CN111337686A - Method for detecting staphylococcus aureus in food by combining immune enrichment with MALDI-TOF MS and application - Google Patents
Method for detecting staphylococcus aureus in food by combining immune enrichment with MALDI-TOF MS and application Download PDFInfo
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- 235000013305 food Nutrition 0.000 title claims abstract description 45
- 238000001514 detection method Methods 0.000 claims abstract description 36
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Abstract
The invention relates to a method for quickly and accurately detecting staphylococcus aureus in food by combining an immune enrichment technology and MALDI-TOF MS (matrix-assisted laser Desorption-time of flight mass spectrometry). Compared with the traditional method, the detection method greatly shortens the screening and culturing time of the target bacteria, and the immune enrichment technology related to the detection method has the characteristics of good specificity, high selectivity and the like, and can specifically enrich the target bacteria from a sample to be detected; meanwhile, the MALDI-TOF MS biological mass spectrometer has the characteristics of high sensitivity, quick and accurate detection result and the like, and the MALDI-TOF MS biological mass spectrometer and the TOF MS biological mass spectrometer are combined for use to detect the staphylococcus aureus in food, have the characteristics of good specificity, high selectivity, strong sensitivity and quick and accurate detection result, and can be used for quickly and accurately identifying the staphylococcus aureus in food samples such as milk and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting staphylococcus aureus in food by combining immune enrichment with MALDI-TOF MS and application thereof.
Background
Staphylococcus aureus (Staphylococcus aureus) is an important bacterium causing food-borne diseases, and it is widely found in nature, and thus has a high probability of causing food contamination. Staphylococcus aureus can secrete more than 20 toxic proteins, causing multiple serious food toxicities. In recent years, food poisoning events caused by food-borne pathogenic bacteria such as staphylococcus aureus are frequent, so that rapid and effective detection and control of staphylococcus aureus are urgently needed.
The enrichment technology of Immunomagnetic beads (IMB) is a technology which utilizes the specific binding of antigen and antibody to separate and enrich target antigen in a short time under the action of a magnetic field. The method has the characteristics of good selectivity and strong specificity, and can quickly separate and enrich target bacteria from a complex system, so that the method is widely applied to separating and enriching specific microorganisms, proteins, trace toxic and harmful substances and the like.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a soft ionization technology which analyzes ions by the fact that the mass-to-charge ratio (M/Z) of the ions is in direct proportion to the flight time of the ions, measures the molecular weight of sample molecules and analyzes mixed biological macromolecules, can inquire and identify proteins in a database through measured protein peptide fingerprint, is the most important identification method in the research of proteomics at present, and therefore can be used for whole-cell protein fingerprint analysis of staphylococcus aureus and further identification of the staphylococcus aureus.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for detecting staphylococcus aureus in food by combining immune enrichment with MALDI-TOF MS and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the method combines an immune enrichment technology with good selectivity and strong specificity with a biological mass spectrometer with high sensitivity and good accuracy, namely MALDI-TOF-MS, and performs specific enrichment on the staphylococcus aureus in the food and whole-cell protein fingerprint analysis, thereby being capable of efficiently, quickly and accurately identifying the staphylococcus aureus in the food.
