阅读说明：本技术 一种金黄色葡萄球菌肠毒素b标签肽及其应用 (Staphylococcus aureus enterotoxin B tag peptide and application thereof ) 是由 张京顺 蔡增轩 黄百芬 于 2020-04-27 设计创作，主要内容包括：本发明属于金黄色葡萄球菌肠毒素B检测的技术领域,本发明提供了一种金黄色葡萄球菌肠毒素B标签肽及其应用。本发明提供的金黄色葡萄球菌肠毒素B标签肽的氨基酸序列如SEQ ID No.1所示,本发明还提供了所述标签肽的内标肽,所述内标肽是将所述标签肽中的亮氨酸用<Sup>13</Sup>C和<Sup>15</Sup>N同位素标记后的肽段。本发明采用高效液相色谱-质谱联用技术进行分析测定,从而测得食品中金黄色葡萄球菌肠毒素B的含量,该方法具有良好的线性、灵敏度、回收率,可以直接定性和定量测定食品中的金黄色葡萄球菌肠毒素B。(The invention belongs to the technical field of detection of staphylococcus aureus enterotoxin B, and provides a staphylococcus aureus enterotoxin B tag peptide and application thereof. The amino acid sequence of the staphylococcus aureus enterotoxin B tag peptide is shown in SEQ ID No.1, the invention also provides an internal standard peptide of the tag peptide, and the internal standard peptide uses leucine in the tag peptide 13 C and 15 n isotope labeled peptide segment. The method adopts a high performance liquid chromatography-mass spectrometry combined technology for analysis and determination, so as to determine the content of the staphylococcus aureus enterotoxin B in the food.)

1. A staphylococcus aureus enterotoxin B tag peptide is characterized in that the amino acid sequence of the tag peptide is shown as SEQID No. 1.

2. The internal standard peptide of a staphylococcus aureus enterotoxin B tag peptide of claim 1, wherein the internal standard peptide is leucine of the tag peptide¹³C and¹⁵n isotope labeled peptide segment.

3. The use of a staphylococcal enterotoxin B tag peptide of claim 1 in the detection of staphylococcal enterotoxin B in a food product.

4. The method for detecting staphylococcus aureus enterotoxin B in food by using the staphylococcus aureus enterotoxin B tag peptide as claimed in claim 1, which is characterized by comprising the following steps:

(1) sample pretreatment: removing impurities, precipitating, washing and dissolving the sample in sequence to obtain a pretreated sample;

(2) preparing a sample to be tested: adding the internal standard peptide into the pretreated sample to carry out denaturation treatment, enzymolysis treatment and termination treatment in sequence to obtain a sample to be detected;

(3) and (3) detection: detecting a sample to be detected by adopting a high performance liquid chromatography-mass spectrometry combined technology, and calculating to obtain the peak area ratio of the tag peptide to the internal standard peptide;

(4) drawing a standard curve: replacing the sample to be detected in the step (2) with a series of standard solutions of the tag peptide, detecting and calculating according to the method in the step (3), and obtaining a standard curve with the peak area ratio of the tag peptide and the internal standard peptide as a vertical coordinate and the concentration of the tag peptide as a horizontal coordinate, wherein the series of standard solutions are prepared by adding the internal standard peptide into the standard solutions of the tag peptide and then diluting with a formic acid aqueous solution;

(5) calculating the content of the staphylococcus aureus enterotoxin B in the sample: and (4) substituting the peak area ratio of the tag peptide to the internal standard peptide obtained in the step (3) into the standard curve obtained in the step (4) to obtain the concentration of the staphylococcus aureus enterotoxin B, and calculating to obtain the content of the staphylococcus aureus enterotoxin B in the sample.

5. The method for detecting staphylococcus aureus enterotoxin B in food as claimed in claim 4, wherein the step of removing impurities is to adjust the pH of a sample to 3.5-4.0 by using acetic acid, perform solid-liquid separation, take a supernatant 1, adjust the pH of the supernatant 1 to 7.0-8.0 by using a sodium hydroxide solution, perform solid-liquid separation, and take a supernatant 2 for later use.

