Staphylococcal enterotoxin B tag peptide and application thereof

A staphylococcus enterotoxin and golden yellow technology, applied in the direction of peptide, decapeptide, chemical analysis, etc., can solve the problems of quantitative determination of enterotoxin, false negative, etc., to avoid the risk of exposure to high concentrations of SEB toxin, Good linearity and guaranteed accuracy

Active Publication Date: 2020-08-11
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods can only be used as a qualitative detection tool and cannot be used for the quantitative determination of enterotoxins in food, and sometimes lead to the risk of false negative results
Because enterotoxins are generally heat-resistant, after heat treatment of foods contaminated by Staphylococcus aureus, although the bacteria have been killed and the toxin coding genes may also be destroyed, the enterotoxins produced by them are still pathogenic

Method used

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  • Staphylococcal enterotoxin B tag peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Create a standard curve:

[0074] Accurately draw 25 μL, 50 μL, 100 μL, 200 μL, 400 μL, and 800 μL of the standard solution of SEB tag peptide with a concentration of 20 nM and place them in 1.5 mL plastic centrifuge tubes, add 100 μL of 20 nM internal standard peptide solution, and then use 0.1% formic acid aqueous solution Make up the volume to 1mL, and prepare a series of standard solutions of tagged peptides with concentrations of 0.5nM, 1.0nM, 2.0nM, 4.0nM, 8.0nM, and 16.0nM, respectively.

[0075] A series of standard solutions of prepared tag peptides were detected by high performance liquid chromatography-mass spectrometry.

[0076] The detection conditions of high performance liquid chromatography in this implementation are: chromatographic column: Acquity UPLC BEH Peptide300C18 column (2.1×100mm, 1.7μm); column temperature: 40°C; injection volume: 5μL; mobile phase A: 0.1% formic acid-water , mobile phase B: 0.1% formic acid-acetonitrile; gradient elution: 0m...

Embodiment 2

[0080] Determination of SEB content in milk:

[0081] Weigh 25g of milk sample, adjust the pH to 3.8 with hydrochloric acid, and centrifuge at 10000 rpm for 15min at 4°C. Transfer the supernatant to a new centrifuge tube, adjust the pH to about 7.5 with 5M sodium hydroxide solution, and centrifuge at 10,000 rpm for 15 min at 4°C. The supernatant was removed, and the same volume of 20% trichloroacetic acid solution was added. After standing at 4°C for 30 minutes, it was centrifuged at 10,000 rpm for 10 minutes at 4°C. Discard the supernatant, wash the precipitate with 5 mL of ice ethanol (pre-cooled to 4°C), vortex and mix well, and centrifuge at 10,000 rpm for 10 min at 4°C, discard the supernatant, and repeat this washing operation twice. The washed precipitate was dried under nitrogen flow (temperature below 30° C.). Dissolve the precipitate with 5 mL of 100 mM Tris-HCl (pH 8.5) buffer to obtain a pretreated sample.

[0082] Pipette 200 μL of the pretreated sample, add 10...

Embodiment 3

[0085] Determination of SEB content in milk powder:

[0086] Reconstitute the milk powder into milk according to the brewing ratio on the manufacturer's package, and weigh 25g of milk after mixing homogeneously. The sample to be tested was prepared according to the same method as in Example 2, and the sample to be tested was detected according to the high performance liquid chromatography-mass spectrometry technology and detection conditions provided in Example 1. Test results: the milk powder provided in this example does not contain Staphylococcus aureus enterotoxin B.

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Abstract

The invention belongs to the technical field of detection of staphylococcal enterotoxin B, and provides a staphylococcal enterotoxin B tag peptide and application thereof. The amino acid sequence of the staphylococcal enterotoxin B tag peptide provided by the invention is represented by SEQ ID No.1. The invention further provides an internal standard peptide of the tag peptide; and the internal standard peptide is a peptide fragment after leucine in the tag peptide is labelled by <13>C and <15>N isotopes. According to the staphylococcal enterotoxin B tag peptide and application thereof in theinvention, assay determination is carried out by adoption of a high performance liquid chromatography-mass spectrometry combined technology; therefore, the content of staphylococcal enterotoxin B in afood is measured; and the method has good linearity, sensitivity and recovery rate, and can be used for directly measuring staphylococcal enterotoxin B in the food qualitatively and quantitatively.

Description

technical field [0001] The invention relates to the technical field of detection of Staphylococcus aureus enterotoxin B, in particular to a staphylococcus aureus enterotoxin B tag peptide and its application. Background technique [0002] Staphylococcus aureus enterotoxin B (SEB) is an exotoxin produced by Staphylococcus aureus. It is a highly heat-resistant enterotoxin and belongs to the microbial protein family of "pyrotoxin superantigen". It is a single-chain polypeptide protein containing 239 amino acid residues with one disulfide bond and a molecular weight of 28,336 Da. SEB is one of the most common toxins associated with food poisoning. In addition, due to the stable nature of SEB, the semi-lethal dose LD50 is about 20ng / kg, it is easy to prepare, and can be dispersed in the form of aerosols. Water sources and food are easily polluted by it. The US Centers for Disease Control and Prevention listed it as a possible biological warfare agent. One of the main toxins. W...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31G01N33/68
CPCC07K14/31G01N33/68G01N33/6848G01N2410/00G01N2458/15
Inventor 张京顺蔡增轩黄百芬
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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