Reagent combination for depression detection, manufacturing method, detection system and application thereof

Abstract

The application relates to a reagent combination for detecting depression, a manufacturing method, a detection system and application thereof, and belongs to the technical field of instrument detection. The reagent combination for detecting depression comprises a calibrator, a quality control sample and an extraction solution of a to-be-detected compound serving as a biomarker; wherein the to-be-detected compound is at least three kinds selected from 5-hydroxyanthranilic acid, methionine, gamma-aminobutyric acid, 3,4-dihydroxyphenylacetic acid, 2-picolinic acid, quinolinic acid, kynurenine, 5-hydroxytryptamine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, dopamine and norepinephrine. The reagent combination for detecting depression and the detection system can significantly improve the accuracy of diagnosis, are convenient to detect, have good repeatability of detection data, are high in specificity, and are accurate in clinical diagnosis results.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of depression detection, and particularly relates to a reagent combination for depression detection, a manufacturing method, a detection system and application thereof. BACKGROUND

[0002] The high rhythm of modern society leads to an increasing number of mental disorders, which may be due to abnormalities in the patient's social, psychological, genetic and physical conditions, and a serious impairment of social adaptation function, resulting in the patient's inability to objectively recognize his or her own mental health status. Mental disorders mainly include six diseases, namely schizophrenia, schizoaffective disorder, paranoid psychosis, bipolar disorder, epilepsy-induced mental disorder and mental retardation with mental disorder. Depression is one of the most common mental illnesses, has a high disability rate, and can affect a person's mood, thinking and behavior. At present, there are at least 320 million patients with various types of depression worldwide, which is the fourth largest disease in the world.

[0003] The official clinical diagnostic criteria for depression mainly include the World Health Organization International Classification of Diseases, Eleventh Edition (ICD-11), the American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), and the Chinese Classification and Diagnostic Criteria for Mental Disorders, Third Edition (CCMD-3). Traditional diagnostic methods, such as depression scales, mainly rely on clinical doctors to assess the patient's symptoms and signs, and refer to standardized diagnostic tools, such as the American Psychiatric Diagnostic and Statistical Manual and the International Classification of Diseases. However, this diagnostic method has the following problems: First, the diagnosis of depression is subjective. Diagnosis mainly relies on the subjective judgment of clinical doctors, including the interpretation of patient self-reports and the assessment of symptoms. Due to the diversity of depression symptoms and individual differences, different doctors may have different diagnostic results, leading to inconsistency and subjectivity in diagnosis. Second, the current diagnostic method for depression may not accurately capture the symptoms of depression in certain groups of people, such as children and adolescents who are younger and may express depression symptoms differently, making it unsuitable for children and adolescents. In addition, the diagnosis of depression is usually based on patient self-reports, but depression patients may have biases in self-reports due to psychological, social or cognitive factors. For example, some patients may underestimate or hide their symptoms due to self-abasement, self-blame or inhibition, leading to missed or misdiagnosed diagnosis. Finally, the diagnosis of depression is mainly based on the patient's current symptoms, but the course of depression is usually complex, involving long-term emotional, cognitive and behavioral changes. The current diagnostic method may not fully consider the long-term changes in the patient's course, which may lead to missed or misdiagnosed diagnosis of depression.

[0004] In recent years, with the continuous development and cross-fusion of genomics, transcriptomics, proteomics and polypeptidomics technologies, specific biomarkers closely related to the occurrence and development of depression for electrochemical sensing technology have been continuously screened and discovered. These biochemical substances that can mark the changes or possible changes in the system, organs, tissues, cells and subcellular structures or functions during the occurrence and development of depression and during the treatment of antidepressants are defined as depression markers (DM). However, there are few depression markers used as diagnostic indicators for clinical depression, and most of the research on biomarkers closely related to depression and their detection methods remains at the basic research stage.

[0005] Sun B. L. from Lanzhou University proposed a construction strategy for electrochemical sensors based on the detection of biomolecules closely related to depression, and proposed several schemes such as electrochemical sensors, electrochemical molecularly imprinted sensors and electrochemical immunosensors. He successfully constructed a competitive electrochemical immunosensor for sensitive analysis of human apolipoprotein A4 (Apo-A4) and electrochemical sensors for cortisol and heat shock protein 70. The constructed electrochemical sensors showed good selectivity, high sensitivity, wide linear range and ultra-low detection limit. However, his research did not provide validation results with confirmed data of depression, and was limited to simple sample analysis, and has not been truly applied to the analysis and evaluation of clinical samples. The feasibility and reliability of his method need to be further verified; the specific limit of the marker as a diagnostic indicator for depression diagnosis and evaluation needs to be standardized.