Moreover, the steps are as follows:
⑴ collecting and processing the sample;
⑵, specific enrichment of bacteria, namely preparing immunomagnetic beads by using anti-staphylococcus aureus antibodies, and specifically enriching staphylococcus aureus to prepare a bacteria-immunomagnetic bead compound;
⑶ selectively enriching the bacteria, namely inoculating the thallus-immunomagnetic bead compound in the step ⑵ to a staphylococcus aureus selectively enriched culture medium for enrichment for 12-15h, and selectively enriching the bacteria obtained by immune enrichment;
⑷ MALDI-TOF MS data acquisition and analysis:
① spotting
Treating the enriched sample by a formic acid/ethanol extraction method, adding 1 mu L of supernatant into a sample application target, parallelly dispensing 4 holes in each sample obtained by each treatment method, covering 1 mu L of saturated matrix CHCA after sample liquid drops are dried, and performing MALDI-TOF MS identification after drying;
② MALDI-TOF MS data acquisition and analysis
Collecting data of the sample by MALDI-TOF MS;
the analysis adopts a linear mode; laser energy: 60-90 Hz; collecting mass-to-charge ratio range m/z: 2000-20000; each sample is bombarded by laser for 100 times; importing the collected data into a database for analysis; the E.coli ATCC 8739 standard was used for calibration before each test within the mass range of the data collected;
⑸ judging result, comparing with database, displaying the result as positive and negative, recording the proportion of positive result in each sample, analyzing data, and displaying the detection result in multiple modes;
wherein, a negative control is set for each detection, and the negative control is immunomagnetic beads which do not react with the sample to be detected.
Moreover, the immune enrichment technology with good selectivity and strong specificity is combined with MALDI-TOF MS biological mass spectrometry with good accuracy and high sensitivity to be used for detecting staphylococcus aureus in food.
Moreover, the conditions for specific enrichment in step ⑵ are:
the detection system for immune enrichment of staphylococcus aureus is 1mL, and specifically comprises the following components:
600 mu L of 250 mu g/mL immunomagnetic beads after magnetic separation and 1mL of food sample liquid to be detected;
the reaction conditions are as follows: the mixture is evenly mixed and reacted for 30min by reversing at room temperature;
in addition, in the step ⑴, the sample is collected and processed according to the method for collecting and preparing the sample in the national standard food microbiology detection of food safety of GB 4789.10 Staphylococcus aureus test.
The specific steps of step ⑵ are as follows:
the treatment per 1mg of magnetic beads was as follows:
① mixing the magnetic beads uniformly, placing 1mg in a 1.5ml centrifuge tube, and washing with 500 μ L2-morpholine ethanesulfonic acid-Tween for 2 times;
the formula of the 2-morpholine ethanesulfonic acid-Tween is as follows: tween-20 with 10mM of 2-morpholine ethanesulfonic acid, pH 6.0 and volume percentage of 0.05 percent;
② adding 200 μ L of newly prepared N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride aqueous solution with concentration of 5mg/mL and N-hydroxysuccinimide aqueous solution with concentration of 5mg/mL respectively, mixing, reacting at 37 deg.C for 30min to activate hydroxyl groups on the surface of the magnetic microsphere, and removing unreacted N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride and N-hydroxysuccinimide by magnetic separation;
③ adding 500 μ L2-morpholine ethanesulfonic acid-Tween suspension magnetic beads, transferring to a new centrifuge tube, washing with 500 μ L2-morpholine ethanesulfonic acid for 3 times, and removing supernatant after magnetic separation;
④ adding 31.2 μ L of anti-Staphylococcus aureus antibody into the magnetic beads with the supernatant removed in step ③, adjusting the system to 500 μ L with 2-morpholine ethanesulfonic acid, and reacting at 37 deg.C for 3 h;
⑤ removing unconjugated antibody by magnetic separation, adding 1ml PBST buffer solution with pH7.4 and mass concentration of 1% BSA, resuspending, reversing at 37 deg.C, mixing uniformly, reacting for 30min, and blocking free radicals on the surface of magnetic beads;
⑥ adding 500 μ L PBST with pH7.4 and 1% BSA by mass concentration for washing 4 times, and finally adding 500 μ L PBST with pH7.4 and 0.02% NaN by mass concentration3And suspending magnetic beads in PBST buffer solution with the mass concentration of 0.5% BSA to obtain a thallus-immunomagnetic bead compound, and storing in a refrigerator at 4 ℃ for later use.
The method is applied to detecting staphylococcus aureus in food.
