CN111366726A - Method for detecting salmonella in food by combining immune enrichment with MALDI-TOF MS and application - Google Patents
Method for detecting salmonella in food by combining immune enrichment with MALDI-TOF MS and application Download PDFInfo
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Abstract
The invention relates to a method for quickly and accurately detecting salmonella 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, the MALDI-TOF MS biological mass spectrometer and the TOF MS biological mass spectrometer are combined for use, the detection method is used for detecting the salmonella in the food, and has the advantages of good specificity, high selectivity, strong sensitivity and quick and accurate detection result, and when the bacteria concentration in a sample is 3CFU/mL, the MALDI-TOF MS can quickly and accurately identify the bacteria concentration in the sample after enrichment culture for 9 hours.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting salmonella in food by combining immune enrichment with MALDI-TOF MS and application thereof.
Background
Salmonella (Salmonella) is one of the pathogenic bacteria causing food-borne diseases in nature and frequently causes bacterial food poisoning worldwide. According to the statistics of the world health organization in 2018, about 1/10 people suffer from food-borne diseases every year, about 30000 people lose lives, and salmonella is one of pathogenic factors; meanwhile, according to the monitoring of food-borne diseases at home and abroad in recent years, the salmonella is still one of the main microbial pathogenic bacteria of the food-borne diseases, wherein the pathogenic mortality rate of the salmonella in 2017 of European Union countries is 33.3%, and the pathogenic mortality rate of the salmonella in 2015 is 0.72%. Therefore, there is an urgent need for effective monitoring and accurate identification of salmonella in food products.
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 biomacromolecules, 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 salmonella and further identification of the salmonella.
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 salmonella 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 carries out specific enrichment on the salmonella in the food and whole-cell protein fingerprint analysis, thereby being capable of efficiently, quickly and accurately identifying the salmonella 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-salmonella antibodies, and specifically enriching salmonella to prepare a thallus-immunomagnetic bead compound;
⑶ selectively enriching the bacteria, namely inoculating the thallus-immunomagnetic bead compound in the step ⑵ to a salmonella selective enrichment 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 no result as negative, recording the proportion of positive result in each sample, analyzing data, and displaying the detection result in multiple ways;
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 combination of the immune enrichment technology with good selectivity and strong specificity and the MALDI-TOF MS biological mass spectrum with good accuracy and high sensitivity is used for detecting the salmonella in the food.
Moreover, the conditions for specific enrichment in step ⑵ are:
the detection system for the immunity enrichment salmonella is 1mL, and specifically 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: room temperature for 30 min;
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 inspection for salmonella of GB 4789.4.
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 anti-salmonella antibody into the magnetic beads with 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.
Use of a method as described above for the detection of salmonella in a food product.
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 the salmonella in the food, the detection process does not need to carry out complex treatment and long-time enrichment culture of the target bacteria on a sample to be detected, but the immune enrichment technology is firstly utilized to carry out specific enrichment on the target bacteria and then the selective enrichment culture is carried out, 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 the salmonella in the 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 the 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 salmonella in a 1mL sample is 3CFU/mL, the selective enrichment can be carried out for 9h after immune enrichment, and the salmonella 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 the salmonella in the food, so that the MALDI-TOF MS biological mass spectrometer has the advantages of good specificity, high selectivity, strong sensitivity and quick and accurate detection result.
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 diagram showing the results of detecting the specificity of Salmonella in a mixed bacteria system according to the present invention; wherein, A is a standard strain sample, B is a salmonella-golden grape mixed strain sample, and C is a salmonella-large intestine mixed strain sample;
FIG. 3 is a graph showing the results of detecting the specificity of different Salmonella bacteria in the present invention; wherein, A is a capture rate result graph, B is a sample whole cell protein fingerprint graph, wherein S.T is Salmonella typhimurium (Salmonella typhimurium), Salmonella enterica (Salmonella enterica) and S.H Salmonella hendelbergii (Salmonella Heidelberg);
FIG. 4 is a graph showing the result of the fingerprint of the whole cell protein of bacteria in different food samples according to the present invention;
FIG. 5 is a graph showing the results of analysis of a sample having a bacteria concentration of 3CFU/mL in a food sample according to the present invention; wherein A is a sample for increasing the bacteria for 6h, B is a sample for increasing the bacteria for 9h, C is a sample for increasing the bacteria for 12h, and D is a sample for increasing the bacteria for 15 h;
FIG. 6 is a graph showing the results of analysis of samples having a microbial concentration of 48CFU/mL in food samples according to the present invention;
FIG. 7 shows that the concentration of bacteria in the food sample of the present invention is 1 × 102CFU/mL sample analysis result graph;
FIG. 8 shows the concentration of bacteria in the food sample of the present invention is 1 × 103CFU/mL sample analysis results.
