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Botulinum toxin specific substrate peptide, detection kit and detection method

A detection kit, botulinum toxin technology, applied in the field of analysis and detection, can solve the problems of cumbersome operation, large coefficient of variation of results, long test period, etc.

Active Publication Date: 2022-05-27
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the mouse animal experiment is still the national standard method for the detection of botulinum toxin, but the method is cumbersome to operate, the coefficient of variation of the result is large, the test period is long, the demand for animals is large, and the cost is high. Therefore, it is necessary to find an alternative method for animal experiments. The research is mainly carried out from the aspects of immunology, cytology, mass spectrometry, etc.
The current commercial botulinum toxin detection kit is only type A botulinum toxin detection ELISA kit

Method used

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  • Botulinum toxin specific substrate peptide, detection kit and detection method
  • Botulinum toxin specific substrate peptide, detection kit and detection method
  • Botulinum toxin specific substrate peptide, detection kit and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0096] Example 1 Determination and synthesis of botulinum toxin serotype-specific substrate peptides

[0097] Through screening and optimization, the present invention obtains four substrate peptides specific for botulinum toxin A, B, E, and F serotypes, respectively, and the amino acid sequences of these four substrate peptides are as shown in SEQ ID NO.1-4 respectively. said.

[0098] The above four substrate peptides are synthesized (synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.), and the synthesized substrate peptides are as follows:

[0099] Type A: Acetyl-RGSNKKPIDAGNQRATRXLGGR-NH 2 ; Wherein, X represents norleucine;

[0100] Type B: LSELDDRAADLQAGASQFESSAAKLKRKYWWKNLK;

[0101] Type E: WWWAKLGQEIDRTNRQKDXIMAKADSNKR-NH 2 ; Wherein, X represents homoarginine;

[0102] Type F: TSNRRLQQTQAQVDEVVDIRMVNVDKVLERDQKLSELDDRADAL.

[0103] After the above-mentioned four substrate peptides are specifically cleaved by their corresponding serotypes of botulinum toxin...

Embodiment 2

[0107] Example 2 Development of botulinum toxin detection kit

[0108] This embodiment provides a botulinum toxin detection kit, which includes reagent 1, reagent 2, reagent 3, reagent 4, reagent 5, reagent 6, and reagent 7;

[0109] Among them, reagent 1 is a PBST solution; reagent 2 is a sample enrichment reagent; reagent 3 is a 1-2 mg / mL BSA solution; reagent 4 is a reaction buffer; reagent 5 is four substrate peptides synthesized in Example 1 (sequences such as SEQ ID NO.1-4); reagent 6 is a saturated solution of α-hydrogen-4-hydroxycinnamic acid (CHCA); reagent 7 is a standard calibration solution of mass spectrometer.

[0110] The composition of the above reaction buffer is as follows: 0.05M Hepes, 25mM DTT, 20μM ZnCl 2 , prepared with ultrapure water.

[0111] The above-mentioned sample enrichment reagent is used to enrich the trace amount of botulinum toxin in the sample, and it contains streptavidin-biotin magnetic bead conjugated antibody. The preparation method is...

Embodiment 3

[0116] Example 3 Establishment of botulinum toxin detection method

[0117] 1. Determination of sample dilution and detection limit

[0118] The botulinum toxin type B sample toxin concentrations were 300 ng / mL, 150 ng / mL, and 30 ng / mL, respectively, diluted 1:1, 1:4, and 1:20 by volume with PBST, and then 20 μL of streptavidin was added. The biotinylated monoclonal antibody was coupled to the magnetic beads and incubated at room temperature for 1 h. After subsequent washing, blocking and enzymatic cleavage reactions, mass spectrometry detection was performed. The optimal dilution factor was determined based on the peak response value of the enzyme digestion mass spectrometer.

[0119] The result is as figure 2 As shown, the peak response value of the enzyme digestion mass spectrum decreases with the increase of the dilution ratio. When the maximum dilution factor is 1:20, the peak of the enzyme-cleavage mass spectrum can still be detected, that is, the detection line of ...

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Abstract

The invention relates to the technical field of analysis and detection, in particular to a botulinum toxin specific substrate peptide, a detection kit and a detection method. The invention provides a botulinum toxin specific substrate peptide. The botulinum toxin specific substrate peptide is one or more of polypeptides with amino acid sequences as shown in SEQ ID NO.1-4. The specific substrate peptide is combined with a serotype specific target peptide fragment generated by cutting the specific substrate peptide through the botulinum toxin, so that relatively specific, sensitive and accurate identification of the botulinum toxin and the serotype of the botulinum toxin can be realized. The invention also provides a detection kit and a detection method based on the specific substrate peptide, by using the kit and the method, the botulinum toxin and the serotype thereof can be rapidly, sensitively and accurately identified with high throughput, and a technical support is provided for botulinum toxin detection.

Description

technical field [0001] The invention relates to the technical field of analysis and detection, in particular to a botulinum toxin-specific substrate peptide, a detection kit and a detection method. Background technique [0002] Botulinum toxin (botulinum neurotoxin) is a natural protein toxin known to be the most toxic to humans produced by Clostridium botulinum, with a median lethal dose of 0.1-1.0ng / kg. Botulinum toxin is divided into seven serotypes: A, B, C, D, E, F, and G. Among them, A, B, E, and F are toxic to humans, C and D can cause animal poisoning, and G rarely seen. The two types of botulinum toxin A and B are widely used clinically in many fields such as neurology, plastic surgery, and rehabilitation. Clostridium botulinum poisoning can cause foodborne outbreaks. The identification and quality control of botulinum toxin serotypes play a very important role in the fields of medical treatment and epidemic prevention and control. [0003] At present, the mouse...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N15/11G01N27/64G01N33/68
CPCC07K14/00G01N33/6848G01N33/6818Y02A50/30
Inventor 肖迪李天一王磊
Owner ICDC CHINA CDC
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