Identification method and kit for characteristic fingerprint marker of biotoxin infected cell

文档序号：466075 发布日期：2021-12-31 浏览：2次中文

阅读说明：本技术 生物毒素侵染细胞的特征指纹标志物的鉴定方法及试剂盒 (Identification method and kit for characteristic fingerprint marker of biotoxin infected cell ) 是由张峰陈凤明国伟姚桂红于 2021-09-23 设计创作，主要内容包括：本发明公开了生物毒素侵染细胞的特征指纹标志物的鉴定试剂盒及方法,其中,确定待测物是否被生物毒素侵染的方法包括：对所述待测物进行提取处理,以便得到提取液；对所述提取液进行液相色谱-质谱检测,筛选出脂质代谢标志物,并依据该标记物确定所述待测物中是否含有生物毒素。该方法通过液相色谱-质谱检测,基于脂质代谢标志物,能准确的判断待测物是否被生物毒素侵染,并且,操作简单,灵敏度和准确率高。(The invention discloses an identification kit and a method for a characteristic fingerprint marker of biotoxin infected cells, wherein the method for determining whether an object to be detected is infected by biotoxins comprises the following steps: extracting the object to be detected to obtain an extracting solution; and performing liquid chromatography-mass spectrometry detection on the extracting solution, screening out a lipid metabolism marker, and determining whether the substance to be detected contains biotoxin or not according to the marker. The method can accurately judge whether the object to be detected is infected by biotoxin or not through liquid chromatography-mass spectrometry detection based on the lipid metabolism markers, and has the advantages of simple operation and high sensitivity and accuracy.)

1. A method of determining whether a test agent is infected with a biological toxin, comprising:

extracting the object to be detected to obtain an extracting solution;

and performing liquid chromatography-mass spectrometry detection on the extracting solution, screening out a lipid metabolism marker, and determining whether the substance to be detected contains biotoxin or not according to the marker.

2. The method according to claim 1, wherein the biological toxin is at least one selected from the group consisting of mycotoxins, plant toxins, animal toxins and microbial toxins, preferably mycotoxins, more preferably the mycotoxins are at least one of aflatoxins B1, fumonisins B1, ochratoxins a and vomitoxin.

3. The method according to claim 2, wherein the lipid metabolism marker of fumonisin B1 is selected from at least one of the following:

(1) fumonisin B1 marker 1: 512.5028 m/z;

(2) fumonisin B1 marker 2: 540.5343 m/z;

(3) fumonisin B1 marker 3: 813.6830 m/z;

(4) fumonisin B1 marker 4: 722.5258 m/z;

(5) fumonisin B1 marker 5: 738.5056 m/z;

(6) fumonisin B1 marker 6: 787.6675m/z of the raw materials,

optionally, the lipid metabolism marker of aflatoxin B1 is selected from at least one of:

(1) aflatoxin B1 marker 1: triglyceride TG (18:1(9Z)/20:1(11Z)/22:1 (13Z));

(2) aflatoxin B1 marker 2: diglyceride DG (18:2(9Z,12Z)/20:0/0: 0);

(3) aflatoxin B1 marker 3: triglyceride TG (18:1(9Z)/20:1(11Z)/20:1 (11Z));

(4) aflatoxin B1 marker 4: triglyceride TG (14:0/16:0/18: 0);

(5) aflatoxin B1 marker 5: triglyceride TG (18:0/18:1(9Z)/20:1(11Z)),

optionally, the marker of ochratoxin a is selected from at least one of:

(1) phosphatidic acid PA (13:0/20:1 (11Z));

(2) phosphatidylserine PS (O-20:0/19:1(9Z)),

optionally, the lipid metabolism marker of the vomitoxin is selected from at least one of:

(1) phosphatidic acid PA (13:0/20:1 (11Z));

(2) lecithin PC (10:0/21: 0);

(3) penthorum chinense pursh B;

(4) lecithin PC (16:0/22:5(4Z,7Z,10Z,13Z, 16Z));

(5) diglyceride DG (O-16:0/18:1 (9Z)).

4. The method of claim 1, wherein the liquid chromatography-mass spectrometry is ultra performance liquid chromatography-time of flight-ion mobility spectrometry.

