阅读说明：本技术 一种代谢谱检测试剂盒及使用方法与应用 (Metabolism spectrum detection kit, use method and application ) 是由 邬建敏 陈晓明 蒋巍 张涵 余捷凯 张秋美 于 2021-07-27 设计创作，主要内容包括：本发明公开了一种代谢谱检测试剂盒及使用方法与应用,包括代谢质谱芯片、预处理试剂、校准品、质控品与生物标志物组合标签,本发明的代谢谱检测试剂盒包括1份代谢质谱芯片、3份预处理试剂、1份校准品、1份质控品与1份生物标志物组合标签。本发明的代谢谱检测试剂盒将代谢质谱芯片与高通量质谱技术相结合,在无基质喷涂的情况下批量获取多个生物样本中的代谢质谱图,试剂盒成本低,通量高,检测速度快,在自动化检测模式下可一次性获得48～102个样本的代谢谱数据,检测速度在5s/单样本左右,适合大样本的快速检测。(The invention discloses a metabolic spectrum detection kit, a using method and application, and the metabolic spectrum detection kit comprises a metabolic mass spectrum chip, a pretreatment reagent, a calibrator, a quality control product and a biomarker combined label, and comprises 1 part of the metabolic mass spectrum chip, 3 parts of the pretreatment reagent, 1 part of the calibrator, 1 part of the quality control product and 1 part of the biomarker combined label. The metabolic spectrum detection kit disclosed by the invention combines a metabolic mass spectrum chip with a high-flux mass spectrum technology, obtains metabolic mass spectrograms in a plurality of biological samples in batch under the condition of no matrix spraying, is low in cost, high in flux and high in detection speed, can obtain metabolic spectrum data of 48-102 samples at one time under an automatic detection mode, is about 5 s/single sample in detection speed, and is suitable for rapid detection of large samples.)

1. A metabolic profile detection kit is characterized by comprising a metabolic mass spectrum chip, a pretreatment reagent, a calibrator, a quality control product and a biomarker combined label; the metabolic mass spectrum chip is used as a carrier for metabolic spectrum detection and has a multi-site parallel detection function; the pretreatment reagent is used for pretreatment of metabolic molecules in a biological sample; the calibrator is used for molecular weight calibration of a metabolic spectrum, and the quality control material is used for quality control in sample pretreatment and detection processes; the biomarker combination tag has a characteristic molecular mass to charge ratio in the molecular weight range of 20-1000 Da.

2. The metabolic spectrum detection kit according to claim 1, wherein the main body material of the metabolic mass spectrometry chip comprises one of a silicon-based material, a silicon-based material covered with silicon oxide, and a silicon-based material covered with silicon nitride, and the metabolic mass spectrometry chip has a plurality of detection sites thereon and is uniformly distributed on the same horizontal plane of the metabolic mass spectrometry chip; the detection site is in the shape of a round hole with the diameter of 0.5-5mm or a square hole with the side length of 0.5-5 mm; the detection sites comprise sample detection sites, calibrator detection sites and quality control product detection sites, the calibrator detection sites are located in a central hole of the chip, the interior of the detection sites is hydrophilic, and the exterior of the detection sites is hydrophobic.

3. The metabolic profiling detection kit according to claim 1, wherein the pretreatment reagent comprises reagent A, reagent B, reagent C and reagent D; wherein, the reagent A comprises one or a mixture of more of methanol, acetonitrile, ethanol, acetone or isopropanol, the reagent B comprises one of methyl tert-butyl ether, trichloromethane or dichloromethane, the reagent C is ultrapure water, the reagent D is a mixture of isopropanol and ultrapure water, and the volume ratio of the isopropanol in the reagent D is 20-80%.

4. The metabolic profiling detection kit according to claim 1, wherein the calibrator comprises a pure metabolite molecule or a mixture of metabolite molecules extracted from a biological sample; wherein the pure product comprises: alanine m/z-88.0804, proline m/z-114.0561, valine m/z-116.0717, taurine m/z-124.0074, malic acid m/z-133.0142, glutamine m/z-145.0619, L-histidine m/z-154.0622, phenylalanine m/z-164.0717, uric acid m/z-167.0211, indigo m/z-262.0748, palmitic acid m/z-255.2330 and sodium tetraphenylborate m/z-319.1664, wherein the concentration of each component is 0.1-1 mg/mL; the metabolic molecule mixture extracted from the biological sample covers the molecular weight range of 20-1000 Da.