6. The method for detecting the staphylococcus aureus enterotoxin B in the food according to claim 4, wherein the precipitation is carried out by adding trichloroacetic acid solution into the supernatant 2 according to the volume ratio of 1 (0.8-1.2), standing, carrying out solid-liquid separation, and taking the precipitate for later use; the mass concentration of the trichloroacetic acid solution is 15-25%.

7. The method for detecting staphylococcus aureus enterotoxin B in food according to claim 4, wherein the washing reagent is glacial ethanol, and the dissolving reagent is Tris-HCl buffer.

8. The method for detecting staphylococcus aureus enterotoxin B in food as claimed in claim 4, wherein the reagents for denaturation treatment comprise a Rapidest SF solution, a dithiothreitol solution and an iodoacetamide solution, the mass concentration of the Rapidest SF solution is 0.5-1.5%, the concentration of the dithiothreitol solution is 50-150 mM, the concentration of the iodoacetamide solution is 100-300 mM, and the volume ratio of the pretreated sample to the Rapidest SF solution, the dithiothreitol solution and the iodoacetamide solution is (100-300): 20:20: 20; the enzyme subjected to enzymolysis is recombinant human trypsin, the concentration of the recombinant human trypsin is 50-150 mu g/ml, and the volume ratio of the pretreated sample to the recombinant human trypsin is (100-300): 20; the reagent for terminating the treatment is formic acid, and the volume ratio of the pretreatment sample to the formic acid is (100-300): 10.

9. the method for detecting staphylococcus aureus enterotoxin B in food as claimed in claim 4, wherein the detection conditions of the high performance liquid chromatography are Acquisty UPLC BEH Peptide300C18 column 2.1 × 100mm, 1.7 μm, column temperature 35-45 ℃, injection volume 5 μ L, mobile phase A of 0.1% formic acid-water, mobile phase B of 0.1% formic acid-acetonitrile, gradient elution of 0min-1min, 5% mobile phase B, 1min-3min, 5% mobile phase B linear change to 25% mobile phase B, 3min-3.5min linear change to 40% mobile phase B, 3.5min-4.5min linear change to 100% mobile phase B, 4.5min-5.8min, 100% mobile phase B maintenance, 5.8min-6.0min linear decrease to 5% mobile phase B, 6.0min-8.0min, 5% mobile phase B maintenance, and electric flow rate 0 mL/3.0 minSpraying mode: ESI⁺(ii) a Mass spectrum scanning mode: monitoring multiple reactions; capillary voltage 3.5 kV; ion source temperature: 125-175 ℃; desolventizing temperature: 325 to 375 ℃; desolventizing air flow rate: 800L/h; taper hole air flow rate: 50L/h; sample introduction amount: 5 μ L.

10. A kit for detecting staphylococcus aureus enterotoxin B in food is characterized by comprising staphylococcus aureus enterotoxin B tag peptide and internal standard peptide thereof, wherein the amino acid sequence of the tag peptide is shown as SEQ ID No.1, and the internal standard peptide is used for brightening acid in the tag peptide¹³C and¹⁵n isotope labeled peptide segment.

Technical Field

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 application thereof.

Background

Staphylococcus aureus enterotoxin b (seb), an exotoxin produced by staphylococcus aureus, is a highly heat-resistant enterotoxin, and belongs to the family of microbial proteins of the "pyrogen toxin superantigen". It is a single-chain polypeptide protein containing 239 amino acid residues, has a disulfide bond and has a molecular weight of 28,336 Da. SEB is one of the most common toxins associated with food poisoning. In addition, because of the stable nature of SEB, a semi-lethal LD50 dose of about 20ng/kg, ease of preparation, ability to be dispensed as an aerosol, and susceptibility to contamination of water and food sources, is listed by the U.S. centers for disease control as one of the major toxins that may be used as biological warfare agents. With the increasingly extensive and intensive research on enterotoxins and the development of modern scientific techniques, methods for detecting enterotoxins have been improved. Mainly comprises an animal experiment method based on toxic symptom observation, a molecular biology method based on enterotoxin coding gene detection and an immunology method based on enterotoxin antigen-antibody reaction. However, these methods are only a qualitative detection tool and cannot be used for quantitative determination of enterotoxins in food products, and sometimes lead to the risk of false negative results. Because of the general heat resistance of enterotoxin, the enterotoxin produced by food contaminated with staphylococcus aureus can be pathogenic after heat treatment, although the bacteria are killed and the toxin-encoding gene is destroyed. Thus, in the health examination of food poisoning incidents, the detection of enterotoxins is a determinant of the contamination of food by staphylococcus aureus. It follows that there is a need to develop a rapid, sensitive, selective and accurate method for the direct qualitative detection and quantification of enterotoxins in food products.

