, and achieve high accuracy of detection results, low cost and accurate results Effect

Active Publication Date: 2020-12-04
WUHAN KERNEL BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The applicant once tried to detect the content of crystal protein in Bti by referring to the detection method of toxin protein in Bt wettable powder standard HG 3617-1999, and found that with the increase of Bti sample sampling amount, the content of Bti toxin protein became lower and lower, even No crystal protein band occurs, indicating that the existing Bt toxin protein detection method cannot accurately quantify the Bti toxin protein, so it is urgent to establish a method for measuring the crystal protein content of Bti products with high accuracy and little external influence factors. So as to better control the quality of Bti products

Method used

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  • A quantitative detection method for Bacillus thuringiensis subsp. israeli toxin protein
  • A quantitative detection method for Bacillus thuringiensis subsp. israeli toxin protein
  • A quantitative detection method for Bacillus thuringiensis subsp. israeli toxin protein

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Experimental program
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Effect test

Embodiment 1

[0021] Method summary: The raw powder of Bacillus thuringiensis subsp. , followed by SDS-PAGE to separate the toxin protein from other miscellaneous proteins according to the difference in the relative molecular mass of the protein, and finally scan the protein region with an electrophoretic image scanner, and quantify it according to the intensity value of the protein band region.

[0022] 1. Reagents and solutions

[0023] Tetramethylethylenediamine (TEMED).

[0024] 0.55mol / L sodium hydroxide solution: Weigh 2.2g of sodium hydroxide and dilute to 100mL with water.

[0025] 10% ammonium persulfate solution (10% AP): Weigh 0.1 g of ammonium persulfate and dilute to 1 mL with water.

[0026] 10% sodium dodecyl sulfate solution (10% SDS): Weigh 10.0 g of sodium dodecyl sulfate and dilute to 100 mL with water.

[0027] 0.1mol / L phosphate buffer solution (pH8.0): Weigh 1.77g of anhydrous potassium dihydrogen phosphate into a beaker, add 6mL of 0.1mol / L anhydrous potassium dihy...

Embodiment 2

[0064] 1. Explore the method of eliminating interfering substances in Bti samples

[0065] The sugars, flocculants and other substances in the Bti sample interfered with the formation of the toxin protein-SDS micelles. As the sampling amount of the Bti sample increased, the interfering substances also gradually increased. When a certain amount was reached, the toxin protein-SDS micelles It cannot be formed at all, resulting in a blank protein band phenomenon, resulting in the inability to detect the toxin protein in the Bti sample. Therefore, it is first necessary to remove the interfering substances in the Bti sample in order to accurately quantify the toxin protein in the Bti sample.

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Abstract

The invention discloses a quantitative detection method of Bti (Bacillus thuringrensis subsp.Israelensis) protein. The emthdo is characterized in that on the basis of the existing Bti protein contentdetection method, a special sample pretreatment method is used; the problem of toxin protein quantitative detection interference by impurities in a test specimen is solved. Meanwhile, an SDS-PAGE gelrecipe is optimized; the migration rate, the resolution and the protein belt form of two kinds of protein belts in the gel electrophoresis map are further improved, so that the detected result is moreaccurate. The method can be used for the quality control of the Bti products, and has the advantages that the operation is simple; the implementation is easy; the speed is high; the cost is low; theexternal influence is small, and the like.

Description

technical field [0001] The invention belongs to the field of quantitative detection of toxin proteins, and in particular relates to a quantitative detection method of toxin proteins of Bacillus thuringiensis subspecies Israel. Background technique [0002] Bacillus thuringiensis (Bacillus thuringiensis, referred to as Bt), is currently the most widely used microbial insecticide, its main insecticidal component is parasporal crystals, Bacillus thuringensis subsp.Israelensis (Bti for short) It is a pathogenic bacterium that is highly virulent to young mosquito pests discovered by Goldberg and Margalit in 1977. The main active ingredient is also parasporal crystals, which are composed of a variety of toxin proteins, including two toxins with molecular weights of 128KD and 65KD Protein is the main ingredient. [0003] Currently the most effective method for quality inspection and control of Bt products is bioassay, but because bioassay is affected by the quality, quantity and e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447
CPCG01N27/44747
Inventor 胡凌云金海燕刘荷梅刘华梅
Owner WUHAN KERNEL BIO-TECH CO LTD
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