5. The method according to claim 4, wherein the column of the liquid chromatography-mass spectrometry is an ACQUITY CSH C18 column

6. The method according to claim 5, wherein the chromatographic mobile phase of the liquid chromatography-mass spectrometry is: mobile phase a aqueous acetonitrile solution containing 10mM ammonium formate and 0.1% formic acid, preferably in a volume ratio of acetonitrile to water of 6: 4;

mobile phase B: a mixed solution of isopropanol and acetonitrile containing 10mM ammonium formate and 0.1% formic acid, wherein the volume ratio of isopropanol to acetonitrile is 9:1,

optionally, the elution of the chromatogram of the liquid chromatography-mass spectrometry is a gradient elution, preferably, the gradient elution conditions are 0-2min, mobile phase B: 40% -43%; 2.0-2.1min, mobile phase B: 43% -50%; 2.1-12.0min, 50% -54%; 12.0-12.1min, 54% -70%; 12.1-18.0min, 70% -99%; 99-40% for 18.0-18.1 min; 18.1-20.0min, 40%.

7. The method of claim 5, wherein the chromatographic conditions of the liquid chromatography-mass spectrometry are:

temperature of the chromatographic column: 55 ℃;

the room temperature of the sample is 10 ℃;

the flow rate was 300. mu.L/min.

8. The method of claim 4, wherein the mass spectrometric conditions of the liquid chromatography-mass spectrometry are:

the collection mode is HDMSE;

the ionization mode is ESI +/ESI-; the voltage of the taper hole is 30V;

the desolvation temperature is 550 ℃;

the desolventizing gas flow rate is 900L/hr;

the collection range is 50-1200 m/z.

9. The method according to claim 1, wherein the analyte has a molecular weight of not more than 1200Da,

optionally, the test agent is derived from a mammal, preferably from the liver and kidney of a mammal.

10. A kit for determining whether a test substance is infected with a biotoxin, comprising the reagents, standards, auxiliary materials, or a combination of at least one of them used in the method for detecting a fingerprint marker characteristic of biotoxin-infected cells of claims 1-9.

11. The kit of claim 10, wherein the standards comprise lipid metabolite standards and mycotoxin standards, wherein the lipid metabolite standards are selected from at least one of N-ceramide-D-erythro-sphingosylphosphorylcholine, C14-dihydroceramide, and C16-dihydroceramide, and C22 sphingomyelin; the mycotoxin standard is selected from at least one of aflatoxin B1, fumonisin B1, vomitoxin and ochratoxin.

Technical Field

The invention relates to the field of analytical chemistry, in particular to an identification kit and a method for a characteristic fingerprint marker of biotoxin infected cells, and more particularly relates to a method for determining whether an object to be detected is infected by biotoxins and a kit for determining whether the object to be detected is infected by the biotoxins.

Background

The detection and identification of the variety and content change of the cell metabolites have important significance for understanding the cytotoxicity mechanism of the biotoxin, and are important references and bases for establishing the toxin limit standard. However, there is no method for qualitatively detecting cell metabolites.

Thus, methods for determining whether a test substance is infected with a biotoxin are under further investigation.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a method for determining whether an analyte is infected with a biotoxin, which is simple to operate and has high sensitivity and accuracy.

Thus, according to one aspect of the invention, there is provided a method of determining whether a test agent is infected with a biological toxin. According to an embodiment of the invention, the method comprises: extracting the object to be detected to obtain an extracting solution; and performing liquid chromatography-mass spectrometry detection on the extracting solution, and determining whether the substance to be detected contains biotoxin or not based on the lipid metabolism marker.

According to the method for determining whether the object to be detected is infected by the biotoxin, whether the object to be detected is infected by the biotoxin can be accurately determined through liquid chromatography-mass spectrometry detection based on the lipid metabolism markers, and the method is simple to operate and high in sensitivity and accuracy.

In addition, the method for determining whether the analyte is infected by the biotoxin according to the above embodiment of the present invention may further have the following additional technical features:

according to an embodiment of the present invention, the biotoxin is at least one selected from the group consisting of mycotoxins, plant toxins, animal toxins and microbial toxins, preferably mycotoxins, more preferably the mycotoxins are at least one of aflatoxin B1, fumonisin B1, ochratoxin a and vomitoxin.