Disclosure of Invention

The invention aims to provide a staphylococcus aureus enterotoxin B tag peptide and application thereof, which can be used for confirming and detecting the pollution level of staphylococcus aureus B in food.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a staphylococcus aureus enterotoxin B tag peptide, and the amino acid sequence of the tag peptide is shown in SEQ ID No. 1.

The invention also provides an internal standard peptide of the staphylococcus aureus enterotoxin B tag peptide, which uses leucine in the tag peptide¹³C and¹⁵n isotope labeled peptide segment.

The invention also provides application of the staphylococcus aureus enterotoxin B tag peptide in detecting staphylococcus aureus enterotoxin B in food.

The invention also provides a method for detecting staphylococcus aureus enterotoxin B in food by using the staphylococcus aureus enterotoxin B tag peptide, which comprises the following steps:

(1) sample pretreatment: removing impurities, precipitating, washing and dissolving the sample in sequence to obtain a pretreated sample;

(2) preparing a sample to be tested: adding the internal standard peptide into the pretreated sample to carry out denaturation treatment, enzymolysis treatment and termination treatment in sequence to obtain a sample to be detected;

(3) and (3) detection: detecting a sample to be detected by adopting a high performance liquid chromatography-mass spectrometry combined technology, and calculating to obtain the peak area ratio of the tag peptide to the internal standard peptide;

(4) drawing a standard curve: replacing the sample to be detected in the step (2) with a series of standard solutions of the tag peptide, detecting and calculating according to the method in the step (3), and obtaining a standard curve with the peak area ratio of the tag peptide and the internal standard peptide as a vertical coordinate and the concentration of the tag peptide as a horizontal coordinate, wherein the series of standard solutions are prepared by adding the internal standard peptide into the standard solutions of the tag peptide and then diluting with a formic acid aqueous solution;

(5) calculating the content of the staphylococcus aureus enterotoxin B in the sample: and (4) substituting the peak area ratio of the tag peptide to the internal standard peptide obtained in the step (3) into the standard curve obtained in the step (4) to obtain the concentration of the staphylococcus aureus enterotoxin B, and calculating to obtain the content of the staphylococcus aureus enterotoxin B in the sample.

Preferably, the step of removing impurities is to adjust the pH value of a sample to 3.5-4.0 by using acetic acid, perform solid-liquid separation, take a supernatant 1, adjust the pH value of the supernatant 1 to 7.0-8.0 by using a sodium hydroxide solution, perform solid-liquid separation, and take a supernatant 2 for later use.

Preferably, the precipitation is carried out by adding trichloroacetic acid solution into the supernatant 2 according to the volume ratio of 1 (0.8-1.2) for precipitation, standing, carrying out solid-liquid separation, and taking the precipitate for later use; the mass concentration of the trichloroacetic acid solution is 15-25%.

Preferably, the washed reagent is glacial ethanol, and the dissolved reagent is Tris-HCl buffer.

Preferably, the reagent for the denaturation treatment comprises a Rapidest SF solution, a dithiothreitol solution and an iodoacetamide solution, wherein the mass concentration of the Rapidest SF solution is 0.5-1.5%, the concentration of the dithiothreitol solution is 50-150 mM, the concentration of the iodoacetamide solution is 100-300 mM, and the volume ratio of the pretreated sample to the Rapidest SF solution, the dithiothreitol solution and the iodoacetamide solution is (100-300): 20:20: 20; the enzyme subjected to enzymolysis is recombinant human trypsin, the concentration of the recombinant human trypsin is 50-150 mu g/ml, and the volume ratio of the pretreated sample to the recombinant human trypsin is (100-300): 20; the reagent for terminating the treatment is formic acid, and the volume ratio of the pretreatment sample to the formic acid is (100-300): 10.

