CN113984948A - Combined diagnosis model for helicobacter pylori infection based on VOC marker and establishment method and application thereof - Google Patents
Combined diagnosis model for helicobacter pylori infection based on VOC marker and establishment method and application thereof Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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Abstract
The invention discloses a helicobacter pylori infection joint diagnosis model based on a VOC marker and an establishment method and application thereof. The headspace in the cell culture process of the tested person and the metabolic volatile organic compounds in the culture medium are compared with the joint detection model of the invention, and then the screening and the diagnosis of the helicobacter pylori can be rapidly completed.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a helicobacter pylori infection combined diagnosis model based on a VOC marker and an establishment method and application thereof.
Background
The helicobacter pylori is mainly distributed inGram-negative helicobacter pylori of the pylorus region of the human stomach is also the only microorganism discovered at present and can live in the stomach of the human body, and the helicobacter pylori is reported to be an important cause of chronic gastritis, gastric ulcer and even gastric cancer. The current detection methods for helicobacter pylori infection mainly comprise: (1) tissue section staining examination, wherein a gastric mucosa specimen of a subject needs to be taken, the specificity is high, but the operation of a professional is needed, and the subject is damaged; (2) bacteria culture, helicobacter pylori culture need specific gas, temperature environment and special culture medium, the operation of the culture and inspection process is complex, the clinical popularization is not facilitated, and the detection result is easily influenced by the operation of the culture process and the environmental control; (3) the detection of serum HP antibody and the detection of excrement HP antigen is unstable due to the continuous existence of immune reaction and the difference of strains among different individuals; (4)13C、14the C expiration detection method is simple and easy to operate, but has high false positive probability.
Disclosure of Invention
The invention aims to solve the problems and provide a combined diagnosis model of helicobacter pylori infection based on a VOC marker and an establishment method and application thereof, and provides a new scientific basis for early discovery and early treatment of helicobacter pylori.
The purpose of the invention is realized by the following technical scheme:
a combined diagnosis model of helicobacter pylori infection based on VOC markers is characterized in that the VOC markers in the air above the cell/bacteria culture bottles of a gastric mucosa epithelial cell group and a helicobacter pylori group and the VOC markers in a cell/bacteria culture medium are respectively obtained by adopting a gas chromatography-mass spectrometry combined method, components with obvious differences are screened out by comparing the composition of metabolites of the helicobacter pylori group and the gastric mucosa epithelial cell group and the content change of each component relative to a blank medium, and a combined detection and analysis model is established;
wherein the VOC marker in the air at the top of the culture flask comprises acetone, butanone, toluene, dimethyl disulfide, propylene glycol methyl ether acetate, lauric acid, myristic acid and palmitic acid; the VOC marker in the cell/bacteria culture medium comprises acetone, butanone, isopropanol, pentanone, dimethyl disulfide, 2- (perfluorohexyloctane) ethanol, 2,4, 6-trimethylpyridine, tetradecane, methyl nonyl ketone and undecanol.
The joint detection model of the invention is as follows: two or more of dimethyl disulfide, lauric acid, myristic acid and palmitic acid appear in the VOC component of the headspace of the cell/bacteria culture bottle of the subject, and two or more of dimethyl disulfide, 2- (perfluorohexyloctane) ethanol, methyl nonyl ketone and undecanol appear in the VOC component of the headspace microextraction of the cell/bacteria culture medium, so that the helicobacter pylori infection can be diagnosed; compared with the VOC component in the headspace of the culture bottles of the blank control group, the peak area of the acetone in the cell/bacteria culture bottle of the subject is improved by more than 50%, the peak area of the butanone is improved by more than 2 times, the peak area of the toluene is improved by more than 60%, and the peak area of the propylene glycol monomethyl ether acetate is basically unchanged or slightly reduced. Compared with VOC components subjected to headspace solid phase microextraction of a blank culture medium, the peak area of acetone is increased by more than 2 times, the peak area of butanone is increased by more than 5 times, the peak area of isopropanol is increased by more than 50%, the peak area of pentanone is increased by more than 1.5 times, the peak area of 2- (perfluorohexyloctane) ethanol is increased by more than 3.5 times, the peak area of 2,4, 6-trimethylpyridine is increased by more than 1.2 times, tetradecane is not detected, and the helicobacter pylori infection is diagnosed in an auxiliary way;
the combined detection model for helicobacter pylori infection provided by the invention is used for comparing acetone, butanone, toluene, dimethyl disulfide, propylene glycol methyl ether acetate, lauric acid, myristic acid and palmitic acid in the headspace air of a culture bottle of cells/bacteria to be detected, acetone, butanone, isopropanol, pentanone, dimethyl disulfide, 2- (perfluorohexyloctane) ethanol, 2,4, 6-trimethylpyridine, tetradecane, methyl nonyl ketone and undecanol in a cell/bacteria culture medium with the combined detection model provided by the invention, and can be used for detecting helicobacter pylori.