5. The metabolic profiling detection kit according to claim 1, wherein the quality control product is a biological sample, and is prepared by mixing 30-50 human saliva samples, urine samples or serum samples, and the volume of the sample is 20 μ L/bottle.

6. The metabolic profiling detection kit according to claim 1, wherein the biomarker combination label comprises: serine, cytosine, uracil, creatinine, proline, valine, succinic acid, nicotinic acid, pyroglutamic acid, malic acid, adenine, benzyl formic acid, guanine, xanthine, gentisic acid, hydroxytyrosol, y-aminobutyric acid, betaine, piperidine acid, aspartic acid, glutamine, glutamic acid, histidine or uric acid.

7. A method of using a metabolic profiling kit, comprising the metabolic profiling kit of any one of claims 1 to 6 for the detection of a biological sample comprising saliva, urine, serum, plasma or tissue extract.

8. The use method of the metabolic profiling kit according to claim 7, wherein the metabolic profiling kit is used for extraction and detection of metabolic molecules in saliva and urine, and comprises the following steps:

(1) pre-cooling the pretreatment reagent for 10min before use, pretreating the biological sample by using a reagent A in batch, wherein the volume ratio of the reagent A to the biological sample is 2: 1-10: 1, the pretreatment process comprises oscillating for 5-30min, and centrifuging to obtain supernatant;

(2) adding a reagent C into the supernatant obtained in the step (1), wherein the ratio of the addition amount to the supernatant is 1: 5-1: 1, so as to obtain a sample solution;

(3) the same steps (1) and (2) are adopted to finish the pretreatment of the quality control product to obtain a quality control product solution;

(4) adding an equivalent amount of a reagent A and an equivalent amount of a reagent C into a calibrator, wherein the volume of the reagent A is 10-30 mu L, and performing vortex oscillation dissolution to obtain a calibrator solution;

(5) spotting 0.05-5 muL of the calibrator solution obtained in the step (4) to a calibrator detection site of a metabolic mass spectrometry chip, spotting 0.05-5 muL of the quality control solution obtained in the step (3) to a quality control detection site of the metabolic mass spectrometry chip, spotting 0.05-5 muL of the sample solution obtained in the step (2) to a sample detection site of the metabolic mass spectrometry chip in the kit, and drying for 10-30min after spotting is finished;

(6) sending the metabolic mass spectrum chip obtained in the step (5) into a mass spectrometer, and starting a data acquisition process of the mass spectrometer; firstly, a spectrogram of a calibrator solution is collected, the spectrogram is subjected to molecular weight calibration, a quality control product is detected after the calibration, the pretreatment process is confirmed to meet the quality inspection requirement, and then spectrograms of other samples are automatically collected.

9. The use method of the metabolic profiling kit according to claim 7, wherein the metabolic profiling kit is used for extracting and detecting metabolic molecules in serum, plasma and tissue extracting solution, and comprises the following steps:

(1) pre-cooling a pretreatment reagent for 10min before use, pretreating a biological sample by using a reagent A in batch, wherein the volume ratio of the reagent A to the biological sample is 2: 1-10: 1, the pretreatment process comprises oscillating for 5-30min, and centrifuging to obtain a supernatant to obtain a sample solution 1;

(2) adding a reagent B into the liquid obtained after the supernatant liquid is obtained in the step (1), wherein the volume ratio of the added amount of the reagent B to the original biological sample is 1: 2-1: 20, and performing vortex vibration for 5-15min until the precipitate in the tube is broken; then adding a reagent C with the volume being 1/3-1/2 corresponding to the volume of the reagent B, and oscillating for 5-10 min;

(3) layering the solution in the step (2) by using a centrifugal method, taking the organic phase and drying the organic phase to obtain dry powder; then re-dissolving with a reagent D with a volume 1 time that of the biological sample to obtain a sample solution 2;

(4) mixing the sample solution 1 obtained in the step (1) and the sample solution 2 obtained in the step (3) in equal volume to obtain a sample solution;