[0006] Chinese patent CN105759065B proposes the use of blood metabolic markers in the preparation of reagents or instruments for diagnosing depression. The blood metabolic markers are: methionine, nicotinic acid, phosphoserine, lactic acid, glutamic acid, hypoxanthine, choline, tryptophan, 5-hydroxytryptamine, propionyl carnitine, glutaryl carnitine, estradiol, cortisol and estriol. The patent extracts the metabolic products in the depression detection kit with a solvent (methanol-water solution) and 13C stable isotope internal standard solution, extract metabolites on dry blood spots; LC-MS / MS-based targeted metabolomics analysis method, 14 metabolites screened in Example 1 are quantitatively determined in the dry blood spot metabolite extraction solution, and the concentrations of the 14 metabolites are compared with the normal values (0.02-7800 micromole) of the concentrations thereof. If methionine, nicotinic acid, phosphoserine, lactic acid, glutamic acid, hypoxanthine, choline, tryptophan, propionyl carnitine, estradiol, corticosteroid and estriol are significantly increased (>1.2 times of the normal value), and 5-hydroxytryptamine and glutaroylcarnitine are significantly reduced (<0.8 times of the normal value), then it is determined as depression. In addition, 100 people who come to the clinic are diagnosed by a professional doctor through a questionnaire scale, which is used as the gold standard for diagnosis, and compared with the kit method. The accuracy of the claimed metabolomics marker kit for diagnosing depression reaches 93.0%, the specificity is 86.8%, and the sensitivity is 96.8%.

[0007] For the above prior art, most of them are not used in clinical practice and have not been verified in practice. Even if they are used in clinical practice, the detection is complicated and 14 metabolites need to be detected at the same time. The judgment standard is very simple and there is only one situation. If the detection results of the 14 metabolites are not within the claimed range, the result cannot be judged, so the scope of application is also limited. In addition, the specificity is claimed to be 86.8%, and the feasibility and reliability of the product need to be further improved. SUMMARY

[0008] Therefore, the main purpose of the present application is to provide a reagent combination, detection system and application for detecting depression, so as to at least partially solve the above technical problems.

[0009] In order to achieve the above purpose, as a first aspect of the present application, a reagent combination for detecting depression is provided, which comprises a calibrator, a quality control and an extraction solution of a to-be-detected compound as a biomarker. The to-be-detected compound is at least 3 kinds selected from 5-hydroxyanthranilic acid, methionine, gamma-aminobutyric acid, 3,4-dihydroxyphenylacetic acid, 2-pyridinecarboxylic acid, quinolinic acid, kynurenine, 5-hydroxytryptamine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, dopamine and norepinephrine, homocysteine.

[0010] As a second aspect of the present application, a manufacturing method of a reagent combination for detecting depression is also provided, which comprises the following steps:

[0011] All or part of the reagents in the reagent combination for detecting depression are obtained by self-preparation or purchase, and are combined into the reagent combination for detecting depression.

[0012] As a third aspect of the present application, a detection system for detecting depression is also provided, comprising:

[0013] An extraction reagent of the compound to be detected as a biomarker, the extraction reagent comprising the reagent combination for detecting depression as described above;

[0014] A detection device for detecting the sample of the testee treated by the extraction reagent to obtain the content of the compound to be detected.

[0015] As a fourth aspect of the present application, the use of biomarkers in the preparation of a kit for detecting depression is also provided, the biomarkers being at least 3 selected from 5-hydroxyanthranilic acid, methionine, gamma-aminobutyric acid, 3,4-dihydroxyphenylacetic acid, 2-picolinic acid, quinolinic acid, kynurenine, 5-hydroxytryptamine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, dopamine, norepinephrine, homocysteine.