According to an embodiment of the invention, said lipid metabolism marker of fumonisin B1 is selected from at least one of the following: (1) fumonisin B1 marker 1: 512.5028 m/z; (2) fumonisin B1 marker 2: 540.5343 m/z; (3) fumonisin B1 marker 3: 813.6830 m/z; (4) fumonisin B1 marker 4: 722.5258 m/z; (5) fumonisin B1 marker 5: 738.5056 m/z; (6) fumonisin B1 marker 6: 787.6675 m/z.

According to an embodiment of the invention, said lipid metabolism marker of aflatoxin B1 is selected from at least one of the following: (1) aflatoxin B1 marker 1: triglyceride TG (18:1(9Z)/20:1(11Z)/22:1 (13Z)); (2) aflatoxin B1 marker 2: diglyceride DG (18:2(9Z,12Z)/20:0/0: 0); (3) aflatoxin B1 marker 3: triglyceride TG (18:1(9Z)/20:1(11Z)/20:1 (11Z)); (4) aflatoxin B1 marker 4: triglyceride TG (14:0/16:0/18: 0); (5) aflatoxin B1 marker 5: triglyceride TG (18:0/18:1(9Z)/20:1 (11Z)).

According to an embodiment of the invention, the marker of ochratoxin a is selected from at least one of: (1) phosphatidic acid PA (13:0/20:1 (11Z)); (2) phosphatidylserine PS (O-20:0/19:1 (9Z)).

According to an embodiment of the invention, the lipid metabolism marker of the vomitoxin is selected from at least one of the following: (1) phosphatidic acid PA (13:0/20:1 (11Z)); (2) lecithin PC (10:0/21: 0); (3) penthorum chinense pursh b (thonninin b); (4) lecithin PC (16:0/22:5(4Z,7Z,10Z,13Z, 16Z)); (5) diglyceride DG (O-16:0/18:1 (9Z)).

According to an embodiment of the invention, the liquid chromatography-mass spectrometry is ultra performance liquid chromatography-time of flight-ion mobility spectrometry.

According to an embodiment of the present invention, the column of the liquid chromatography-mass spectrometry is an ACQUITY CSH C18 column.

according to an embodiment of the present invention, the elution of the liquid chromatography-mass spectrometry chromatogram is a gradient elution, preferably, the gradient elution condition is 0-2min, mobile phase B: 40% -43%; 2.0-2.1min, mobile phase B: 43% -50%; 2.1-12.0min, 50% -54%; 12.0-12.1min, 54% -70%; 12.1-18.0min, 70% -99%; 99-40% for 18.0-18.1 min; 18.1-20.0min, 40%.

According to the embodiment of the invention, the molecular weight of the analyte is not more than 1200 Da.

According to an embodiment of the invention, the analyte is derived from a mammal, preferably from the liver and kidney of a mammal.

According to another aspect of the invention, there is provided a kit for determining whether a test agent is infected with a biotoxin. According to an embodiment of the invention, the kit comprises a reagent, a standard, an auxiliary material or a combination of at least one of the foregoing reagents, standards, auxiliary materials or combinations thereof used in the method for detecting a characteristic fingerprint marker of a biotoxin infected cell. Therefore, the kit can accurately judge whether the object to be detected is infected by biotoxin or not through liquid chromatography-mass spectrometry detection based on the lipid metabolism marker, and is simple to operate and high in sensitivity and accuracy. In addition, it should be noted that the kit has all the technical features and technical effects of the aforementioned method for determining whether the analyte is infected by the biotoxin, and details are not repeated here.