Table 1 is a graph of the results of the capture rate measurements for different food samples according to the present invention.
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 carries out specific enrichment on the salmonella in the food and whole-cell protein fingerprint analysis, thereby being capable of efficiently, quickly and accurately identifying the salmonella 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-salmonella antibodies, and specifically enriching salmonella to prepare a thallus-immunomagnetic bead compound;
⑶ selectively enriching the bacteria, namely inoculating the thallus-immunomagnetic bead compound in the step ⑵ to a salmonella selective enrichment 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 no result as negative, recording the proportion of positive result in each sample, analyzing data, and displaying the detection result in multiple ways;
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 combination of the immune enrichment technology with good selectivity and strong specificity and the MALDI-TOFMS biological mass spectrum with good accuracy and high sensitivity is used for detecting the salmonella in the food.
Preferably, the conditions for specific enrichment in step ⑵ are:
the detection system for the immunity enrichment salmonella is 1mL, and specifically 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: room temperature for 30 min;
preferably, the step ⑴ is performed according to the method for collecting and preparing samples in the national food safety standard food microbiology detection salmonella test of GB 4789.4.
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 anti-salmonella antibody into the magnetic beads with 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 mu.L of PB 7.4 pH containing 1% BSA by massST washing 4 times, and adding 500 μ L of NaN with pH7.4 and mass concentration of 0.02%3And 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.
Use of a method as described above for the detection of salmonella in a food product.
Specifically, the relevant preparations, examples and assays may be as follows:
example 1: operational steps of immune enrichment technology combined with MALDI-TOF MS method for rapidly detecting salmonella 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 anti-salmonella antibody into ③, adjusting system to 500 μ L with 2-morpholine ethanesulfonic acid (MES), placing at 37 deg.C, reversing, mixing and reacting 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 salmonella selective enrichment culture 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 no result as negative, recording the proportion of positive result in each sample, analyzing data, and displaying the detection result in multiple ways;
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-TOF MS method for rapidly detecting salmonella in food
The invention discloses a method for rapidly detecting salmonella 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: room temperature for 30 min.
Example 3: specificity analysis
⑴ detection of specificity of Salmonella in mixed bacteria system
The salmonella detection specificity analysis in the mixed bacteria system of the method for quickly detecting salmonella in food is to prepare bacterial suspensions of staphylococcus aureus, escherichia coli and salmonella according to the experimental method, then uniformly mix the staphylococcus aureus, the escherichia coli and the salmonella respectively according to the proportion of 1:1, react with immunomagnetic beads under the optimal reaction condition, carry out magnetic separation, take 100 mu L of supernatant to coat a flat plate, calculate IMB capture rate according to the formula 1, take 100 mu L of supernatant to respectively inoculate a salmonella chromogenic medium and a staphylococcus aureus identification medium, and observe the medium phenomenon after culturing at the constant temperature of 37 ℃ for 12-15 h. And identifying and analyzing the thallus-immunomagnetic bead compound by MALDI-TOFMS according to the above experimental method. Wherein, the bacterial colony of the salmonella is light purple in the salmonella chromogenic medium, and the escherichia coli is blue; staphylococcus aureus is hemolyzed in blood plate culture medium, and the rest bacteria are not hemolyzed.
C0Bacterial colony count of original bacteria liquid (CFU/mL);
C1colony count of supernatant after immunomagnetic bead capture (CFU/mL)
The result is shown in fig. 2, the peak intensities of the fingerprint spectra of the samples obtained by the mixed bacteria reaction systems with different proportions are different, but the distribution of the characteristic peaks is approximately the same, and the identification coincidence rate of the fingerprint data of the samples of the mixed bacteria reaction systems with different proportions and the comparison of the database is more than 81%, and the result shows that the detection sample is salmonella, namely the immune enrichment combined MALDI-TOFMS has specificity for detecting salmonella in the mixed bacteria reaction system.
⑵ specificity of detection of different Salmonella
The different salmonella detection specificities of the method for quickly detecting the salmonella in the food are that 1mL of the salmonella is 1 × 102～1×104CFU/mL Salmonella enteritidis(Salmonella enterica, S.E), Salmonella typhimurium (S.T) and Salmonella haidebergensis (Salmonella Heidelberg, S.H) were reacted with immunomagnetic beads respectively under optimum reaction conditions, and IMB capture rate was calculated according to the method described in (1), after overnight culture of the cell-immunomagnetic bead complex, 1mL of the bacterial solution was magnetically separated, treated with formic acid-ethanol method, and then identified and analyzed by MALDI-TOF MS.