[0016] Based on the above technical solutions, the reagent combination for detecting depression, the detection system and the application of the present application have at least one of the following beneficial effects compared with the prior art:

[0017] (1) New potential biomarkers are screened out, providing new evidence for the diagnosis and treatment of depression;

[0018] (2) Multi-index joint diagnosis significantly improves the accuracy of diagnosis compared with single-index content diagnosis;

[0019] (3) Convenient detection, only a small amount of testee blood, such as fingertip blood or venous blood, is needed, and the result can be obtained after simple detection;

[0020] (4) Accurate and objective, compared with the subjective symptom and sign scale, the detection data has good repeatability and high specificity (greater than or equal to 90%), and the diagnosis result is accurate. BRIEF DESCRIPTION OF DRAWINGS

[0021] The method and device of the present application will be further described below in combination with the drawings and examples:

[0022] Figures 1-13 are the relative content distributions (content ratios) of the 13 biomarkers in the plasma samples of different groups, respectively;

[0023] Figures 14-55 is the ROC curve diagram of various combinations of the 13 preferred biomarkers of the present application for diagnosing major depressive disorder and healthy controls. DETAILED DESCRIPTION

[0024] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed explanations will be given below in combination with specific examples and with reference to the accompanying drawings.

[0025] In the present application, some terms have the following meanings:

[0026] Depression is a common mental disorder that involves long periods of feeling low, losing interest or pleasure in activities, and often affecting an individual's work, study and social functions.

[0027] Standard, i.e. standard article, is a standard content in content determination. The standard includes a stoichiometric standard and a drug testing standard.

[0028] Quality control product is a stable substance for checking the performance of analytical instruments or methods. The quality control product includes a standard and a substance similar to the actual sample matrix.

[0029] Extract, chemical reagent or chemical reagent containing a standard for quantitative determination; used for separating target analytes and biological matrix, reducing the interference of biological matrix on the quantitative determination of target analytes.

[0030] Isotope internal standard is a "stable isotope labeled compound" unique to mass spectrometry method, which is used as an internal standard for mass spectrometric detection. The stable isotope labeled compound is used as an internal standard in mass spectrometric detection. The response abundance ratio (peak area ratio) of the measured substance and the internal standard is used for linear regression. Through accurate mass spectrometric measurement of isotope abundance and accurate measurement of the added internal standard, quantitative determination of the measured substance is achieved.

[0031] Biomarker generally refers to a certain characteristic biochemical index that can be objectively measured and evaluated in a general physiological or pathological or therapeutic process, and through its measurement, the current progress of the organism in the biological process can be known. In the present application, the preferred compound is a metabolite that is differentially present (i.e. increased or decreased) in the biological sample of a subject or a group of subjects having a first phenotype (e.g. suffering from a disease) compared to a subject having a biological phenotype. The biomarker is preferably differentially present (i.e. p value less than 0.05, determined using Welch's T test or Wilcoxon rank sum test) at a statistically significant level.

[0032] A "metabolite" or "small molecule" refers to an organic and inorganic molecule that is present in a cell. The term does not include macromolecules, such as large proteins, large nucleic acids, or large polysaccharides. Small molecules in a cell are typically found in solution in the cytoplasm or other organelles (e.g., mitochondria), where they form a pool of intermediates that can be further metabolized or used to make macromolecules. The term "small molecule" includes signaling molecules and intermediates in chemical reactions that convert energy in food into usable forms. Examples of small molecules include sugars, fatty acids, amino acids, nucleotides, intermediates formed in cellular processes, and other small molecules found within cells.

[0033] Specificity, in the context of a medical test for diagnosing a disease, refers to the ability to correctly identify those who are not diseased.

[0034] Sensitivity, in the context of a medical test for diagnosing a disease, refers to the ability to correctly identify those who are diseased.

[0035] Kit, a box for containing chemical reagents for detecting chemical components, drug residues, virus species, etc. Some kits also include sample plates for facilitating sample drop and result presentation. The kit is produced to enable the experimenter to get rid of the tedious reagent preparation and optimization process, so the kit generally comes with a corresponding instruction manual, and the user can get satisfactory results without or with only a small amount of optimization according to the instruction manual.

[0036] The present inventors have conducted a large amount of research on the diagnosis of depression, and through searching, it has been found that there are several metabolic pathways related to depression, and there are about three to four thousand small molecule compounds involved. Although there are many articles, reports, and patent results related to the detection and diagnosis methods of depression, there are very few schemes that are actually used in clinical practice. There are few reagent kit products for detecting small molecule biomarkers of depression on the market, and the existing technologies reported in the public domain are still in the state of clinical improvement and cannot enter the practical state. After in-depth research on the detection method, repeated optimization of the data and clinical verification, the present inventors have proposed several reagent combinations for detecting depression, manufacturing methods, detection systems, and applications thereof, which can quickly and accurately diagnose whether the tested object has depression through the simplest detection means. The improvement of the detection means is applied in another case.