(5) the same steps (1) to (4) are adopted to finish the pretreatment of the quality control product to obtain a quality control product solution;

(6) adding an equivalent amount of a reagent A and an equivalent amount of a reagent D into a calibrator, wherein the volume of the reagent A and the reagent D is 10-30 mu L, and performing vortex oscillation dissolution to obtain a calibrator solution;

(7) spotting 0.05-5 muL of the calibrator solution obtained in the step (6) to a calibrator detection site of a metabolic mass spectrometry chip, spotting 0.05-5 muL of the quality control solution obtained in the step (5) to a quality control detection site of the metabolic mass spectrometry chip, spotting 0.05-5 muL of each tube of the sample solution obtained in the step (4) to a sample detection site of the metabolic mass spectrometry chip in the kit, and drying for 10-30min after spotting is finished;

(8) sending the metabolic mass spectrum chip obtained in the step (7) into a mass spectrometer, and starting a data acquisition process of the mass spectrometer; firstly, a spectrogram of a calibrator solution is collected, the spectrogram is subjected to molecular weight calibration, a quality control product is detected after the calibration, the pretreatment process is confirmed to meet the quality inspection requirement, and then spectrograms of other samples are automatically collected.

10. The use method of the metabolic profiling detection kit according to claim 8, wherein the mass spectrometer is a time-of-flight mass spectrometer.

11. Use of a metabolic profiling kit according to any one of claims 1 to 6 or mass spectrometry data generated by the use of the method according to any one of claims 7 to 10 in tumor-assisted identification or chronic disease monitoring management; wherein the tumor is selected from lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, esophageal cancer or oral cancer, and chronic diseases selected from diabetes, gestational diabetes, hypertension, hyperlipidemia or Alzheimer's disease.

Technical Field

The invention belongs to the technical field of mass spectrometry, and relates to a metabolic spectrum detection kit which is low in cost, high in flux, high in detection speed and simple in pretreatment method, and a using method and application thereof.

Background

The metabolome composed of amino acid, fatty acid, urea, glucose, choline and derivatives thereof is positioned at the downstream of a system biological network, can reflect the common 'terminal point' caused by genome and proteome changes, provides biological terminal information, embodies the phenotypic changes, physiological and pathological conditions of organisms, thereby predicting the disease progress more sensitively and being a sensitive biomarker. At present, the metabolic abnormality, hyperglycemia, hyperlipidemia, chronic nephropathy, nonalcoholic hepatitis, cancer and other diseases of the newborn are closely related to the metabolite composition and the abnormal pathway in the human body.

The research of metabonomics has been one of the most leading hotspots in the fields of chemistry, biology, medicine and the like. The mass spectrometry is an optimal technical platform suitable for detecting the metabolic small molecules, can be qualitative and quantitative, has very strong advantages in the aspects of detection sensitivity, specificity, analysis speed, simultaneous detection of multiple indexes and the like, and particularly can obtain a metabolic spectrum consisting of a plurality of metabolic molecules in a biological sample at one time to reflect the occurrence and development conditions of diseases.

Gas mass spectrometry (GC-MS), liquid mass spectrometry (LC-MS) and matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) are common omics analysis mass spectrometry technology platforms. However, disadvantages of these omic analysis mass spectrometry techniques include:

1) GC-MS needs to enable an analyte to form gaseous molecules in a combustion gasification mode, so that the GC-MS is suitable for volatile or semi-volatile small molecule detection, a large number of metabolic molecules can meet the detection requirements only through oximation, methylation and other derivatization steps, and the derivatization steps add complexity to analysis and detection;

2) LC-MS is most commonly used in metabonomics analysis, but has higher requirements on sample pretreatment, low detection flux and high detection cost, is not suitable for rapid and efficient detection of a large number of clinical samples, and is difficult to popularize clinically;

3) MALDI-MS is a high-throughput mass spectrometry detection technology, which has been widely used in the detection of macromolecules such as proteins, polypeptides, nucleic acids, etc., and the microorganism identification technology under the platform has been gradually applied in clinic. The patent CN109541012A filed by the applicant can realize the rapid identification of the microorganism by using a universal nano chip. However, due to the problems of matrix background interference, nonuniform crystallization and the like, the conventional MALDI-MS can hardly realize accurate detection of the metabolic molecules in a small molecular section (0-400Da) and reflect the composition of the metabolic molecules, and the reliability of the result is not high.