The invention has the advantages and positive effects that:
1. according to the invention, the immune enrichment technology is combined with MALDI-TOF MS to rapidly detect staphylococcus aureus in food, complex treatment and long-time enrichment culture of target bacteria are not required to be carried out on a sample to be detected in the detection process, and the immune enrichment technology is firstly utilized to carry out specificity enrichment on the target bacteria and then selectively enrich the bacteria, so that the culture time of the bacteria is greatly shortened.
2. The invention utilizes the immune enrichment technology to combine with MALDI-TOF MS to rapidly detect staphylococcus aureus in food, the MALDI-TOF MS biological mass spectrometer used in the detection process has the characteristics of good sensitivity, accurate result and the like, and a related database can automatically analyze data and display a comparison result. And the target bacteria after selective enrichment is identified by using MALDI-TOF MS, the identification process is quick, the identification result is visual and accurate, the specificity is good, the detection limit is low, and the detection can be detected to be 3CFU/mL at least.
3. The method has the advantages of high specificity and accuracy, simple and quick detection process and accurate identification result. According to research results, when the concentration of staphylococcus aureus in 1mL of sample is 3CFU/mL, selective enrichment can be carried out for 9h after immune enrichment, and the staphylococcus aureus can be quickly and accurately identified by MALDI-TOF MS. Compared with the traditional identification method, the method greatly shortens the time required by identification, and the method has the advantages of visual and accurate result interpretation, high accuracy, high sensitivity, good repeatability and low detection limit, and can detect 3CFU/mL at least.
4. Compared with the traditional method, the detection method greatly shortens the screening and culturing time of the target bacteria, and the immune enrichment technology related to the detection method has the characteristics of good specificity, high selectivity and the like, and can specifically enrich the target bacteria from a sample to be detected; meanwhile, the MALDI-TOF MS biological mass spectrometer has the characteristics of high sensitivity, quick and accurate detection result and the like, and the MALDI-TOF MS biological mass spectrometer and the TOF MS biological mass spectrometer are combined for use to detect staphylococcus aureus in food, so that the MALDI-TOF MS biological mass spectrometer has the advantages of good specificity, high selectivity, strong sensitivity, quick and accurate detection result and the like.
Drawings
FIG. 1 is a graph showing the result of analyzing a sample of a negative control in the present invention;
FIG. 2 is a graph showing the result of detecting the specificity of Staphylococcus aureus in a mixed bacteria system according to the present invention; wherein A is a staphylococcus aureus standard strain sample, and B is a salmonella-staphylococcus aureus mixed strain sample;
FIG. 3 is a graph showing the result of the bacterial whole cell protein fingerprint when the bacterial concentration in the milk sample is 61 CFU/mL; wherein, A is a staphylococcus aureus standard strain sample, and B is a sample with the bacterial concentration of 61CFU/mL in a milk sample.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The method combines an immune enrichment technology with good selectivity and strong specificity with a biological mass spectrometer with high sensitivity and good accuracy, namely MALDI-TOF-MS, and performs specific enrichment on the staphylococcus aureus in the food and whole-cell protein fingerprint analysis, thereby being capable of efficiently, quickly and accurately identifying the staphylococcus aureus in the food.
Preferably, the steps are as follows:
⑴ collecting and processing the sample;
⑵, specific enrichment of bacteria, namely preparing immunomagnetic beads by using anti-staphylococcus aureus antibodies, and specifically enriching staphylococcus aureus to prepare a bacteria-immunomagnetic bead compound;
⑶ selectively enriching the bacteria, namely inoculating the thallus-immunomagnetic bead compound in the step ⑵ to a staphylococcus aureus selectively enriched culture medium for enrichment for 12-15h, and selectively enriching the bacteria obtained by immune enrichment;
⑷ MALDI-TOF MS data acquisition and analysis:
① spotting
Treating the enriched sample by a formic acid/ethanol extraction method, adding 1 mu L of supernatant into a sample application target, parallelly dispensing 4 holes in each sample obtained by each treatment method, covering 1 mu L of saturated matrix CHCA after sample liquid drops are dried, and performing MALDI-TOF MS identification after drying;
② MALDI-TOF MS data acquisition and analysis
Collecting data of the sample by MALDI-TOF MS;
the analysis adopts a linear mode; laser energy: 60-90 Hz; collecting mass-to-charge ratio range m/z: 2000-20000; each sample is bombarded by laser for 100 times; importing the collected data into a database for analysis; the E.coli ATCC 8739 standard was used for calibration before each test within the mass range of the data collected;
⑸ judging result, comparing with database, displaying the result as positive and negative, recording the proportion of positive result in each sample, analyzing data, and displaying the detection result in multiple modes;
wherein, a negative control is set for each detection, and the negative control is immunomagnetic beads which do not react with the sample to be detected.