[0037] Specifically, the present application discloses a reagent combination for detecting depression, which comprises a calibrator, a quality control product, and an extraction solution of a to-be-detected compound as a biomarker.

[0038] The to-be-detected compounds are preferably at least 3 of 5-hydroxyanthranilic acid (5-HAA), methionine (MET), gamma-aminobutyric acid (ABU), 3,4-dihydroxyphenylacetic acid (DPA), 2-pyridinecarboxylic acid (PICA), quinolinic acid (QUIN), kynurenine (KYN), 5-hydroxytryptophan (5-HTP), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), dopamine (DA), norepinephrine (NE), and homocysteine (HCY), and more preferably any 3-6 of them.

[0039] Figures 1-13 The relative content distribution (content ratio) of the 13 biomarkers in the plasma samples of different groups is shown in the figures. It can be seen from the figures that these biomarkers are very helpful for distinguishing and detecting patients with severe depression and healthy people.

[0040] The to-be-detected compounds are preferably at least 3 of 5-hydroxyanthranilic acid (5-HAA), methionine (MET), gamma-aminobutyric acid (ABU), 3,4-dihydroxyphenylacetic acid (DPA), 2-pyridinecarboxylic acid (PICA), quinolinic acid (QUIN), kynurenine (KYN), 5-hydroxytryptophan (5-HTP), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), dopamine (DA), norepinephrine (NE), and homocysteine (HCY), and more preferably any 3-6 of them.

[0041] The to-be-detected compounds are preferably at least 3 of 5-hydroxyanthranilic acid (5-HAA), methionine (MET), gamma-aminobutyric acid (ABU), 3,4-dihydroxyphenylacetic acid (DPA), 2-pyridinecarboxylic acid (PICA), quinolinic acid (QUIN), kynurenine (KYN), 5-hydroxytryptophan (5-HTP), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), dopamine (DA), norepinephrine (NE), and homocysteine (HCY), and more preferably any 3-6 of them.

[0042] By using the above-mentioned reagent combination for detecting depression, the content of the to-be-detected compounds can be accurately detected by means of chromatography, high-performance liquid chromatography (HPLC), or a combination thereof, and then the risk of severe depression of the detected person can be calculated by using the fitting formula discovered by the inventor.

[0043] The reagent combination for detecting depression can further comprise an isotopic internal standard for mass spectrometric detection, so that the content of the to-be-detected compounds can be accurately detected by mass spectrometry or chromatography-mass spectrometry.

[0044] The reagent combination is, for example, a kit provided to medical diagnostic personnel in the form of a kit (Kit) to facilitate their accurate and rapid operation and diagnosis.

[0045] In a preferred embodiment, the sample for detection can be prepared as follows: 50-100 μL of plasma obtained from the intravenous blood of the subject is added with isotopic internal standards corresponding to several compounds, mixed, 8-15 μL of extraction solution is added, mixed, left for 10 min, centrifuged, 50-100 μL of supernatant is taken, 50-100 μL of dilution solution is added, mixed for 2 min; standby, used for detection of the instrument.

[0046] The application also provides a manufacturing method of the reagent combination for detecting depression, comprising the following steps:

[0047] The reagents for detecting depression as described above are obtained by self-preparation or purchase, and are combined into a reagent combination for detecting depression, such as a kit (Kit).

[0048] The application also discloses a detection system for detecting depression, comprising:

[0049] The extraction reagent of the to-be-detected compound as a biomarker; wherein the to-be-detected compound is selected from at least 3 of 5-hydroxyanthranilic acid, methionine, γ-aminobutyric acid, 3,4-dihydroxyphenylacetic acid, 2-picolinic acid, quinolinic acid, kynurenine, 5-hydroxytryptamine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, dopamine, norepinephrine and homocysteine, and is further preferably selected from any 3-6 of them.

[0050] The detection device is used for detecting the sample treated by the above extraction reagent, to obtain the content of the to-be-detected compound.

[0051] The to-be-detected compound is further preferably selected from at least 3 of 5-hydroxyanthranilic acid, γ-aminobutyric acid, 3,4-dihydroxyphenylacetic acid, 2-picolinic acid, quinolinic acid, kynurenine, 3-hydroxykynurenine, 5-hydroxytryptamine, 3-hydroxyanthranilic acid and homocysteine, and is further preferably selected from any 3-6 of them.

