A kind of staphylococcus aureus enterotoxin a tag peptide and its application

A Staphylococcus gut, golden yellow technology, applied in the direction of peptides, decapeptides, markers used in chemical analysis, etc., can solve the problems of false positives, false negatives, etc., achieve good linearity, ensure accuracy, ensure feasibility and safety sexual effect

Active Publication Date: 2021-10-12
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ELISA is qualitative while RPLA is only semi-quantitative
Furthermore, immunoassay methods based on antigen-antibody reactions are prone to false-negative or false-positive results due to cross-reactivity or interference of complex food matrix components

Method used

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  • A kind of staphylococcus aureus enterotoxin a tag peptide and its application
  • A kind of staphylococcus aureus enterotoxin a tag peptide and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Create a standard curve:

[0075] Accurately draw 25 μL, 50 μL, 100 μL, 200 μL, 400 μL, and 800 μL of the standard solution of SEA tag peptide with a concentration of 20 nM into 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.

[0076] A series of standard solutions of prepared tag peptides were detected by high performance liquid chromatography-mass spectrometry.

[0077] The detection conditions of high performance liquid chromatography in this implementation are: chromatographic column: Acquity UPLC BEH Peptide 300C18 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: 0min-1min, 5% ...

Embodiment 2

[0081] Determination of SEA content in milk:

[0082] 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. Remove the supernatant, add the same volume of 20wt% trichloroacetic acid solution, let stand at 4°C for 30min, then centrifuge at 10000 rpm for 10min 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.). The precipitate was dissolved with 5 mL of 100 mM Tris-HCl (pH 8.5) buffer to obtain a pretreated sample.

[0083] Pipette 200 μL of the pretreated sample, add 100 μL of 20 nM internal stan...

Embodiment 3

[0086] Determination of SEA content in milk powder:

[0087] 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 A.

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Abstract

The invention belongs to the technical field of detection of staphylococcus aureus enterotoxin A, and the invention provides a staphylococcus aureus enterotoxin A tag peptide and application thereof. The amino acid sequence of the Staphylococcus aureus enterotoxin A tag peptide provided by the present invention is shown in SEQ ID No.1, and the corresponding internal standard peptide was designed and synthesized, and the internal standard peptide is the amino acid in the tag peptide use 13 C and 15 N isotope-labeled peptide segment, the amino acid is the leucine at the 3rd and 8th positions in the amino acid sequence of the tag peptide, and the present invention uses high performance liquid chromatography-mass spectrometry for analysis and determination, so that Determine the content of Staphylococcus aureus enterotoxin A in food. The method has good linearity, sensitivity and recovery, and can directly determine the qualitative and quantitative determination of Staphylococcus aureus enterotoxin A in food.

Description

technical field [0001] The invention relates to the technical field of detection of staphylococcus aureus enterotoxin A, in particular to a staphylococcus aureus enterotoxin A tag peptide and an application thereof. Background technique [0002] Staphylococcal enterotoxin A (SEA) is one of the most common toxins associated with food poisoning. SEA-contaminated milk and its by-products have caused widespread outbreaks of gastroenteritis, and although little or no S. aureus pathogen was detected in some cases of food poisoning, SEA was often present in these samples. SEA is an exotoxin produced by Staphylococcus aureus, which is stable under high temperature conditions. Due to individual differences in sensitivity to SEA, the toxic dose of SEA in humans is still unknown. However, for public health, it is necessary to monitor SEA residues in milk and dairy products in terms of food safety. [0003] Due to the different complexity of food matrices, the detection method of SEA...

Claims

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Application Information

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