Therefore, the metabolic spectrum detection technology based on the high-throughput mass spectrum platform is always in a limited state, and a reliable metabolic spectrum detection kit cannot be constructed.

Disclosure of Invention

In order to solve the defects of the prior art, the invention discloses a metabolic spectrum detection kit, a using method and application thereof. The kit has low cost, high flux, high detection speed and simple pretreatment method; the method can be used for regulating and controlling detection parameters in real time so as to ensure the stability and reliability of detection results.

In order to achieve the purpose, the invention adopts the following technical scheme:

a metabolic profile detection kit comprises a metabolic mass spectrum chip, a pretreatment reagent, a calibrator, a quality control product and a biomarker combination label; the metabolic mass spectrum chip is used as a carrier for metabolic spectrum detection and has a multi-site parallel detection function; the pretreatment reagent is used for pretreatment of metabolic molecules in a biological sample; the calibrator is used for molecular weight calibration of a metabolic spectrum, and the quality control material is used for quality control in sample pretreatment and detection processes; the biomarker combination tag has a characteristic molecular mass to charge ratio in the molecular weight range of 20-1000 Da.

In the technical scheme, the metabolic spectrum detection kit comprises 1 part of a metabolic mass spectrometry chip, 3 parts of a pretreatment reagent, 1 part of a calibrator, 1 part of a quality control material and 1 part of a biomarker combination label. The metabolic spectrum detection kit disclosed by the invention combines a metabolic mass spectrum chip with a high-flux mass spectrum technology, obtains metabolic mass spectrograms in a plurality of biological samples in batch under the condition of no matrix spraying, is low in cost, high in flux and high in detection speed, can obtain metabolic spectrum data of 48-102 samples at one time under an automatic detection mode, is about 5 s/single sample in detection speed, and is suitable for rapid detection of large samples.

As a preferred scheme of the invention, the main body material of the metabolic mass spectrometry chip comprises one of a silicon-based material, a silicon-based material covered with silicon oxide and a silicon-based material covered with silicon nitride, and the metabolic mass spectrometry chip is provided with a plurality of detection sites which are uniformly distributed on the same horizontal plane of the metabolic mass spectrometry chip; the detection site is in the shape of a round hole with the diameter of 0.5-5mm or a square hole with the side length of 0.5-5 mm; the detection sites comprise sample detection sites, calibrator detection sites and quality control product detection sites, the calibrator detection sites are located in a central hole of the chip, the interior of the detection sites is hydrophilic, and the exterior of the detection sites is hydrophobic.

Preferably, the main material of the metabolic mass spectrometry chip is monocrystalline silicon.

Preferably, the detection site is in the shape of a circular hole with a diameter of 1.5-3.5 mm.

As a preferable scheme of the invention, the pretreatment reagent comprises a reagent A, a reagent B, a reagent C and a reagent D; wherein, the reagent A comprises one or a mixture of more of methanol, acetonitrile, ethanol, acetone or isopropanol, the reagent B comprises one of methyl tert-butyl ether, trichloromethane or dichloromethane, the reagent C is ultrapure water, the reagent D is a mixture of isopropanol and ultrapure water, and the volume ratio of the isopropanol in the reagent D is 20-80%.

In the technical scheme, the calibrator comprises a pure metabolic molecule or a metabolic molecule mixture extracted from a biological sample; wherein the pure product comprises: alanine m/z-88.0804, proline m/z-114.0561, valine m/z-116.0717, taurine m/z-124.0074, malic acid m/z-133.0142, glutamine m/z-145.0619, L-histidine m/z-154.0622, phenylalanine m/z-164.0717, uric acid m/z-167.0211, indigo m/z-262.0748, palmitic acid m/z-255.2330 and sodium tetraphenylborate m/z-319.1664, wherein the concentration of each component is 0.1-1 mg/mL; the metabolic molecule mixture extracted from the biological sample covers the molecular weight range of 20-1000 Da.