Preferably, the immune enrichment technology with good selectivity and strong specificity is combined with MALDI-TOFMS biological mass spectrometry with good accuracy and high sensitivity to be used for detecting staphylococcus aureus in food.
Preferably, the conditions for specific enrichment in step ⑵ are:
the detection system for immune enrichment of staphylococcus aureus is 1mL, and specifically comprises the following components:
600 mu L of 250 mu g/mL immunomagnetic beads after magnetic separation and 1mL of food sample liquid to be detected;
the reaction conditions are as follows: mixing at room temperature for 30 min;
preferably, the step ⑴ is performed according to the method for collecting and preparing samples in the national standard food microbiology detection of food safety of GB 4789.10 for Staphylococcus aureus test.
Preferably, the specific steps of step ⑵ are as follows:
the treatment per 1mg of magnetic beads was as follows:
① mixing the magnetic beads uniformly, placing 1mg in a 1.5ml centrifuge tube, and washing with 500 μ L2-morpholine ethanesulfonic acid-Tween for 2 times;
the formula of the 2-morpholine ethanesulfonic acid-Tween is as follows: tween-20 with 10mM of 2-morpholine ethanesulfonic acid, pH 6.0 and volume percentage of 0.05 percent;
② adding 200 μ L of newly prepared N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride aqueous solution with concentration of 5mg/mL and N-hydroxysuccinimide aqueous solution with concentration of 5mg/mL respectively, mixing, reacting at 37 deg.C for 30min to activate hydroxyl groups on the surface of the magnetic microsphere, and removing unreacted N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride and N-hydroxysuccinimide by magnetic separation;
③ adding 500 μ L2-morpholine ethanesulfonic acid-Tween suspension magnetic beads, transferring to a new centrifuge tube, washing with 500 μ L2-morpholine ethanesulfonic acid for 3 times, and removing supernatant after magnetic separation;
④ adding 31.2 μ L of anti-Staphylococcus aureus antibody into the magnetic beads with the supernatant removed in step ③, adjusting the system to 500 μ L with 2-morpholine ethanesulfonic acid, and reacting at 37 deg.C for 3 h;
⑤ removing unconjugated antibody by magnetic separation, adding 1ml PBST buffer solution with pH7.4 and mass concentration of 1% BSA, resuspending, reversing at 37 deg.C, mixing uniformly, reacting for 30min, and blocking free radicals on the surface of magnetic beads;
⑥ adding 500 μ L PBST with pH7.4 and 1% BSA by mass concentration for washing 4 times, and finally adding 500 μ L PBST with pH7.4 and 0.02% NaN by mass concentration3And suspending magnetic beads in PBST buffer solution with the mass concentration of 0.5% BSA to obtain a thallus-immunomagnetic bead compound, and storing in a refrigerator at 4 ℃ for later use.
The method is applied to detecting staphylococcus aureus in food.