preferably, the detection conditions of the high performance liquid chromatography comprise an acquisition UPLC BEH Peptide300C18 column 2.1 × 100mm and 1.7 mu m, a column temperature of 35-45 ℃, a sample injection volume of 5 mu L, a mobile phase A of 0.1% formic acid-water, a mobile phase B of 0.1% formic acid-acetonitrile, gradient elution of 0min-1min and 5% mobile phase B, linear change of 1min-3min and 5% mobile phase B to 25% mobile phase B, linear change of 3min-3.5min to 40% mobile phase B, linear change of 3.5min-4.5min to 100% mobile phase B, linear change of 4.5min-5.8min, retention of 100% mobile phase B, linear decrease of 5.8min-6.0min to 5% mobile phase B, mass spectrum of 6.0min-8.0min, retention of 5% mobile phase B, a flow rate of 0.3mL/min, and an electrospray mode⁺(ii) a Mass spectrum scanning mode: monitoring multiple reactions; capillary voltage 3.5 kV; ion source temperature: 125-175 ℃; desolventizing temperature: 325 to 375 ℃; desolventizing air flow rate: 800L/h; taper hole air flow rate: 50L/h; sample introduction amount: 5 μ L.

The invention also provides a kit for detecting staphylococcus aureus enterotoxin B in food, the kit comprises staphylococcus aureus enterotoxin B tag peptide and internal standard peptide thereof, the amino acid sequence of the tag peptide is shown as SEQ ID No.1, and the internal standard peptide is used for brightening acid in the tag peptide¹³C and¹⁵n isotope labeled peptide segment.

Compared with the prior art, the invention has the following beneficial effects:

(1) the method designs and synthesizes stable isotope labeling internal standard peptide aiming at the label peptide by screening the label peptide of the SEB, and realizes the quantitative detection of the SEB by utilizing the analysis technology of high performance liquid chromatography tandem mass spectrometry.

(2) The method selects the tag peptide in the SEB enzymolysis product as a detection object, is suitable for detecting the denatured protein, can meet the simultaneous detection of denatured and non-denatured proteins in a sample, and ensures the accuracy of the method.

(3) According to the invention, the SEB label peptide is selected as a direct detection target object, so that the dependence of the existing detection technology on the SEB toxin standard substance which is difficult to obtain is avoided, the risk that an analyst contacts the high-concentration SEB toxin is also avoided, and the feasibility and the safety of the method are ensured.

Detailed Description

The invention provides a staphylococcus aureus enterotoxin B tag peptide and application thereof, wherein the sequence of the tag peptide is shown in SEQ ID No. 1. The selection of the tag peptide is a key step in the targeted proteomics detection technology, and is directly related to the specificity and accuracy of the quantitative method. The invention comprehensively considers and verifies factors such as specificity, enzymolysis stability, mass spectrum response signal intensity and recovery rate of candidate polypeptide, finally selects VTAQELDYLTR (SEQ ID No.1) as a label peptide of SEB toxin, takes an amino acid sequence of the label peptide as a template, and introduces the SEB toxin¹³C、¹⁵N stable isotope labeled amino acid, and chemical synthesis method for obtaining isotope labeled peptide VTAQE (L-¹³C₆,¹⁵N)DY(L-¹³C₆,¹⁵N) TR as internal standard. The invention adopts the high performance liquid chromatography-mass spectrometry combined technology for analysis and determination, thereby measuring the content of the staphylococcus aureus enterotoxin B in the food.

The invention provides a staphylococcus aureus enterotoxin B tag peptide, and the amino acid sequence of the tag peptide is shown in SEQ ID No. 1.

In the invention, the tag peptide is obtained by screening an enzymolysis product of staphylococcus aureus enterotoxin B, and has good specificity and stability.

The invention also provides an internal standard peptide of the staphylococcus aureus enterotoxin B tag peptide, which uses leucine in the tag peptide¹³C and¹⁵n isotope labeled peptide segment.

The invention also provides application of the staphylococcus aureus enterotoxin B tag peptide in detecting staphylococcus aureus enterotoxin B in food.