As a preferable scheme of the invention, the quality control product is a biological sample, and is prepared by mixing 30-50 human saliva samples, urine samples or serum samples, and the volume is 20 mu L/bottle.

As a preferred embodiment of the present invention, the biomarker combination tag comprises: serine, cytosine, uracil, creatinine, proline, valine, succinic acid, nicotinic acid, pyroglutamic acid, malic acid, adenine, benzyl formic acid, guanine, xanthine, gentisic acid, hydroxytyrosol, y-aminobutyric acid, betaine, piperidine acid, aspartic acid, glutamine, glutamic acid, histidine or uric acid.

The calibrator and the quality control materials in the metabolic spectrum detection kit need to be stored at the temperature of-20 ℃, and the metabolic mass spectrometry chip and the pretreatment reagent can be dried and stored at normal temperature. The pretreatment reagents were pre-chilled in crushed ice for 10min prior to use.

The invention also provides a use method of the metabolic profiling detection kit, which comprises the step of using the metabolic profiling detection kit for detecting biological samples, wherein the biological samples comprise saliva, urine, serum, plasma or tissue extract.

As a preferable scheme of the invention, the metabolic spectrum detection kit is used for extracting and detecting metabolic molecules in saliva and urine, and comprises the following steps:

(1) pre-cooling the pretreatment reagent for 10min before use, pretreating the biological sample by using a reagent A in batch, wherein the volume ratio of the reagent A to the biological sample is 2: 1-10: 1, the pretreatment process comprises oscillating for 5-30min, and centrifuging to obtain supernatant;

(2) adding a reagent C into the supernatant obtained in the step (1), wherein the ratio of the addition amount to the supernatant is 1: 5-1: 1, so as to obtain a sample solution;

(3) the same steps (1) and (2) are adopted to finish the pretreatment of the quality control product to obtain a quality control product solution;

(4) adding an equivalent amount of a reagent A and an equivalent amount of a reagent C into a calibrator, wherein the volume of the reagent A is 10-30 mu L, and performing vortex oscillation dissolution to obtain a calibrator solution;

(5) spotting 0.05-5 muL of the calibrator solution obtained in the step (4) to a calibrator detection site of a metabolic mass spectrometry chip, spotting 0.05-5 muL of the quality control solution obtained in the step (3) to a quality control detection site of the metabolic mass spectrometry chip, spotting 0.05-5 muL of the sample solution obtained in the step (2) to a sample detection site of the metabolic mass spectrometry chip in the kit, and drying for 10-30min after spotting is finished;

(6) sending the metabolic mass spectrum chip obtained in the step (5) into a mass spectrometer, and starting a data acquisition process of the mass spectrometer; firstly, a spectrogram of a calibrator solution is collected, the spectrogram is subjected to molecular weight calibration, a quality control product is detected after the calibration, the pretreatment process is confirmed to meet the quality inspection requirement, and then spectrograms of other samples are automatically collected.

As a preferable scheme of the invention, the metabolic profile detection kit is used for extracting and detecting metabolic molecules in serum, plasma and tissue extracting solutions, and comprises the following steps:

(1) pre-cooling a pretreatment reagent for 10min before use, pretreating a biological sample by using a reagent A in batch, wherein the volume ratio of the reagent A to the biological sample is 2: 1-10: 1, the pretreatment process comprises oscillating for 5-30min, and centrifuging to obtain a supernatant to obtain a sample solution 1; (2) adding a reagent B into the liquid obtained after the supernatant liquid is obtained in the step (1), wherein the volume ratio of the added amount of the reagent B to the original biological sample is 1: 2-1: 20, and performing vortex vibration for 5-15min until the precipitate in the tube is broken; then adding a reagent C with the volume being 1/3-1/2 corresponding to the volume of the reagent B, and oscillating for 5-10 min;

(3) layering the solution in the step (2) by using a centrifugal method, taking the organic phase and drying the organic phase to obtain dry powder; then re-dissolving with a reagent D with a volume 1 time that of the biological sample to obtain a sample solution 2;

(4) mixing the sample solution 1 obtained in the step (1) and the sample solution 2 obtained in the step (3) in equal volume to obtain a sample solution;