The method for establishing the helicobacter pylori infection joint diagnosis model based on the VOC marker enriches volatile organic compounds in cell metabolites through cell/bacteria culture and gas and culture medium headspace solid phase microextraction technology optimization. Separating and detecting the extracted organic matters by using a gas chromatography-mass spectrometry coupling technology, screening out volatile organic metabolites highly related to helicobacter pylori, and performing substance retrieval and analysis by using a NIST14 spectral library carried by a mass spectrum. And (3) carrying out comparative analysis on volatile metabolites detected in cell/bacterium headspace air and a culture medium and volatile organic compounds of a blank culture medium control group, retrieving VOC markers from two dimensions of organic compound components and content, and establishing a helicobacter pylori joint detection model.
The joint detection method specifically comprises the following steps:
1) on-line enrichment of cell/bacteria culture flasks, and headspace in culture flasks of blank control group without cell/bacteria medium for 30min using a solid phase microextraction core of 75 μm Car/PDMS;
2) collecting culture solution of gastric mucosa epithelial cells and helicobacter pylori and blank culture solution without cells/bacteria with the same volume, centrifuging at a rotating speed of 8000rpm/min, collecting supernatant, performing headspace solid phase microextraction by using a 75-micron Car/PDMS extraction core, oscillating a sample in a closed box, uniformly heating at 60 ℃, and extracting for 30 min;
3) separating and detecting the sample by using a gas chromatography-mass spectrometer: enriching the sample on extraction core, desorbing at GCMS sample inlet at 260 deg.C for 5min without split-flow mode, and separating with chromatographic column DB-WAX of 0.25 μm × 30 and × 0.25 mm; temperature rising procedure: the initial temperature is 40 ℃, the holding time is 5min, the temperature is increased to 250 ℃ at the speed of 10 ℃/min, and the holding time is 5 min. Scanning by a mass spectrometer in a full range of 40-400amu, wherein the electron bombardment energy is 70eV, the ion source temperature of a quadrupole mass spectrometer is 250 ℃, the carrier gas is high-purity helium, and the flow rate is 1 ml/min; the detected substances are analyzed by using a NIST14 spectrum library carried by a mass spectrum;
4) respectively comparing and analyzing VOC components in headspace air of the helicobacter pylori and gastric mucosal epithelial cells and VOC types and content change percentages of the VOC components relative to respective blank groups, and screening out components with obvious differences in metabolites of the helicobacter pylori and gastric mucosal epithelial cells.
Specifically, two or more of dimethyl disulfide, lauric acid, myristic acid, and palmitic acid appear in the VOC components of the headspace air of the cell/bacteria culture bottle of the subject, and two or more of dimethyl disulfide, 2- (perfluorohexyloctane) ethanol, methyl nonyl ketone, and undecanol appear in the VOC components of the headspace microextraction of the cell/bacteria culture medium, and thus, helicobacter pylori infection can be diagnosed; compared with the VOC component in the headspace of the culture bottles of the blank control group, the peak area of the acetone in the cell/bacteria culture bottle of the subject is improved by more than 50%, the peak area of the butanone is improved by more than 2 times, the peak area of the toluene is improved by more than 60%, and the peak area of the propylene glycol monomethyl ether acetate is basically unchanged or slightly reduced. Compared with VOC components subjected to headspace solid phase microextraction of a blank culture medium, the peak area of acetone is increased by more than 2 times, the peak area of butanone is increased by more than 5 times, the peak area of isopropanol is increased by more than 50%, the peak area of pentanone is increased by more than 1.5 times, the peak area of 2- (perfluorohexyloctane) ethanol is increased by more than 3.5 times, the peak area of 2,4, 6-trimethylpyridine is increased by more than 1.2 times, fourteen compounds are not detected, and the adjuvant diagnosis of helicobacter pylori infection is assisted.