According to an embodiment of the invention, the standards comprise a lipid metabolite standard and a mycotoxin standard, wherein the lipid metabolite standard is selected from at least one of N-ceramide-D-erythro-sphingosylphosphorylcholine, C14-dihydroceramide and C16-dihydroceramide and C22 sphingomyelin; the mycotoxin standard is selected from at least one of aflatoxin B1, fumonisin B1, vomitoxin and ochratoxin.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows the basic peaks (ESI) after intervention with different mycotoxins according to one embodiment of the invention⁺) A schematic diagram;

FIG. 2 shows the fundamental peaks (ESI) after intervention with different mycotoxins according to one embodiment of the invention^-) A schematic diagram;

FIG. 3 shows a schematic diagram of a mobility separation capability verification according to one embodiment of the present invention;

FIG. 4 shows a schematic representation of Extracted Ion Chromatograms (EICs) in Hepg2 extracts (as exemplified by m/z 722.52) following intervention with different concentrations of fumonisin B1 according to one embodiment of the present invention;

FIG. 5 shows a schematic of the detection of control groups and groups infected with mycotoxins according to one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

According to one aspect of the invention, a method is provided for determining whether a test agent is infected with a biological toxin. According to an embodiment of the invention, the method comprises: extracting the object to be detected to obtain an extracting solution; and performing liquid chromatography-mass spectrometry detection on the extracting solution, and determining whether the substance to be detected contains biotoxin or not based on the lipid metabolism marker.

According to the embodiment of the invention, the method can realize the detection of the cells of the livestock and the escherichia coli and the detection of the human cells, has important significance on the metabolism research of the human cells under the intervention of biotoxin, and has important application value in establishing infection models of food, medicines, cosmetics and other harmful substances in the environment.

According to an embodiment of the invention, the biological toxin is selected from at least one of mycotoxins, plant toxins, animal toxins and microbial toxins. The method provided by the embodiment of the invention is suitable for detecting infection of various toxins. According to a preferred embodiment of the invention, the biological toxin is a mycotoxin, more preferably, the mycotoxin is at least one of aflatoxin B1, ochratoxin a, fumonisin B1 and vomitoxin. Mycotoxin is an important toxin infecting cells, is qualitatively detected, and has important application value for establishing an infection model of food, medicine, cosmetics and other harmful substances in the environment.

According to an embodiment of the invention, the marker of ochratoxin a is selected from at least one of: (1) phosphatidic acid PA (13:0/20:1 (11Z)); (2) phosphatidylserine PS (O-20:0/19:1 (9Z)).

According to the embodiment of the invention, the extraction processing of the object to be detected comprises the following steps: extracting cells of a substance to be detected, washing the cells with a phosphate buffer solution for 3 times, adding pancreatin, digesting the cells for 1 to 2min at 37 ℃, discarding the pancreatin, slightly washing the cells with a Du's phosphate buffer solution for one time, replacing the Du's phosphate buffer solution with a new one, blowing the cells down, centrifuging the cells for 2 to 3 times at 4 ℃, discarding the Du's phosphate buffer solution, adding dichloromethane with the solid volume of 4 to 5 times: methanol (2:1, v/v), sonicated for 10min, centrifuged for 15min (12,000rpm,5min,4 ℃), nitrogen blown dry, isopropanol/methanol/water (2:1:1, v/v/v) redissolved, and filtered through a 0.22 μm filter to give an extract.

According to an embodiment of the invention, the liquid chromatography-mass spectrometry is ultra performance liquid chromatography-time of flight-ion mobility spectrometry. Ion Mobility Spectrometry (IMS) has shown broad application prospects in the analysis of complex metabolites. Some ions with the same mass to charge ratio and the same or similar retention time may have different headgroups, different fatty acid acyl attachment modes, and different positions of the double bonds of the fatty acid acyl. The IMS separates ions according to size and shape without depending on chromatographic conditions according to different collision cross sections when the ions collide with buffer gas in the drift tube, and has the advantages of simple operation, accurate identification, easy realization of automation and the like. However, the increase in mobility dimension reduces sensitivity and makes subsequent dosing very challenging. The inventor adopts ultra-high performance liquid chromatography, the analysis time is short, the solvent consumption is small, and the detection speed is high; in addition, the method increases the dimension of ion mobility and improves the separation effect and the reliability of the identification result.

According to an embodiment of the present invention, the column of the liquid chromatography-mass spectrometry is an ACQUITY CSH C18 column. The inventor finds that the CSH C18 chromatographic column has low level positive surface charge, not only improves the loading capacity of lipid metabolites but also enhances the separation capacity of the lipid metabolites in an acidic mobile phase, and has strong detection signals for the lipid metabolites.