(5) the same steps (1) to (4) are adopted to finish the pretreatment of the quality control product to obtain a quality control product solution;

(6) adding an equivalent amount of a reagent A and an equivalent amount of a reagent D into a calibrator, wherein the volume of the reagent A and the reagent D is 10-30 mu L, and performing vortex oscillation dissolution to obtain a calibrator solution;

(7) spotting 0.05-5 muL of the calibrator solution obtained in the step (6) to a calibrator detection site of a metabolic mass spectrometry chip, spotting 0.05-5 muL of the quality control solution obtained in the step (5) to a quality control detection site of the metabolic mass spectrometry chip, spotting 0.05-5 muL of each tube of the sample solution obtained in the step (4) to a sample detection site of the metabolic mass spectrometry chip in the kit, and drying for 10-30min after spotting is finished;

(8) sending the metabolic mass spectrum chip obtained in the step (7) into a mass spectrometer, and starting a data acquisition process of the mass spectrometer; firstly, a spectrogram of a calibrator solution is collected, the spectrogram is subjected to molecular weight calibration, a quality control product is detected after the calibration, the pretreatment process is confirmed to meet the quality inspection requirement, and then spectrograms of other samples are automatically collected.

In a preferred embodiment of the present invention, the mass spectrometer is a time-of-flight mass spectrometer.

The invention also provides the application of the metabolic spectrum detection kit, and the application of the mass spectrum data generated by the metabolic spectrum detection kit or the use method of the invention in tumor-assisted discrimination or chronic disease monitoring management; wherein the tumor is selected from lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, esophageal cancer or oral cancer, and chronic diseases selected from diabetes, gestational diabetes, hypertension, hyperlipidemia or Alzheimer's disease.

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

1. the metabolic spectrum detection kit provided by the invention combines a metabolic mass spectrum chip with a high-flux mass spectrum technology, obtains metabolic mass spectrograms in a plurality of biological samples in batch under the condition of no matrix spraying, has low cost, high flux and high detection speed, can obtain metabolic spectrum data of 48-102 samples at one time under an automatic detection mode, has the detection speed of about 5 s/single sample, and is suitable for the rapid detection of large samples;

2. the metabolic spectrum detection kit provided by the invention comprises a calibrator and a quality control material, and can be used for regulating and controlling detection parameters in real time so as to ensure the stability and reliability of a detection result;

3. the mass spectrum data acquired by the metabolic spectrum detection kit provided by the invention can be suitable for detection of various biological samples, can be used for pretreatment of metabonomics and development of detection methodology, and is a universal metabolic spectrum detection kit;

4. the metabolic spectrum detection kit provided by the invention is widely applied to tumor auxiliary discrimination, chronic disease monitoring management and property judgment, particularly, the modeling sensitivity and specificity are up to 100% through metabolic spectrum detection and modeling of saliva samples of lung cancer patients and healthy people, and the kit has potential application value.

Drawings

FIG. 1 is a representative metabolic profile of a quality control in a kit of the invention.

FIG. 2 is a schematic view of the entire detection process of the kit of the present invention.

FIG. 3 shows the sensitivity, specificity and accuracy indexes of the kit of the present invention when applied to the lung cancer auxiliary discrimination.

FIG. 4 is a diagram of OPLS-DA when the kit of the present invention is applied to the auxiliary discrimination of lung cancer.

Detailed Description

Specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings, and it should be noted that the embodiments described herein are only for illustration and are not intended to limit the present invention. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be understood by those of ordinary skill in the art that these specific details are not required in order to practice the present invention.

The algorithm such as the OPLS-DA analysis and the double-sample t-test mentioned in the present invention can refer to another patent (CN201910577241.5) that the applicant has applied.

The remaining reagents used in the present invention are commercially available.

