阅读说明：本技术 一种金黄色葡萄球菌肠毒素a标签肽及其应用 (Staphylococcus aureus enterotoxin A tag peptide and application thereof ) 是由 张京顺 黄百芬 蔡增轩 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A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A标签肽及其应用。本发明提供的金黄色葡萄球菌肠毒素A标签肽的氨基酸序列如SEQ ID No.1所示,并设计合成了相对应的内标肽,所述内标肽是将所述标签肽中的氨基酸用<Sup>13</Sup>C和<Sup>15</Sup>N同位素标记后的肽段,所述氨基酸为所述标签肽的氨基酸序列中的第3和第8位置上的亮氨酸,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A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A。(The invention belongs to the technical field of staphylococcus aureus enterotoxin A detection, and provides a staphylococcus aureus enterotoxin A tag peptide and application thereof. The amino acid sequence of the staphylococcus aureus enterotoxin A tag peptide is shown in SEQ ID No.1, and corresponding internal standard peptide is designed and synthesized, wherein the internal standard peptide is prepared by using amino acid in the tag peptide 13 C and 15 the N isotope labeled peptide segment, the amino acid is leucine at the 3 rd and 8 th positions in the amino acid sequence of the label peptide, and the content of the staphylococcus aureus enterotoxin A in the food is determined by adopting a high performance liquid chromatography-mass spectrometry combined technology for analysis and determination. The method has good linearity, sensitivity and recovery rate, and can be used for directly qualitatively and quantitatively determining Staphylococcus aureus enterotoxin A in food.)

1. A staphylococcus aureus enterotoxin A 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 a tag peptide of claim 1, wherein the internal standard peptide is obtained by substituting amino acids in the tag peptide with amino acids¹³C and¹⁵n isotope labeled peptide segment, wherein the amino acid is leucine at the 3 rd and 8 th positions in the amino acid sequence of the tag peptide.

3. The use of a staphylococcal enterotoxin a tag peptide of claim 1 to detect staphylococcal enterotoxin a in milk and dairy products.

4. The method for detecting staphylococcus aureus enterotoxin a in milk and dairy products by using the staphylococcus aureus enterotoxin a tag peptide of 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 A 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 A, and calculating to obtain the content of the staphylococcus aureus enterotoxin A in the sample.

5. The method for detecting staphylococcus aureus enterotoxin A in milk and dairy products according to 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 staphylococcus aureus enterotoxin A in milk and dairy products according to claim 4, wherein the precipitation is carried out by adding trichloroacetic acid solution into supernatant 2 according to a 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 A in milk and dairy products 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 A in milk and dairy products according to 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 A in milk and dairy products as claimed in claim 4, wherein the detection conditions of the high performance liquid chromatography are Acquisty UPLC BEH Peptide 300C18 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 retention, 5.8min-6.0min mass spectrum linear decrease to 5% mobile phase B, 6.0min-8.0min, 5% mobile phase ESI retention, 0.5 min flow rate retention of 0% mobile phase B, electrospray flow rate of 3 mL/mist 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.

10. A kit for detecting staphylococcus aureus enterotoxin A in milk and dairy products is characterized by comprising staphylococcus aureus enterotoxin A tag peptide and internal standard peptide thereof, wherein the amino acid sequence of the tag peptide is shown in SEQ ID No.1, and the internal standard peptide is obtained by using leucine at the 3 rd and 8 th positions in the amino acid sequence of the tag peptide¹³C and¹⁵n isotope labeled peptide segment.

Technical Field

The invention relates to the technical field of staphylococcus aureus enterotoxin A detection, in particular to a staphylococcus aureus enterotoxin A tag peptide and application thereof.

Background

Staphylococcus aureus enterotoxin a (sea) is one of the most common toxins associated with food poisoning. SEA-contaminated milk and its by-products can cause extensive gastroenteritis outbreaks, and SEA is often present in these samples, although few or no Staphylococcus aureus pathogens are detected in some cases of food poisoning. SEA is an exotoxin produced by staphylococcus aureus and is stable under high temperature conditions. Because of the varying sensitivity of individuals to SEA, the toxic dose of SEA to humans remains unknown. But for public health it is essential to monitor SEA residues in milk and dairy products in terms of food safety.

The SEA detection method requires higher sensitivity and specificity due to the different complexity of the food matrix. With the use of antibodies specific for staphylococcal enterotoxin, immunoassay methods such as enzyme linked immunosorbent assay (ELISA) and reverse passive latex agglutination assay (RPLA) have been widely used. However, ELISA is qualitative, whereas 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. Therefore, in order to obtain good reproducibility between laboratories, a validated quantitative method is required. In food poisoning associated with SEA, other bacterial toxins may also be suspected causative agents, as they may also cause acute emesis or severe illness. Since these bacterial toxins are resistant to extreme pH conditions, even pasteurization, it is necessary to establish an efficient method for the precise identification and accurate quantification of SEA.