According to an embodiment of the invention, the chromatography mobile phase of the liquid chromatography-mass spectrometry is: mobile phase a aqueous acetonitrile solution containing 10mM ammonium formate and 0.1% formic acid, preferably in a volume ratio of acetonitrile to water of 6: 4; mobile phase B: a mixed solution of isopropanol and acetonitrile containing 10mM ammonium formate and 0.1% formic acid, wherein the volume ratio of isopropanol to acetonitrile is 9: 1. the inventor finds that the addition of formic acid in the mobile phase is beneficial to protonation of positive ions and maintains the ionization state of a sample in the mobile phase; the addition of ammonium formate can increase the polarity of the mobile phase, adjust the ionic strength in the solution and facilitate the full separation of lipid metabolites.

According to an embodiment of the invention, the elution of the chromatogram of the liquid chromatography-mass spectrum is a gradient elution, preferably the gradient elution conditions are: 0-2min, mobile phase B: 40% -43%; 2.0-2.1min, mobile phase B: 43% -50%; 2.1-12.0min, 50% -54%; 12.0-12.1min, 54% -70%; 12.1-18.0min, 70% -99%; 99-40% for 18.0-18.1 min; 18.1-20.0min, 40%. This facilitates sufficient separation of the lipid metabolism marker and improves sensitivity.

According to an embodiment of the invention, the chromatographic conditions of the liquid chromatography-mass spectrometry are: temperature of the chromatographic column: 55 ℃; the room temperature of the sample is 10 ℃; the flow rate was 300. mu.L/min. The higher the column temperature, the faster the peak, the shorter the retention time, but some components may not be separated enough; the column temperature is reduced, the flow rate is accelerated, but the column pressure is possibly overhigh, and through a large number of experiments, the inventor finds that the chromatographic column temperature is 55 ℃, the flow rate is 300 mu L/min, and the peak-out time and the retention time of the lipid metabolite are appropriate. In addition, biological samples are typically stored cryogenically.

According to an embodiment of the invention, the mass spectrometric conditions of the liquid chromatography-mass spectrometry are: the collection mode is HDMSE; the ionization mode being ESI⁺/ESI^-(ii) a The voltage of the taper hole is 30V; the desolvation temperature is 550 ℃; the desolventizing gas flow rate is 900L/hr; the collection range is 50-1200 m/z. The inventors have found that the conformational space width of lipids is relatively compressed in the negative ionization mode, and thus the ESI⁺Was used for subsequent studies.

According to the embodiment of the invention, the molecular weight of the analyte is not more than 1200 Da.

According to an embodiment of the invention, the analyte is derived from a mammal, preferably from the liver of a mammal. Therefore, the liver is an important digestive organ of mammals, contains abundant enzymes, is active in substance metabolism and is suitable for detection of biotoxins.

According to another aspect of the invention, there is provided a kit for determining whether a test agent is infected with a biotoxin. According to an embodiment of the invention, the kit comprises reagents, standards, auxiliary materials or a combination of at least one of them used in the aforementioned method for detecting a characteristic fingerprint marker of biotoxin-infected cells. Therefore, the kit can accurately judge whether the object to be detected is infected by biotoxin or not through liquid chromatography-mass spectrometry detection based on the lipid metabolism marker, and is simple to operate and high in sensitivity and accuracy. In addition, it should be noted that the kit has all the technical features and technical effects of the aforementioned method for determining whether the analyte is infected by the biotoxin, and details are not repeated here.

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.

Example 1

In this embodiment, the method for determining whether the analyte is infected by the biotoxin is used to detect the lipid metabolite of the cells behind the fumonisin stem prognosis and determine the antagonistic/promoting trend of the stem prognosis at different concentrations, which is specifically as follows:

1. and (3) establishing an intervention system:

(1) taking out Hepg2 cells from liquid nitrogen, rapidly shaking and thawing a freezing tube in warm water at 37 ℃, adding 4-5mL of culture medium, and uniformly mixing;

(2) centrifuging at 1000rpm for 4min, removing supernatant, adding 1-2mL culture medium, and blowing;

(3) adding the cell suspension into a T25 culture bottle or a 60mm culture dish, supplementing a proper amount of culture medium, and adding the medicine after 24 hours of adherence.