The invention provides a method for diagnosing helicobacter pylori infection by detecting the headspace air of a helicobacter pylori culture bottle and a volatile metabolite marker in a culture medium, and provides a principle and frontier research result for an exhalation diagnosis mode of helicobacter pylori infection. The cell metabolite contains a plurality of markers, wherein the volatile organic compound markers are rich in types and easy to obtain, and can be used as a marker for detecting helicobacter pylori. When the stomach is infected by helicobacter pylori, the physiological activity of the stomach produces various metabolites, and compared with the normal gastric mucosa cells which are not infected by the helicobacter pylori, the composition and the content of volatile organic compounds in the stomach are changed. Due to the difference of the boiling point, the saturated vapor pressure and the water solubility of different volatile organic compounds, the volatile organic compound marker related to the helicobacter pylori not only exists in the headspace of the culture bottle, but also a considerable part of the marker is dissolved in the culture medium of cells/bacteria, so that the detection accuracy can be greatly improved by simultaneously analyzing and jointly verifying the headspace of the cell/bacteria culture bottle and the components of the culture medium.
The combined detection model established by combining the helicobacter pylori headspace gas and the volatile metabolite of the culture medium can be widely applied to a plurality of fields of auxiliary diagnosis of the helicobacter pylori, guidance of clinical medication, epidemiological research and the like.
Compared with the prior art, the invention has the following advantages:
1) the detection method provided by the invention is simple to operate, the sample sources are rich, and the samples can be obtained from the gastric juice, saliva, urine and the like of a testee and can be sampled noninvasively;
2) the helicobacter pylori marker screened by the established cell/bacterium culture bottle headspace air and VOC component joint detection model in the culture medium is more comprehensive, and the detection result is stable and reliable;
3) the detection mode of the combination of multiple markers greatly improves the detection accuracy and can provide a certain basis for screening helicobacter pylori infection clinically.
Drawings
FIG. 1 is a gas chromatography mass spectrum and markers in the headspace of a culture flask for helicobacter pylori and gastric mucosal epithelial cells;
FIG. 2 is a gas chromatogram mass spectrogram and a marker obtained by headspace solid-phase microextraction of a culture medium of helicobacter pylori and gastric mucosal epithelial cells;
FIG. 3 is a flow chart of a combined diagnostic model for helicobacter pylori infection based on VOC markers.
Detailed Description
The experimental procedures, for which specific conditions are not specified in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturer, and the reagents are cell-specific.
Reagents and instrumentation: the culture medium for helicobacter pylori comprises deionized water, Brucella broth powder, and antibiotics (vancomycin, amphotericin B, cefsulodin, and trimethoprim); complete medium composition of gastric mucosal epithelial cells, cell culture medium (DMEM, Hyclone), fetal bovine serum (GIBCO), penicillin-streptomycin (PS), pancreatin (Hyclone), 75cm2 cell culture flask (Thermo Fisher), cell culture chamber (Thermo), gas chromatography mass spectrometer-headspace solid phase microextraction (Agilent, 7890B-5977B), 75 μm CAR/PDMS SPME extraction core (SUPELCO, 57344-U), headspace sample injection flask (ampere spectrum, 20ml, 40 ml).
Helicobacter Pylori (HP) is from clinical laboratory center of Shanghai city, and gastric mucosal epithelial cells (GES-1) are from cell bank of Chinese academy.