The invention also provides a method for detecting staphylococcus aureus enterotoxin B in food by using the staphylococcus aureus enterotoxin B tag peptide, which comprises the following steps:

(1) sample pretreatment: removing impurities, precipitating, washing and dissolving the sample in sequence to obtain a pretreated sample;

(2) preparing a sample to be tested: adding the internal standard peptide into the pretreated sample to carry out denaturation treatment, enzymolysis treatment and termination treatment in sequence to obtain a sample to be detected;

(3) and (3) detection: detecting a sample to be detected by adopting a high performance liquid chromatography-mass spectrometry combined technology, and calculating to obtain the peak area ratio of the tag peptide to the internal standard peptide;

(4) drawing a standard curve: replacing the sample to be detected in the step (2) with a series of standard solutions of the tag peptide, detecting and calculating according to the method in the step (3), and obtaining a standard curve with the peak area ratio of the tag peptide and the internal standard peptide as a vertical coordinate and the concentration of the tag peptide as a horizontal coordinate, wherein the series of standard solutions are prepared by adding the internal standard peptide into the standard solutions of the tag peptide and then diluting with a formic acid aqueous solution;

(5) calculating the content of the staphylococcus aureus enterotoxin B in the sample: and (4) substituting the peak area ratio of the tag peptide to the internal standard peptide obtained in the step (3) into the standard curve obtained in the step (4) to obtain the concentration of the staphylococcus aureus enterotoxin B, and calculating to obtain the content of the staphylococcus aureus enterotoxin B in the sample.

According to the invention, after sampling, the sample is pretreated, and the sample is subjected to impurity removal, precipitation, washing and dissolution in sequence to obtain a pretreated sample.

In the invention, acetic acid is preferably adopted to adjust the pH value of the sample to 3.5-4.0 during impurity removal, solid-liquid separation is carried out, supernatant 1 is taken, sodium hydroxide solution is preferably adopted to adjust the pH value of the supernatant 1 to 7.0-8.0, solid-liquid separation is carried out, and supernatant 2 is taken for later use. The invention carries out impurity removal for removing impurity protein in a sample.

In the invention, the pH value of the acetic acid adjusting sample is preferably 3.5-4.0, and is further preferably 3.8; the pH value of the supernatant liquid 1 is preferably adjusted to 7.0-8.0 by the sodium hydroxide solution, and more preferably 7.5.

In the invention, the solid-liquid separation in the impurity removal process is preferably performed in a centrifugal mode, the centrifugal temperature is preferably 3-5 ℃ independently, and the centrifugal temperature is preferably 4 ℃ independently; the centrifugal rotation speed is preferably 8000-15000 rpm independently, and further preferably 10000 rpm independently; the time for centrifugation is preferably 10-20 minutes independently, and more preferably 15 minutes independently.

In the invention, the precipitation is carried out by adding trichloroacetic acid solution into the supernatant 2 according to the volume ratio of 1 (0.8-1.2), more preferably 1:1, precipitating, standing, carrying out solid-liquid separation, and taking the precipitate for later use.

In the invention, the concentration of the sodium hydroxide is preferably 3-8M, and the concentration of the sodium hydroxide is preferably 5M.

In the invention, solid-liquid separation in the precipitation process is preferably centrifugation, and the centrifugation temperature is preferably 3-5 ℃, and more preferably 4 ℃; the rotation speed of the centrifugation is preferably 8000-15000 r/min, and further preferably 10000 r/min; the time for centrifugation is preferably 10 to 20 minutes, and more preferably 15 minutes.

In the present invention, the mass concentration of trichloroacetic acid is preferably 15 to 25%, and more preferably 20%.

In the invention, the standing temperature is preferably 2-8 ℃, and more preferably 4 ℃; the standing time is preferably 20 to 40 minutes, and more preferably 30 minutes.

In the invention, solid-liquid separation after standing is preferably centrifugal, and the centrifugal temperature is preferably 4-10 ℃, and more preferably 4 ℃; the rotation speed of the centrifugation is preferably 8000-15000 r/min, and further preferably 10000 r/min; the time for centrifugation is preferably 5 to 15 minutes, and more preferably 10 minutes.

In the present invention, the precipitate is preferably cooled to 4 ℃ before the washing.