[0052] The to-be-detected compound is further preferably selected from at least 3 of 2-picolinic acid, quinolinic acid, kynurenine, 5-hydroxytryptamine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid, and is further preferably selected from any 3-4 of them.

[0053] Through the above reagent combination for detecting depression, the content of the to-be-detected compound can be accurately detected by a detection device, and then the risk of severe depression of the subject is calculated based on the fitting formula obtained by the inventors through in-depth research.

[0054] The reagent combination for detecting depression can further include an isotope internal standard for mass spectrometric detection, so that the content of the compound to be detected can be accurately detected by mass spectrometry or chromatography, and the detection equipment is mass spectrometry or chromatography equipment.

[0055] The detection equipment can further include a data processing module, which can be a part of the detection equipment or a separate computing device independent of the detection equipment, so that the data processing module can receive the detection results of the detection equipment and determine the risk of severe depression of the testee based on a fitting formula obtained by fitting a multiple linear equation or a power exponential equation to the detected biomarker concentrations in advance.

[0056] The fitting formula obtained by fitting a multiple linear equation or a power exponential equation to the detected biomarker concentrations in advance can be as follows:

[0057] ln(p / 1-p)=A0+A1×C1+A2×C2+……+A n ×C n ;

[0058] Or,

[0059] Wherein, p is the probability of severe depression, A0, A1, …, A n is a fitting correction coefficient, C1, C2, …, C n is the concentration of each biomarker detected. The fitting formula can be obtained by fitting the correction coefficient of each biomarker in the above formula to a large amount of detection data.

[0060] The fitting formula obtained can be input into the data processing platform of the detection equipment itself or stored in a U disk or other external storage device that can be read by the detection equipment. The specific determination logic is realized by the operation unit of the detection equipment, so that the data processing module is a part of the detection equipment. Alternatively, the fitting formula obtained can be input into a separate external computing device, and the risk of severe depression of the testee can be determined by the external computing device based on the detection result and the fitting formula by inputting the detection result into the external computing device in any manner such as manual input, network transmission or storage medium transmission, and the data processing module is independent of the detection equipment.

[0061] The application also discloses use of the biomarker in preparation of a kit, for example, use of the biomarker in a severe depression detection kit; and use of the biomarker in a detection system, for example, use of the biomarker as a to-be-detected compound of the detection system, and judgment of whether the subject has severe depression based on a detected concentration of the biomarker.

[0062] The application will be further described and illustrated by specific examples. It should be noted that the following examples are only illustrative and are not intended to limit the application.

[0063] For the convenience of the test, the example part will simultaneously detect the content of 5-hydroxyanthranilic acid, methionine, gamma-aminobutyric acid, 3,4-dihydroxyphenylacetic acid, 2-picolinic acid, quinolinic acid, kynurenine, 5-hydroxytryptophan, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, dopamine, norepinephrine, homocysteine and other to-be-detected compounds in blood, and then construct a corresponding model or fitting formula through statistical analysis or algorithm in each example.

[0064] I. The kit is used as an experimental medicine in each example, and the kit comprises:

[0065] (1) Instrument test: polar end-capped C18 chromatographic column, 1% formic acid (volume fraction) and 0.03% trifluoroacetic acid (volume fraction) mixed aqueous solution as mobile phase additive A, and 1% formic acid (volume fraction) and 90% acetonitrile aqueous solution (volume fraction) mixed solution (1% (v / v) formic acid solution) as mobile phase additive B.

[0066] (2) Sample detection: reagents for detecting 5-hydroxyanthranilic acid, methionine, gamma-aminobutyric acid, 3,4-dihydroxyphenylacetic acid, 2-picolinic acid, quinolinic acid, kynurenine, 5-hydroxytryptophan, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, dopamine, norepinephrine and homocysteine in blood plasma.

[0067] ① Calibration: a mixed solution containing 5-hydroxyanthranilic acid, methionine, gamma-aminobutyric acid, 3,4-dihydroxyphenylacetic acid, 2-picolinic acid, quinolinic acid, kynurenine, 5-hydroxytryptophan, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, dopamine, norepinephrine and homocysteine;

[0068] ② Quality control: a plasma solution containing 5-hydroxyanthranilic acid, methionine, gamma-aminobutyric acid, 3,4-dihydroxyphenylacetic acid, 2-picolinic acid, quinolinic acid, kynurenine, 5-hydroxytryptophan, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, dopamine, norepinephrine and homocysteine.