Example 1
Separation and detection of VOC components in the headspace air of helicobacter pylori and gastric mucosal epithelial cell culture flasks
15ml HP medium containing H.pylori and a headspace vial containing 15ml blank HP medium were removed after shaking continuously at 100rpm/min on a shaker at 37 ℃ for 24 h. Aging SPME extraction core at 260 deg.C under continuous purging with high purity nitrogen for 30min to remove impurities, inserting the extraction core into a bottle through a headspace sampling bottle gasket, extracting the cell at the top of the sampling bottle, and extracting in a 37 deg.C HP incubator for 30 min;
resuscitating human gastric mucosal epithelial cells in 15ml complete medium, digesting the adherently grown gastric mucosal epithelial cells with pancreatin, centrifuging, collecting, resuspending, counting at 1 × 106The density of/ml is dispersed in 15ml complete culture medium, and the culture medium is passed through a cell culture bottle, another cell culture bottle is added with 15ml complete culture medium with the same proportion, and the mixture is cultured in a cell culture box for about 24 hours at constant temperature, so that the cell activity is ensured to be more than 90%. Aging SPME extraction core at 260 deg.C under continuous purging with high purity nitrogen for 30min to remove impurities, inserting the extraction core into the culture bottle through the gas exchange membrane of the cell culture bottle, extracting the cell at the top of the cell culture bottle, and extracting at 37 deg.C for 30min in the cell culture box.
The extraction core enriched with helicobacter pylori expiration, helicobacter pylori culture medium headspace air, gastric mucosa epithelial cell expiration and cell complete culture medium headspace air completes the test within 1 h.
The SPME extraction core is desorbed for 5min at the temperature of 260 ℃ of a gas chromatography sample inlet, and is injected in a non-split mode. The chromatographic column is DB-WAX0.25 μm × 30 and × 0.25 mm; temperature rising procedure: the initial temperature is 40 ℃, the holding time is 5min, the temperature is increased to 250 ℃ at the speed of 10 ℃/min, and the holding time is 5 min. Scanning by a mass spectrometer in a full range of 40-400amu, wherein the electron bombardment energy is 70eV, the ion source temperature of a quadrupole mass spectrometer is 250 ℃, the carrier gas is high-purity helium, and the flow rate is 1 ml/min; the detected substances were analyzed using the NIST14 library carried by the mass spectrometer. FIG. 1 shows the GC mass spectrum and markers of H.pylori and gastric mucosal epithelial cells in the headspace of a culture flask.
TABLE 1 markers in the headspace of culture flasks for H.pylori and gastric mucosal epithelial cells
Example 2
Separation and detection of VOC components in culture medium of helicobacter pylori and gastric mucosal epithelial cells
The medium cultured with helicobacter pylori and the blank HP medium, the medium cultured with gastric mucosal epithelial cells and the blank complete medium in example 1 were collected, centrifuged at 8000rpm/min at 4 ℃ for 15min, and the supernatants of the above samples were each taken 8ml, added to 20ml headspace vials, and the caps were screwed down.
The sample is injected and tested by an automatic sample injector, wherein a sample cavity is preheated for 5min at 60 ℃, an SPME extraction core is aged for 30min under the conditions of 260 ℃ and continuous purging of high-purity nitrogen to remove impurities, penetrates through a sample bottle spacer and is inserted into the sample bottle to collect headspace air, and is continuously vibrated and extracted for 30min under the condition of 60 ℃.
The SPME extraction core is desorbed for 5min at the temperature of 260 ℃ of a gas chromatography sample inlet, and is injected in a non-split mode. The chromatographic column is DB-WAX0.25 μm × 30 and × 0.25 mm; temperature rising procedure: the initial temperature is 40 ℃, the holding time is 5min, the temperature is increased to 250 ℃ at the speed of 10 ℃/min, and the holding time is 5 min. Scanning by a mass spectrometer in a full range of 40-400amu, wherein the electron bombardment energy is 70eV, the ion source temperature of a quadrupole mass spectrometer is 250 ℃, the carrier gas is high-purity helium, and the flow rate is 1 ml/min; the detected substances were analyzed using the NIST14 library carried by the mass spectrometer. FIG. 2 is a gas chromatogram mass spectrum and markers obtained by headspace solid-phase microextraction of culture medium of helicobacter pylori and gastric mucosa epithelial cells.