Specifically, the relevant preparations, examples and assays may be as follows:
example 1: operational steps of immune enrichment technology combined with MALDI-TOFMS method for rapidly detecting staphylococcus aureus in food
⑴ magnetic bead activation and immunomagnetic bead preparation
① mixing the magnetic beads uniformly, placing 1mg into a 1.5ml centrifuge tube, and washing with 500 μ L2-morpholine ethanesulfonic acid-Tween (MEST10mM, pH 6.0, 0.05% Tween-20) for 2 times;
② adding 200 μ L of newly prepared N- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) (5mg/mL) and hydroxysuccinimide (NHS) (5mg/mL) respectively, mixing, reacting at 37 deg.C for 30min to activate hydroxyl group on the surface of magnetic microsphere, and removing unreacted EDC and NHS by magnetic separation;
③ adding 500 μ L MEST suspension magnetic beads, transferring to a new centrifuge tube, washing with 500 μ L MEST for 3 times, magnetically separating, and discarding supernatant;
④ adding 31.2 μ L of anti-Staphylococcus aureus antibody into ③, adjusting system to 500 μ L with 2-morpholine ethanesulfonic acid (MES), and reacting at 37 deg.C by reverse mixing for 3 h;
⑤ removing unconjugated antibody by magnetic separation, adding 1ml PBST buffer (pH 7.4, containing 1% BSA) for resuspension, reversing at 37 deg.C, mixing for 30min, and blocking free radicals on the surface of magnetic beads;
⑥ washing with 500. mu.L PBST for 4 times, and adding 500. mu.L PBST buffer (pH 7.4, containing 0.02% NaN)3And 0.5% BSA) were suspended and stored in a refrigerator at 4 ℃ for further use.
⑵ -mesh bacterium enrichment
Taking 600 mu L of prepared immunomagnetic beads for magnetic separation, reacting with 1mL of sample solution at room temperature for 30min, then carrying out magnetic separation, and discarding the supernatant.
⑶ -mesh bacterium selective enrichment
Inoculating the thallus-immunomagnetic bead compound in the step (2) to a staphylococcus aureus selective enrichment medium for enrichment for 12-15h, and performing selective enrichment on bacteria obtained by immune enrichment;
⑷ MALDI-TOF MS detection
① spotting
Treating the enriched sample in the step (3) by a formic acid/ethanol extraction method, taking 1 mu L of supernatant, adding the supernatant into a sample application target, parallelly dispensing 4 holes in the sample obtained by each treatment method, covering 1 mu L of saturated matrix CHCA after sample liquid drops are dried, and performing MALDI-TOF MS identification after drying;
② MALDI-TOF MS data acquisition and analysis
Collecting data of the sample by MALDI-TOF MS;
the analysis adopts a linear mode; laser energy: 60-90 Hz; collecting mass-to-charge ratio range m/z: 2000-20000; each sample is bombarded by laser for 100 times; importing the collected data into a database for analysis; the E.coli ATCC 8739 standard was used for calibration before each test within the mass range of the data collected;
⑸ judging result, comparing with database, displaying the result as positive and negative, recording the proportion of positive result in each sample, analyzing data, and displaying the detection result in multiple modes;
wherein, a negative control is set for each detection, and the negative control is immunomagnetic beads which do not react with the sample to be detected. As shown in FIG. 1, the immunomagnetic bead sample which has not reacted with the sample to be detected has no detection result.
Example 2: establishment of immune enrichment technology combined with MALDI-TOFMS method for rapidly detecting staphylococcus aureus in food
The invention discloses a method for rapidly detecting staphylococcus aureus in food, which is a method based on immune enrichment technology and MALDI-TOF MS (matrix-assisted laser Desorption-time of flight mass spectrometry), wherein the total volume of an immune enrichment reaction system is 1mL, and the method comprises the following steps: 600 mu L of 250 mu g/mL immunomagnetic beads after magnetic separation and 1mL of food sample liquid to be detected;
the reaction conditions are as follows: mix by inversion at room temperature for 30 min.