The result is shown in fig. 3(a), the capture rate of the IMB to different salmonella is greater than 80%, and as can be seen from fig. 3(B), the peak intensities of the fingerprint spectra of 3 different salmonella are different, the distribution of the characteristic peaks is also different, but the obtained spectrum data can be compared with the database to obtain correct identification results, the match rate with the database is greater than 80%, that is, the detection sample is salmonella. Therefore, the identification method in the research can also be used for specific enrichment and identification of S.T, S.E strain and S.H strain.
Example 4: actual sample detection
The actual sample detection of the method for rapidly detecting the salmonella in the food is to dilute 10 times of milk, yolk and egg white and then respectively add known concentrations of 1 × 102、1×103After the CFU/mL standard bacterial solution was reacted with IMB under optimum conditions, the capture rate was calculated and identified and analyzed by MALDI-TOF MS as described in example 3.
As shown in Table 1, bacterial liquids with known concentrations are respectively added into the milk, egg white and yolk sample treatment liquids, and the recovery rate is 88-100 percent through a standard recovery experiment and basically accords with the actual addition amount; as shown in FIG. 4, the peak intensities of the bacterial fingerprints of different actual samples are different, but the distribution of characteristic peaks is basically the same, and the comparison coincidence rate of the fingerprint data and the database is respectively more than 85%, namely, the detection sample is salmonella, which indicates that the detection method can be used for detecting milk, egg white and egg yolk samples.
TABLE 1 Capture rates of different actual samples
Example 5: detection limit and bacterium increasing time exploration
The detection limit and enrichment time of the method for rapidly detecting salmonella in food are researched by adjusting the concentration of salmonella to be lower than 10CFU/mL by using sterile normal saline, inoculating the salmonella into a milk sample treatment solution, carrying out magnetic separation after the salmonella reacts with immunomagnetic beads under the optimal condition, inoculating a bacterium-immunomagnetic bead compound into a salmonella selective enrichment culture medium, carrying out shake culture at 37 ℃, and setting 4 experimental groups with different enrichment times, wherein the experimental groups are respectively 6, 9, 12 and 15 hours. The bacterial liquids with different enrichment times were identified and analyzed by MALDI-TOF MS according to the method described in step (4) of example 1.
The results are shown in FIGS. 5 to 8. As can be seen from FIG. 5, when the concentration of bacteria in the milk sample is 3CFU/mL, the selective enrichment of 9h after the immune enrichment can be accurately identified by MALDI-TOF MS, as shown in FIGS. 6-8, the selective enrichment of 9h of the target bacteria after the immune enrichment can be accurately identified by MALDI-TOF MS with the increase of the concentration of the bacteria, and compared with the national standard food microbiology detection of Salmonella detection for food safety of GB 4789.4, the time consumption of the Salmonella detection method of the present invention is shortened by about 35 h.
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 salmonella 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 mass spectrometer-matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with high sensitivity and good accuracy, specifically enriches the salmonella in the food and analyzes the whole-cell protein fingerprint spectrum, and can efficiently, quickly and accurately identify the salmonella in the food.
2. The method for detecting salmonella in food by combining immune enrichment and MALDI-TOF MS according to claim 1, wherein the immune enrichment 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-salmonella antibodies, and specifically enriching salmonella to prepare a thallus-immunomagnetic bead compound;
⑶ selectively enriching the bacteria, namely inoculating the thallus-immunomagnetic bead compound in the step ⑵ to a salmonella selective enrichment 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 no result as negative, recording the proportion of positive result in each sample, analyzing data, and displaying the detection result in multiple ways;
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 salmonella in food by combining immune enrichment and MALDI-TOF MS according to claim 2, wherein the immune enrichment comprises the following steps: the combination of the immune enrichment technology with good selectivity and strong specificity and the MALDI-TOF MS biological mass spectrum with good accuracy and high sensitivity is used for detecting the salmonella in the food.
4. The method for detecting salmonella in food by combining immune enrichment and MALDI-TOF MS as claimed in claim 2, wherein the conditions for specific enrichment in step ⑵ are as follows:
the detection system for the immunity enrichment salmonella is 1mL, and specifically 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: room temperature for 30 min.
5. The method for detecting the salmonella in the food by combining the immune enrichment with the MALDI-TOF MS as claimed in claim 2, wherein the step ⑴ is to collect and process the sample according to the collection and preparation method of the sample in GB 4789.4 food safety national standard food microbiology detection salmonella test.
6. The method for detecting salmonella in food by combining immune enrichment with MALDI-TOF MS as claimed in 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 anti-salmonella antibody into the magnetic beads with 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 detecting salmonella in a food product.
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