Disclosure of Invention

The invention aims to provide a staphylococcus aureus enterotoxin A tag peptide and application thereof, and the tag peptide can be used for confirming and detecting the pollution level of staphylococcus aureus enterotoxin A in milk and dairy products.

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

the invention provides a staphylococcus aureus enterotoxin A 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 A tag peptide, wherein the internal standard peptide uses amino acid in the tag peptide¹³C and¹⁵n isotope labeled peptide segment, wherein the amino acids are leucine at the 3 rd and 8 th positions in the amino acid sequence of the tag peptide。

The invention also provides application of the staphylococcus aureus enterotoxin A tag peptide in detecting staphylococcus aureus enterotoxin A in milk and dairy products.

The invention also provides a method for detecting staphylococcus aureus enterotoxin A in milk and dairy products by using the staphylococcus aureus enterotoxin A 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 A 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 A, and calculating to obtain the content of the staphylococcus aureus enterotoxin A 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 Peptide 300C18 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 A in milk and dairy products, which comprises staphylococcus aureus enterotoxin A tag peptide and internal standard peptide thereof, wherein the amino acid sequence of the tag peptide is shown as SEQID No.1The internal standard peptide is obtained by using leucine at the 3 rd and 8 th positions in the amino acid sequence of the tag peptide¹³C and¹⁵n isotope labeled peptide segment.

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

(1) the stable isotope labeled internal standard peptide of the SEA labeled peptide is designed and synthesized by screening the SEA labeled peptide, and the quantitative detection of the SEA is realized by utilizing the high performance liquid chromatography-mass spectrometry combined analysis technology.

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

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

Detailed Description

The invention provides a staphylococcus aureus enterotoxin A tag peptide, and 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. According to the invention, SELQGAALGNLK (SEQ ID No.1) is finally selected as the tag peptide of SEA after factors such as specificity, enzymolysis stability, mass spectrum response signal intensity and recovery rate of candidate polypeptide are comprehensively considered and verified. And the amino acid sequence of the tag peptide is taken as a template to introduce stable isotope labeled Fmoc-Leu-OH-¹³C₆,¹⁵N amino acid, and the internal standard peptide SE (L-¹³C₆,¹⁵N)QGAA(L-¹³C₆,¹⁵N) GNLK as an 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 A in the food.

The invention provides a staphylococcus aureus enterotoxin A 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 SEA, and has good specificity and stability.

The invention also provides an internal standard peptide of the staphylococcus aureus enterotoxin A tag peptide, wherein the internal standard peptide uses amino acid in the tag peptide¹³C and¹⁵n isotope labeled peptide segment, wherein the amino acid is leucine at the 3 rd and 8 th positions in the amino acid sequence of the tag peptide.

The invention also provides application of the staphylococcus aureus enterotoxin A tag peptide in detecting staphylococcus aureus enterotoxin A in milk and dairy products.

The invention also provides a method for detecting staphylococcus aureus enterotoxin A in milk and dairy products by using the staphylococcus aureus enterotoxin A 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 A 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 A, and calculating to obtain the content of the staphylococcus aureus enterotoxin A 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 RaPigestSF 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 μ g/mL, and is further preferably 100 μ 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 Peptide 300C18 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 staphylococcus aureus enterotoxin A-labeled peptide is preferably as follows: 601.4 → 189.1 (collision voltage is preferably: 30eV), 601.4 → 217.1 (collision voltage is preferably: 25eV) and 601.4 → 242.2 (collision voltage is preferably:20 eV); the taper hole voltage is preferably: 30V; the quantitative ion pair is preferably: 601.4 → 217.1 (collision voltage is preferably: 25 eV); the monitoring ion pair of the internal standard peptide is preferably as follows: 608.2 → 189.1 (collision voltage is preferably: 30eV), 608.2 → 217.1 (collision voltage is preferably: 25eV) and 608.2 → 750.3 (collision voltage is preferably: 20 eV); the taper hole voltage is preferably: 32V; the quantitative ion pair is preferably: 608.2 → 217.1 (collision voltage is 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, detection and calculation are performed according to the method in step (3), a standard curve with the peak area ratio of the tag peptide to the internal standard peptide as a ordinate and the concentration of the tag peptide as an abscissa is obtained, and 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;

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 formic acid is preferably present at a mass concentration of 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 1-4 nM, and more preferably 2 nM.

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

The invention also provides a kit for detecting staphylococcus aureus enterotoxin A in milk and dairy products, the kit comprises staphylococcus aureus enterotoxin A tag peptide and internal standard peptide thereof, the amino acid sequence of the tag peptide is shown as SEQID No.1, the internal standard peptide is the No. 3 and the No. 3 in the amino acid sequence of the tag peptideFor leucine in position 8¹³C and¹⁵n isotope labeled peptide segment.

The present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.