2. Lipid metabolite extraction:

(1) adding fumonisins with different concentrations for intervention, and culturing at 37 deg.C for 5.0% CO when the cells grow to about 80%₂Taking out from the constant temperature incubator, discarding the culture medium, and washing for 3 times by using phosphate buffer solution.

(2) Adding 1mL of pancreatin into a culture dish, digesting for 1-2min at 37 ℃, discarding the pancreatin, and slightly washing once by using a Duchenne phosphate buffer solution;

(3) replacing with new Du's phosphate buffer solution to blow out the cells and centrifuging at 4 deg.C for 2-3 times, discarding the Du's phosphate buffer solution, adding 4-5 times of solid volume of dichloromethane: methanol (2:1, v/v), and ultrasonic treatment for 10 min;

(4) centrifuge for 15min (12,000rpm,5min,4 ℃), blow-dry with nitrogen, redissolve with isopropanol/methanol/water (2:1:1, v/v/v), and filter through a 0.22 μm filter.

3. Reagents and materials

(1) The chromatographic grade acetonitrile, isopropanol used in the experiment was purchased from Saimerfi, UK, Sigma-Aldrich, methanol, leucine-Naphthalein, USA. Mycotoxin standards used in the experiments, aflatoxin B1, fumonisin B1, vomitoxin, ochratoxin, were purchased from Romer, australia, and lipid standards, N-ceramide-D-erythro-sphingosylphosphorylcholine, C14-dihydroceramide, C16-dihydroceramide, and C22 sphingomyelin, were purchased from Sigma-Aldrich, usa.

(2) Preparation of standard stock solution

Accurately weighing 4 mycotoxin standards 5mg, respectively placing in sample bottles, adding phosphate buffer solution to dissolve fumonisin B1 and vomitoxin, adding methanol to dissolve aflatoxin B1 and ochratoxin, respectively preparing into 2000 μ M stock solutions, and storing in a refrigerator at-20 deg.C for later use.

(3) Preparation of mixed standard working solution

Precisely measuring each 500 mu L of the single-standard stock solution, placing the single-standard stock solution into a 10mL volumetric flask, diluting the single-standard stock solution with phosphate buffer solution to obtain 400 mu M standard solution, and placing the standard solution into a refrigerator at 4 ℃ for storage for later use. According to the actual working requirement, the gradient is diluted into sequence concentration.

3. Detecting the reference substance solution in a high performance liquid chromatography-mass spectrum, wherein the conditions of the chromatogram and the mass spectrum are as follows:

a chromatographic column: an ACQUITY CSH C18 chromatography column;

temperature of the chromatographic column: 55 ℃;

the room temperature of the sample is 10 ℃;

the flow rate is 300 mu L/min;

mobile phase a acetonitrile/water (6: 4) containing 10mM ammonium formate and 0.1% formic acid;

mobile phase B was isopropanol/acetonitrile (9: 1) containing 10mM ammonium formate and 0.1% formic acid;

gradient elution, elution conditions: 0-2min, mobile phase B: 40% -43%; 2.0-2.1min, mobile phase B: 43% -50%; 2.1-12.0min, 50% -54%; 12.0-12.1min, 54% -70%; 12.1-18.0min, 70% -99%; 99-40% for 18.0-18.1 min; 18.1-20.0min, 40%. The details are shown in Table 1

Table 1 mobile phase proportions and flow rate parameter settings

Mass spectrum conditions: mass spectrum: SYNAPT G2 HDMS; an acquisition mode: HDMSE; ionization mode: ESI +/ESI-; cone voltage: 30V; the temperature of the desolvation: 550 ℃; desolventizing air flow rate: 900L/hr; source temperature: 120 ℃; buffer gas: nitrogen and helium; collision cell voltage: 20-50V; the collection range is as follows: 50-1200 m/z.

The peak profile of Hepg2 lipid extract base after different mycotoxin interventions is shown in FIG. 1 (ESI)⁺) And as in FIG. 2 (ESI)^-) It is shown that ESI is due to the relative compression of the conformational space width of the lipid in negative ionization mode⁺Was used for subsequent studies.