TABLE 2 markers obtained by headspace solid-phase microextraction on the culture media
Example 3
Establishment of combined detection model based on air at top of cell/bacteria culture bottle and VOC (volatile organic compounds) in culture medium
The VOC components detected in examples 1 and 2 above and their corresponding peak areas were analyzed, in order to avoid the effect of the difference in the composition of the culture medium for helicobacter pylori and the culture medium for gastric mucosal epithelial cells on the results, the analysis was performed using the relative peak area change rate in the screening process, the air at the top of the flask in which the cells/bacteria were cultured and the volatile metabolic organic matter components in the culture medium were compared with the blank cell/bacteria culture medium, and the analysis was performed using the relative change amount of the peak areas as an index, to screen out the portions having significant differences between helicobacter pylori and gastric mucosal epithelial cells.
FIG. 3 is a flow chart of a joint detection model: two or more of dimethyl disulfide, lauric acid, myristic acid and palmitic acid appear in the VOC component of the headspace of the cell/bacteria culture bottle of the subject, and two or more of dimethyl disulfide, 2- (perfluorohexyloctane) ethanol, methyl nonyl ketone and undecanol appear in the VOC component of the headspace microextraction of the cell/bacteria culture medium, so that the helicobacter pylori infection can be diagnosed; compared with the VOC component in the headspace of the culture bottles of the blank control group, the peak area of the acetone in the cell/bacteria culture bottle of the subject is improved by more than 50%, the peak area of the butanone is improved by more than 2 times, the peak area of the toluene is improved by more than 60%, and the peak area of the propylene glycol monomethyl ether acetate is basically unchanged or slightly reduced. Compared with VOC components obtained by headspace solid phase microextraction of a blank culture medium, in a cell/bacteria culture medium of a subject, the peak area of acetone is increased by more than 2 times, the peak area of butanone is increased by more than 5 times, the peak area of isopropanol is increased by more than 50%, the peak area of pentanone is increased by more than 1.5 times, the peak area of 2- (perfluorohexyloctane) ethanol is increased by more than 3.5 times, the peak area of 2,4, 6-trimethylpyridine is increased by more than 1.2 times, tetradecane is not detected, and the adjuvant diagnosis of helicobacter pylori infection is assisted.
The invention uses the model to carry out the combined detection of the helicobacter pylori at the in vitro cell level, and the detection rate of the helicobacter pylori in the detection of the gastric mucosa epithelial cells and the human gastric cancer cells (803) control group is more than 95 percent.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A combined diagnosis model of helicobacter pylori infection based on VOC markers is characterized in that the VOC markers in the air above a cell/bacteria culture bottle of a gastric mucosa epithelial cell group and the helicobacter pylori group and the VOC markers in a cell/bacteria culture medium are respectively obtained by adopting a gas chromatography-mass spectrometry combined method, components with obvious differences are screened out by comparing the composition of metabolites of the helicobacter pylori group and the gastric mucosa epithelial cell group and the content change of each component relative to a blank culture medium, and a combined detection and analysis model is established;
wherein the VOC marker in the air at the top of the culture flask comprises acetone, butanone, toluene, dimethyl disulfide, propylene glycol methyl ether acetate, lauric acid, myristic acid and palmitic acid; the VOC marker in the cell/bacteria culture medium comprises acetone, butanone, isopropanol, pentanone, dimethyl disulfide, 2- (perfluorohexyloctane) ethanol, 2,4, 6-trimethylpyridine, tetradecane, methyl nonyl ketone and undecanol.
2. The model of claim 1, wherein the VOC component of the headspace air of the cell/bacteria culture flask comprises two or more of dimethyl disulfide, lauric acid, myristic acid, and palmitic acid,
the VOC component extracted in the headspace micro-extraction in the cell/bacteria culture medium comprises two or more than two of dimethyl disulfide, 2- (perfluorohexyloctane) ethanol, methyl nonyl ketone and undecanol.