Example 1 preparation and detection of quality control Material in a Metabolic Spectroscopy assay kit

A preparation and detection method of a quality control material in a metabolic profile detection kit comprises the following steps:

step one, preparing a quality control product;

(1) collecting saliva samples of multiple persons, wherein the collected persons comprise 15-25 males and 15-25 females;

(2) uniformly mixing the collected saliva in equal amount to form a saliva mixture;

(3) adding the saliva mixture into a pretreatment reagent A in the kit according to a ratio of 1:2, wherein the reagent A comprises one of methanol, acetonitrile, ethanol, acetone and isopropanol or a mixture of methanol, acetonitrile, ethanol, acetone and isopropanol, preferably, the ratio of methanol to acetonitrile in the mixed pretreatment reagent A is 1: 1;

(4) and oscillating the saliva sample added with the pretreatment reagent A for 5-10min, then centrifuging at a low temperature and a high speed, and collecting supernatant. As an example, when the saliva mixture used for pretreatment is 20 μ L, 15 to 30 μ L of supernatant can be collected, and pretreatment reagent C with the volume of 1/3 of supernatant can be added to obtain the quality control product in the kit, and 3 parts of the same quality control product are pretreated in parallel.

Step two, quality control product detection:

(1) spotting 0.05-5 μ L of quality control material on a metabolic mass spectrometry chip, and naturally drying at normal temperature for 10-30 min;

(2) and (4) sending the metabolic mass spectrum chip into a mass spectrometer, and starting the data acquisition process of the mass spectrometer.

FIG. 1 is a representative metabolic profile of the control. The abscissa is mass-to-charge ratio (m/z) without units, and the ordinate is peak intensity without units.

(3) Importing the metabolic spectrogram of the quality control product into a quality inspection library, wherein if the data of 3 parallel quality control samples are in the passing area of the quality inspection library, the kit is a qualified kit, and otherwise, the kit is an unqualified kit; the specific method for constructing the quality inspection library and setting the quality inspection threshold specifically refers to another invention patent CN111239237A of the applicant.

Example 2 Metabolic Spectrum detection kit for Mass Spectrometry automated detection of multiple samples

The number of the metabolic spectrum detection kit used for mass spectrum automatic detection of multiple samples is determined according to sample detection flux on a metabolic mass spectrum chip, and the detection flux is 48-126 samples/chip. As an embodiment, when the detection flux on the metabolic mass spectrometry chip is 108, the disposable collectable mass spectrogram is 102 persons, the rest holes are used as detection sites of a calibrator and a quality control product, and the obtained biological sample comprises saliva, urine, serum, plasma and tissue extract, and the specific steps are as follows:

step one, preprocessing a saliva and urine sample;

(1) taking 20 mu L/part of each biological sample, adding the biological samples into a pretreatment reagent A in the kit according to a ratio of 1:2, wherein the reagent A comprises one of methanol, acetonitrile, ethanol, acetone and isopropanol or a mixture of methanol, acetonitrile, ethanol, acetone and isopropanol, preferably, the ratio of methanol to acetonitrile in the mixed pretreatment reagent A is 1: 1;

(2) and oscillating the biological sample added with the pretreatment reagent A for 5-10min, then centrifuging at a low temperature and a high speed, and collecting supernatant. As an example, when the volume of the biological sample mixture for pretreatment is 20. mu.L, 15 to 30. mu.L of supernatant can be collected, and the mixture is supplemented with a volume 1/3 of pretreatment reagent C, and vortexed and uniformly shaken to obtain a sample solution.

Step two, preprocessing serum, plasma and tissue extract;

(1) taking 20 mu L/part of each biological sample, adding the biological samples into a pretreatment reagent A in the kit according to a ratio of 1:2, wherein the reagent A comprises one of methanol, acetonitrile, ethanol, acetone and isopropanol or a mixture of methanol, acetonitrile, ethanol, acetone and isopropanol, preferably, the ratio of methanol to acetonitrile in the mixed pretreatment reagent A is 1: 1;

(2) and oscillating the biological sample added with the pretreatment reagent A for 5-10min, then centrifuging at a low temperature and a high speed, and collecting supernatant. As an example, when the volume of the biological sample mixture for pretreatment is 20. mu.L, 15 to 30. mu.L of supernatant can be collected to obtain a sample solution 1;

(3) adding a pretreatment reagent B into the liquid left after the supernatant liquid is taken out in the step (1), wherein the pretreatment reagent B is one of methyl tert-butyl ether, trichloromethane and dichloromethane, preferably, when the pretreatment reagent is methyl tert-butyl ether, the addition amount is 200 mu L, and vortex vibration is carried out for 5-15min until the precipitate in the tube is cracked; then adding 50-100 mu L of pretreatment reagent C, wherein the component of the pretreatment reagent C is ultrapure water, and oscillating for 5-10 min;