4. Data processing:

(1) raw data were collected using MassLynx version 4.2 software (Waters Corporation, Wilmslow, UK);

(2) progenetics QI further performed peak alignment and calculation of CCS values.

(3) The resulting data were submitted to EZ info 3.0(Waters Corporation, Wilmslow, UK) and SIMCA 14.1 software (Umetrics, Sweden) for statistical analysis.

(4) After filtering out statistically significant feature data, lipid identification was performed using a lipid online search tool (http:// www.lipidmaps.org) to obtain detailed biomarker information.

And (3) integrating the VIP value, the q value, the anova (p), the score information and the ranking to obtain a biomarker (FB1), which is as follows:

(1) cer (d18:0/14:0) (m/z 512.5028) (VIP >1, anova (p) 0, q 0, score 39.2);

(2) cer (d18:0/16:0) (m/z 540.5343) (VIP >1, anova (p) 3.06E-09, q 8.85E-09, score 42.5);

(3) SM (d18:1/24:1(15Z)) (m/Z813.6830) (VIP >1, anova (p) ═ 1.15463E-14, q ═ 1.5098E-14, score 47.6);

(4)1- (8- [5] -ladder-octanyl) -2- (8- [3] -ladder-octanyl) -sn-glycerylphosphohethanolamines (m/z 722.5258) (VIP >1, anova (p) ═ 5.61218E-13, q ═ 5.47066E-13, score 43.6);

(5) PE (14:1(9Z)/22:4(7Z,10Z,13Z,16Z)) (m/Z738.5056) (VIP >1, anova (p) 0, q 0, score 56.1);

(6) SM (d18:1/22:0) (m/z 787.6675) (VIP >1, anova (p) 0, q 0, score 47.9).

FIG. 3 shows the separation of m/z 780.549 in the drift time dimension for retention time of 7.93 minutes, from which the CCS dimension is important in the identification of non-targeted metabolomics. Extracted Ion Chromatograms (EICs) of different concentrations of fumonisins B1 in Hepg2 extracts (in m/z 722.52 for example) are shown in fig. 4. In biomarker-based model identification, control and mycotoxin-infected groups were completely separated, as shown in fig. 5.

Example 2

The method of example 1 is adopted to detect the cell lipid metabolite of the aflatoxin B1 dry prognosis, and judge the antagonism/promotion trend of the dry prognosis with different concentrations, and the difference is that the aflatoxin B1 with different concentrations is added in the step 2 (1) for intervention, and the detected biomarker of the aflatoxin B1 is

(1) Triglyceride TG (18:1(9Z)/20:1(11Z)/22:1 (13Z));

(2) diglyceride DG (18:2(9Z,12Z)/20:0/0: 0);

(3) triglyceride TG (18:1(9Z)/20:1(11Z)/20:1 (11Z));

(4) triglyceride TG (14:0/16:0/18: 0);

(5) triglyceride TG (18:0/18:1(9Z)/20:1 (11Z)).

Example 3

The method of example 1 is adopted to detect cellular lipid metabolites of ochratoxin A stem prognosis, and judge the antagonism/promotion trend of different concentrations of stem prognosis, and the difference is that in step 2 (1), intervention of ochratoxin A with different concentrations is added, and the detected biomarkers of ochratoxin A are as follows:

(1) phosphatidic acid PA (13:0/20:1 (11Z));

(2) phosphatidylserine PS (O-20:0/19:1 (9Z)).

Example 4

The method of example 1 is adopted to detect cellular lipid metabolites of ochratoxin A stem prognosis, and judge the antagonism/promotion trend of different concentrations of stem prognosis, and the difference is that in step 2, (1) different concentrations of vomitoxin intervention are added, and the detected vomitoxin biomarkers are as follows:

(1) phosphatidic acid PA (13:0/20:1 (11Z));

(2) lecithin PC (10:0/21: 0);

(3)Thonningianin B；

(4) lecithin PC (16:0/22:5(4Z,7Z,10Z,13Z, 16Z));

(5) diglyceride DG (O-16:0/18:1 (9Z)).

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

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