In the invention, the washing is preferably carried out by using the glacial ethanol, and the invention has no special requirement on the dosage of the glacial ethanol and can fully wash the glacial ethanol; during washing, the invention adds the glacial ethanol into the sediment, uniformly mixes the mixture by adopting vortex, centrifuges the mixture for 10 minutes at 4 ℃ at 10000 r/min, discards supernatant fluid and repeats the washing step for 2 times.

In the present invention, it is preferable that the washed precipitate is dried after the washing, the drying is performed by blow-drying under a nitrogen flow, and the drying temperature is preferably 25 to 35 ℃, and more preferably 30 ℃.

In the invention, a Tris-HCl solution is adopted for dissolution, and the pH value of the Tris-HCl solution is preferably 8-9, and is further preferably 8.5; the concentration of the Tris-HCl solution is preferably 50-150 mM, and more preferably 100 mM; the dosage of the Tris-HCl solution is preferably 2-10 mL, and more preferably 5 mL.

After a pretreatment sample is prepared, the internal standard peptide is added into the pretreatment sample, and then denaturation treatment, enzymolysis treatment and termination treatment are sequentially carried out to obtain a sample to be detected.

In the invention, the concentration of the internal standard peptide is preferably 15-25 nM, and more preferably 20 nM; the volume ratio of the pretreatment sample to the internal standard peptide is preferably (150-250): 100, and more preferably 200: 100.

In the invention, the reagent for denaturation treatment preferably comprises a Rapidest SF solution, a dithiothreitol solution (DTT) and an iodoacetamide solution (IAA), and the mass concentration of the Rapidest SF solution is preferably 0.5-1.5%, and more preferably 1%; the concentration of the dithiothreitol solution is preferably 50-150 mM, and more preferably 100 mM; the concentration of the iodoacetamide solution is preferably 100-300 mM, and more preferably 200 mM; the volume ratio of the pretreatment sample to the Rapidest SF solution, the dithiothreitol solution and the iodoacetamide solution is preferably (100-300): 20:20:20, more preferably 200: 20:20: 20.

in the invention, the step of denaturation treatment is preferably that after the internal standard peptide is added into the pretreated sample, the Rapidest SF solution and the dithiothreitol solution are added, vortex mixing is carried out, the mixture is placed in a boiling water bath for reaction for 10 minutes, cooling is carried out, then the iodoacetamide solution is added, and the mixture is placed in a dark place for reaction for 30 minutes.

In the invention, the cooling temperature is preferably 20-25 ℃.

In the invention, the enzyme for enzymolysis treatment is preferably recombinant human trypsin, the concentration of the recombinant human trypsin is preferably 50-150 [ mu ] g/ml, and is further preferably 100 [ mu ] g/ml, and the volume ratio of the pretreatment sample to the recombinant human trypsin is preferably (100-300): 20, more preferably 200: 20.

In the present invention, the recombinant human trypsin preferably has an activity of 2500USP units/mg protein or more. The recombinant human trypsin of the present invention is preferably purchased from Shanghai Yaxin Biotechnology, Inc.

In the invention, the step of enzymolysis treatment is preferably to add recombinant human trypsin into the solution after denaturation treatment, mix the solution evenly and perform enzymolysis reaction.

In the invention, the temperature of the enzymolysis reaction is preferably 40-50 ℃, and further preferably 45 ℃; the time of the enzymolysis reaction is preferably 15-20 h, and further preferably 18 h.

In the invention, the reagent for terminating the treatment is preferably formic acid, more preferably pure formic acid, and the volume ratio of the pretreatment sample to the formic acid is preferably (100-300): 10, more preferably 200: 10.

In the present invention, it is preferable to add ultrapure water to the solution after the termination of the treatment to make up the solution system for quantitative calculation.

In the present invention, it is preferable that the solution to which ultrapure water is added is subjected to solid-liquid separation, and the solid-liquid separation is preferably centrifugation, and the centrifugation is preferably performed at 12000 rpm for 10 minutes at 4 ℃, and the supernatant is taken to obtain a sample to be measured.

After the sample to be detected is prepared, the sample to be detected is preferably detected by adopting a high performance liquid chromatography-mass spectrometry combined technology, peak areas of the tag peptide and the internal standard peptide are obtained, and the peak area ratio of the tag peptide to the internal standard peptide is calculated.