(4) layering the solution in the step (3) by using a centrifugal method, taking 150 mu L of organic phase and drying to obtain dry powder; then adding 20-40 mu L of pretreatment reagent D for redissolving to obtain a sample solution 2, wherein the component of the pretreatment reagent D is 20-80% of isopropanol solution as a preferable selection;

(5) and (3) mixing the sample solution 1 in the step (2) and the sample solution 2 in the step (4) in equal volumes to obtain sample solutions.

Step three, detecting mass spectra;

(1) sequentially spotting the biological sample and the quality control substance of each detection object on a metabolic mass spectrometry chip, and naturally drying the biological sample and the quality control substance at normal temperature for 10-30min at 0.05-5 mu L/sample;

(2) if the biological sample is saliva or urine, adding a pretreatment reagent A and a pretreatment reagent C which are equal in volume to 10-30 mu L into a calibrator in the kit, carrying out vortex oscillation to dissolve the pretreatment reagent A and the pretreatment reagent C, and dotting the solution on a calibration detection site on a metabolic mass spectrometry chip, wherein the calibration detection site is usually positioned at the central detection site of the whole metabolic mass spectrometry chip;

(3) if the biological sample is serum, plasma or tissue, adding an equal amount of pretreatment reagent A and pretreatment reagent D into a calibrator in the kit, wherein the volume of the pretreatment reagent A and the pretreatment reagent D is 10-30 mu L, performing vortex oscillation to dissolve the pretreatment reagent A and the pretreatment reagent D, and dotting the solution on a calibration detection site on a metabolic mass spectrometry chip, wherein the calibration detection site is usually positioned at the central detection site of the whole metabolic mass spectrometry chip;

(4) sending the metabolic mass spectrum chip into a mass spectrometer;

(5) detecting a spectrogram of the calibration site, calibrating the molecular weight of the spectrogram, and fixing mass spectrum detection parameters;

(6) detecting a spectrogram of the quality control site, and verifying the performance of the kit;

(7) and setting the mass spectrometry method as an automatic detection method, confirming that the instrument positioning system can position the position of each detection site, and calling the automatic mass spectrometry detection method to finish the automatic detection process of all samples on the whole metabolic mass spectrometry chip.

FIG. 2 is a schematic diagram of the whole detection process of the kit.

Example 3 application of saliva metabolism spectrum detection kit in tumor auxiliary discrimination

The application of the metabolic profiling detection kit in tumor auxiliary discrimination comprises the following specific steps:

collecting saliva samples of healthy controls and cancer patients, and carrying out sample pretreatment in batches;

step two, collecting saliva metabolism spectrum data of each group according to the embodiment 2;

step three, carrying out batch normalization processing on the collected saliva metabolism spectrum data;

and step four, performing K-W inspection on the saliva metabolism spectrum data obtained in the step three, and using the molecules meeting the requirement that p is less than 0.05 (with significance level) to participate in genetic algorithm modeling. The model automatically calculates modeling sensitivity, specificity and cross-validation accuracy. As an example, when saliva samples of lung cancer patients and healthy controls are collected, the biomarker markers involved in noninvasive lung cancer discrimination include several of amino acids, organic acids, and fatty acids, and m/z is a combination of y-aminobutyric acid, proline, betaine, nicotinic acid, pipecolic acid, aspartic acid, glutamine, glutamic acid, histidine, and uric acid.

FIG. 3 is a table of the lung cancer model sensitivity, specificity and accuracy data for this example. FIG. 4 is a graph of OPLS-DA (as done in SIMCA, a commercial software) based on these characteristic peaks.

The result shows that the metabolic spectrum detection kit has a good lung cancer auxiliary discrimination function when being used for pretreatment and detection of saliva samples.

It should be noted that the kit for detecting a metabolic profile is applicable to detection of metabolic molecules in a plurality of biological samples including saliva, urine, serum, plasma and the like of lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, esophageal cancer and oral cancer and sample property discrimination, and is not limited to this embodiment.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.