Example 3: specificity analysis
⑴ specificity of staphylococcus aureus in detection mixed bacteria system
The staphylococcus aureus detection specificity analysis in the mixed bacteria system for the method for quickly detecting staphylococcus aureus in food respectively prepares staphylococcus aureus and salmonella into bacterial suspensions, then the staphylococcus aureus and the salmonella are uniformly mixed according to the proportion of 1:1 and react with immunomagnetic beads under the optimal reaction condition for magnetic separation, 100 mu L of supernatant is taken to be respectively inoculated in a staphylococcus aureus identification culture medium and a salmonella display culture medium, and the culture medium phenomenon is observed after the culture is carried out at the constant temperature of 37 ℃ for 12-15 h. And identifying and analyzing the thallus-immunomagnetic bead compound by MALDI-TOF MS according to the above experimental method. Wherein, the bacterial colony of the salmonella is light purple in the salmonella chromogenic medium, and the staphylococcus aureus does not develop color; staphylococcus aureus has hemolysis in blood plate culture medium, and salmonella has no hemolysis.
The result is shown in fig. 2, the peak intensities of the fingerprint spectra of the samples obtained by the mixed bacteria reaction system are different, but the distribution of the characteristic peaks is approximately the same, the identification coincidence rate of the fingerprint data of the samples of the mixed bacteria system compared with the database is more than 90%, and the result shows that the detected sample is staphylococcus aureus, namely the immune enrichment combined MALDI-TOF MS has specificity for detecting staphylococcus aureus in the mixed bacteria system.
Example 4: actual sample detection
The actual sample detection of the method for rapidly detecting staphylococcus aureus in food is to add standard bacterial liquid with known concentration of 61CFU/mL into 1mL milk sample, react with IMB under the optimal condition, and then perform MALDI-TOF MS identification and analysis according to the method.
As shown in figure 3, when the concentration of bacteria in the milk sample is 61CFU/mL, the bacteria can be accurately identified by MALDI-TOF MS after selective enrichment for 12-15h after immune enrichment, and the identification coincidence rate of the fingerprint data of the sample compared with the database is more than 85%, namely the immune enrichment and MALDI-TOF MS can be used for detecting staphylococcus aureus in the milk sample.
The invention is a method based on immune enrichment technology and MALDI-TOF MS biological mass spectrometry identification, which has strong specificity, high sensitivity, good repeatability, accurate result and high efficiency.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.
Claims (7)
1. A method for detecting staphylococcus aureus in food by combining immune enrichment with MALDI-TOF MS is characterized by comprising the following steps: the method combines an immune enrichment technology with good selectivity and strong specificity with a biological mass spectrometer with high sensitivity and good accuracy, namely matrix assisted laser desorption ionization time of flight mass spectrometry MALDI-TOF MS, and specifically enriches staphylococcus aureus in food and analyzes whole-cell protein fingerprint spectrum, so that the staphylococcus aureus in food can be identified efficiently, quickly and accurately.
2. The method for detecting staphylococcus aureus in food by combining immune enrichment with MALDI-TOF MS according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
⑴ collecting and processing the sample;
⑵, specific enrichment of bacteria, namely preparing immunomagnetic beads by using anti-staphylococcus aureus antibodies, and specifically enriching staphylococcus aureus to prepare a bacteria-immunomagnetic bead compound;
⑶ selectively enriching the bacteria, namely inoculating the thallus-immunomagnetic bead compound in the step ⑵ to a staphylococcus aureus selectively enriched culture medium for enrichment for 12-15h, and selectively enriching the bacteria obtained by immune enrichment;
⑷ MALDI-TOF MS data acquisition and analysis:
① spotting
Treating the enriched sample by a formic acid/ethanol extraction method, adding 1 mu L of supernatant into a sample application target, parallelly dispensing 4 holes in each sample obtained by each treatment method, covering 1 mu L of saturated matrix CHCA after sample liquid drops are dried, and performing MALDI-TOF MS identification after drying;
② MALDI-TOF MS data acquisition and analysis
Collecting data of the sample by MALDI-TOF MS;
the analysis adopts a linear mode; laser energy: 60-90 Hz; collecting mass-to-charge ratio range m/z: 2000-20000; each sample is bombarded by laser for 100 times; importing the collected data into a database for analysis; the E.coli ATCC 8739 standard was used for calibration before each test within the mass range of the data collected;
⑸ judging result, comparing with database, displaying the result as positive and negative, recording the proportion of positive result in each sample, analyzing data, and displaying the detection result in multiple modes;
wherein, a negative control is set for each detection, and the negative control is immunomagnetic beads which do not react with the sample to be detected.