3. The model of claim 2, wherein the peak area of acetone, the peak area of butanone and the peak area of toluene are increased by more than 50%, and the peak area of acetone and the peak area of toluene are increased by more than 60% respectively, compared with the VOC content in the headspace of the culture bottles of the blank control group;
compared with VOC components subjected to headspace solid phase microextraction of a blank culture medium, the cell/bacteria culture medium has the advantages that the acetone peak area is increased by more than 2 times, the butanone peak area is increased by more than 5 times, the isopropanol peak area is increased by more than 50%, the pentanone peak area is increased by more than 1.5 times, the 2- (perfluorohexyloctane) ethanol peak area is increased by more than 3.5 times, and the 2,4, 6-trimethylpyridine peak area is increased by more than 1.2 times.
4. The method for establishing the combined diagnosis model of helicobacter pylori infection based on the VOC marker as claimed in claim 1, wherein the volatile organic compounds in the cell metabolites are enriched by cell/bacteria culture and gas and culture medium headspace solid phase microextraction technology, the extracted organic compounds are separated and detected by gas chromatography-mass spectrometry, the volatile organic metabolites highly related to the helicobacter pylori are screened out, substance retrieval and analysis are performed by NIST14 spectral library carried by mass spectrometry, the volatile metabolites detected in the cell/bacteria headspace and culture medium are compared with the volatile organic compounds in a blank culture medium control group for analysis, the VOC marker is retrieved from two dimensions of organic matter components and content, and the combined detection model of helicobacter pylori is established.
5. The method for establishing the combined diagnostic model of helicobacter pylori infection based on VOC marker as claimed in claim 4, which comprises the following steps:
1) on-line enrichment of cell/bacteria culture flasks using solid phase microextraction cores, and headspace in culture flasks of blank controls of cell/bacteria-free medium;
2) collecting culture solution of gastric mucosa epithelial cells and helicobacter pylori and blank culture solution without cells/bacteria in the same volume, centrifuging at a rotating speed, collecting supernatant, performing headspace solid phase microextraction, and oscillating, heating and extracting a sample in a closed box;
3) separating and detecting the sample by using a gas chromatography-mass spectrometer;
4) respectively comparing and analyzing VOC components in headspace air of the helicobacter pylori and gastric mucosal epithelial cells and VOC types and content change percentages of the VOC components relative to respective blank groups, and screening out components with obvious differences in metabolites of the helicobacter pylori and gastric mucosal epithelial cells.
6. The method for establishing the combined diagnostic model of helicobacter pylori infection based on VOC marker as claimed in claim 5, wherein step 1) uses a solid phase microextraction core of 75 μm Car/PDMS for on-line enrichment for 30 min.
7. The method for establishing the combined diagnostic model of helicobacter pylori infection based on the VOC marker as claimed in claim 5, wherein the step 2) is performed by centrifugation at 8000rpm/min, headspace solid-phase microextraction is performed by using a 75 μm Car/PDMS extraction core, uniform heating is performed at 60 ℃, and extraction is performed for 30 min.
8. The method for establishing the combined diagnosis model of helicobacter pylori infection based on the VOC marker of claim 5, wherein the concrete method in the step 3) is that the sample is enriched on an extraction core, and is desorbed at a GCMS sample inlet at a high temperature of 260 ℃ for 5min without a split-flow mode, and a chromatographic column is DB-WAX0.25 μm x 30 and x0.25 mm; temperature rising procedure: the initial temperature is 40 ℃, the holding time is 5min, the temperature is increased to 250 ℃ at the speed of 10 ℃/min, and the holding time is 5 min.
9. The method for establishing the combined diagnosis model of helicobacter pylori infection based on the VOC marker of claim 8, wherein the mass spectrometer scans in the full range of 40-400amu, the electron bombardment energy is 70eV, the ion source temperature of the quadrupole mass spectrometer is 250 ℃, the carrier gas is high-purity helium, and the flow rate is 1 ml/min; the detected substances were analyzed using the NIST14 library carried by the mass spectrometer.
10. Use of a combined diagnostic model for helicobacter pylori infection based on VOC markers according to claim 1 as a means for determining helicobacter pylori infection.
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