In the invention, the detection conditions of the high performance liquid chromatography are as follows: acquisty UPLC BEH Peptide300C18 column 2.1X 100mm, 1.7 μm; the column temperature is preferably: 35-45 ℃, and more preferably 40 ℃; the sample introduction volume is preferably: 5 mu L of the solution; the mobile phase a is preferably: 0.1% formic acid-water, mobile phase B is preferably: 0.1% formic acid-acetonitrile; the gradient elution is preferably: 0min-1min, 5% mobile phase B; 1min-3min, linearly changing 5% of mobile phase B to 25% of mobile phase B; 3min-3.5min, linear change to 40% mobile phase B; 3.5min-4.5min, linearly changing to 100% mobile phase B; keeping 100% of mobile phase B for 4.5-5.8 min; linearly decreasing to 5% of mobile phase B within 5.8-6.0 min; keeping 5% of mobile phase B for 6.0-8.0 min; the flow rate is preferably 0.3 mL/min.

In the present invention, the conditions of the mass spectrum are: the electrospray mode is preferably: ESI⁺(ii) a The mass spectrum scanning mode is preferably as follows: monitoring multiple reactions; the capillary voltage is preferably 3.5 kV; the ion source temperature is preferably: 125-175 ℃, and more preferably 150 ℃; the desolvation temperature is preferably: 325-375 ℃, and more preferably 350 ℃; the desolventizing gas flow rate is preferably: 800L/h; the taper hole airflow speed is preferably as follows: 50L/h; the sample injection amount is preferably: 5 mu L of the solution; the monitoring ion pair of the SEB tag peptide is preferably as follows: 655.3 → 173.1 (collision voltage is preferably: 28eV), 655.3 → 201.1 (collision voltage is preferably: 25eV) and 655.3 → 552.1 (collision voltage is preferably: 25 eV); the taper hole voltage is preferably: 32V, the quantitative ion pair is preferably: 655.3 → 552.1 (preferred collision voltage: 25eV), the internal standard peptide monitoring ion pair is preferably: 662.4 → 72.2 (the collision voltage is preferably: 40eV), 662.4 → 173.1 (the collision voltage is preferably: 25eV), and 662.4 → 201.1 (the collision voltage is preferably: 25 eV); the taper hole voltage is preferably: 32V, the quantitative ion pair is preferably: 662.4 → 201.1 (impact voltage)Preferably: 25 eV).

In the invention, preferably, the sample to be detected in step (2) is replaced by a series of standard solutions of the tag peptide, the standard curve with the peak area ratio of the tag peptide to the internal standard peptide as the ordinate and the concentration of the tag peptide as the abscissa is obtained by detection and calculation according to the method in step (3), and the series of standard solutions are prepared by adding the internal standard peptide into the standard solution of the tag peptide and then diluting with a formic acid aqueous solution.

In the present invention, the concentration of the standard solution of the tag peptide is preferably 15 to 25nM, and more preferably 20 nM.

In the present invention, the mass concentration of the aqueous formic acid solution is preferably 0.1%.

In the present invention, the concentration of the series of standard solutions of the tag peptide is preferably 0.5nM, 1.0nM, 2.0nM, 4.0nM, 8.0nM, 16.0 nM.

In the invention, the concentration of the internal standard peptide in the series of standard solutions is preferably 1.0-4.0 nM, and more preferably 2.0 nM.

After a standard curve is drawn, substituting the peak area ratio of the tag peptide and the internal standard peptide obtained in the step (3) into the obtained standard curve to obtain the concentration of staphylococcus aureus enterotoxin B, and calculating to obtain the content of SEB in the sample according to the equimolar corresponding relation of the SEB and the SEB tag peptide.

The invention also provides a kit for detecting staphylococcus aureus enterotoxin B in food, the kit comprises staphylococcus aureus enterotoxin B tag peptide and internal standard peptide thereof, the amino acid sequence of the tag peptide is shown as SEQ ID No.1, the internal standard peptide is amino acid in the tag peptide¹³C and¹⁵n isotope labeled peptide segment, wherein the amino acid is leucine.

The following will explain in detail a staphylococcus aureus enterotoxin B-tag peptide and its application provided by the present invention with reference to the examples, but they should not be construed as limiting the scope of the present invention.