3. The method for detecting staphylococcus aureus in food by combining immune enrichment with MALDI-TOF MS according to claim 2, wherein the method comprises the following steps: the immune enrichment technology with good selectivity and strong specificity is combined with MALDI-TOFMS biological mass spectrum with good accuracy and high sensitivity to detect staphylococcus aureus in food.
4. The method for detecting staphylococcus aureus in food by combining immune enrichment with MALDI-TOF MS according to claim 2, wherein the conditions for specific enrichment in step ⑵ are as follows:
the detection system for immune enrichment of staphylococcus aureus is 1mL, and specifically comprises the following components:
600 mu L of 250 mu g/mL immunomagnetic beads after magnetic separation and 1mL of food sample liquid to be detected;
the reaction conditions are as follows: the reaction was mixed by inversion at room temperature for 30 min.
5. The method for detecting staphylococcus aureus in food by combining immune enrichment with MALDI-TOF MS as claimed in claim 2, wherein the step ⑴ is carried out by collecting and processing samples according to the method for collecting and preparing samples in GB 4789.10 national food safety Standard food microbiology detection staphylococcus aureus test.
6. The method for detecting staphylococcus aureus in food by combining immune enrichment with MALDI-TOF MS according to claim 2, wherein the specific steps of the step ⑵ are as follows:
the treatment per 1mg of magnetic beads was as follows:
① mixing the magnetic beads uniformly, placing 1mg in a 1.5ml centrifuge tube, and washing with 500 μ L2-morpholine ethanesulfonic acid-Tween for 2 times;
the formula of the 2-morpholine ethanesulfonic acid-Tween is as follows: tween-20 with 10mM of 2-morpholine ethanesulfonic acid, pH 6.0 and volume percentage of 0.05 percent;
② adding 200 μ L of newly prepared N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride aqueous solution with concentration of 5mg/mL and N-hydroxysuccinimide aqueous solution with concentration of 5mg/mL respectively, mixing, reacting at 37 deg.C for 30min to activate hydroxyl groups on the surface of the magnetic microsphere, and removing unreacted N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride and N-hydroxysuccinimide by magnetic separation;
③ adding 500 μ L2-morpholine ethanesulfonic acid-Tween suspension magnetic beads, transferring to a new centrifuge tube, washing with 500 μ L2-morpholine ethanesulfonic acid for 3 times, and removing supernatant after magnetic separation;
④ adding 31.2 μ L of anti-Staphylococcus aureus antibody into the magnetic beads with the supernatant removed in step ③, adjusting the system to 500 μ L with 2-morpholine ethanesulfonic acid, and reacting at 37 deg.C for 3 h;
⑤ removing unconjugated antibody by magnetic separation, adding 1ml PBST buffer solution with pH7.4 and mass concentration of 1% BSA, resuspending, reversing at 37 deg.C, mixing uniformly, reacting for 30min, and blocking free radicals on the surface of magnetic beads;
⑥ adding 500 μ L PBST with pH7.4 and 1% BSA by mass concentration for washing 4 times, and finally adding 500 μ L PBST with pH7.4 and 0.02% NaN by mass concentration3And suspending magnetic beads in PBST buffer solution with the mass concentration of 0.5% BSA to obtain a thallus-immunomagnetic bead compound, and storing in a refrigerator at 4 ℃ for later use.
7. Use of a method according to any one of claims 1 to 6 for the detection of staphylococcus aureus in a food product.
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