[0069] 3. Internal standard solution: 5% methanol aqueous solution containing 5-hydroxyanthranilic acid-D5, methionine-D3, gamma-aminobutyric acid-D3, 3,4-dihydroxyphenylacetic acid-D3, 2-pyridinecarboxylic acid-D4, quinolinic acid- 13 C3 15 N, kynurenine- 13 C6, 5-hydroxytryptophan-D4, 3-hydroxykynurenine- 13 C6, 3-hydroxyanthranilic acid-D3, dopamine-D4, norepinephrine-D5, homocysteine-D4;

[0070] 4. Extraction solution: 5-sulfosalicylic acid aqueous solution with a mass fraction of 2% dithiothreitol (DTT) and a volume fraction of 15-30%;

[0071] 5. Dilution solution: a mixed solution of 0.2% formic acid (volume fraction) and 0.006% trifluoroacetic acid (volume fraction).

[0072] Of course, the kit form can not be used, but the reagents purchased or prepared by oneself can also be used as long as the purpose of the present application can be achieved.

[0073] II. Detection process

[0074] (1) Experimental subjects

[0075] Through cooperation with two municipal hospitals with certain geographical distance in Jiangsu, the following experimental data were collected and sorted. Among the total of 400 subjects, the age range was 18-66 years old, including 150 cases of depressive patients (MDD) and 120 healthy controls (HC). The detailed information of the subjects is shown in Table 1:

[0076] Table 1 Detailed information of subjects

[0077]

[0078] a: Chi-square test; b: K-W test

[0079] It should be noted that since depression is still diagnosed by means of scales, etc., there is no gold standard for other instrument detection at present, and therefore only severe patients with clear manifestations are selected for the above-mentioned depressive patients to ensure the accuracy of the results. In future research, the study of mild and moderate patients will be gradually carried out to explore more scientific, objective and gold standard detection methods.

[0080] In addition, the above detection has informed the tested subjects who collected the samples and obtained their consent.

[0081] (2) Sample collection

[0082] Collect venous blood into vacuum blood collection tubes containing anticoagulants. Mix well by slowly inverting, store at 2-8℃, complete centrifugal separation within 30 min to obtain plasma, transfer the plasma to EP tubes, and store at -80℃ for later use.

[0083] (3) Sample pretreatment

[0084] Accurately pipette 50 μL of plasma into a 1.5 mL EP tube, add 20 μL of internal standard, then add 8 μL of 30% (volume fraction) 5-sulfosalicylic acid solution containing 2% (mass fraction) DTT, high-frequency oscillate for 5 min for protein precipitation; centrifuge at 11000 rpm for 10 min at 4℃; take 50 uL of supernatant and place it in a 96-well plate, then add 50 μL of a mixture of 0.2% formic acid water (volume fraction) and 0.015% trifluoroacetic acid (volume fraction) to each empty position, low-frequency oscillate for 2 min, and then detect on the instrument.

[0085] (4) UPLC-MS / MS detection parameter settings

[0086] Separation was performed using a Thermofisher aQ column, with a column temperature of 35℃, an injection volume of 5 uL, mobile phase A: 0.0075% trifluoroacetic acid-0.1% formic acid water, mobile phase B: 0.1% formic acid water 90% acetonitrile water, and the mobile phase gradient was set as shown in Table 2:

[0087] Table 2 Mobile phase gradient settings

[0088]

[0089] The mass spectrometry device parameter settings are shown in Table 3:

[0090] Table 3 Mass spectrometry device parameter settings

[0091] Capillary voltage 1.0 kv Ion source temperature 150℃ Desolvation temperature 450℃ Desolvation flow 650 L / h

[0092] (5) Data analysis and diagnosis

[0093] A. Based on the software provided with the liquid chromatography tandem mass spectrometer (such as MassLynx software), a calibration curve was established, and the corresponding factors of each compound in the on-machine liquid were substituted into the linear equation to output the content of each compound in the plasma;

[0094] B. For the combined index, the content of a single compound was introduced into the self-built system, and the combined index was calculated according to the previously constructed data model;

[0095] (6) Actual measurement data

[0096] The reagent is used for distinguishing healthy controls from severe depression; the contents of biomarkers in the blood of 120 healthy people and 150 severe depression people are detected, one-way analysis of variance is performed, the content differences of selected biomarkers in healthy people and severe depression patients are evaluated, a multiple logistic regression diagnostic model is established, and ROC analysis is further performed to quantify the diagnostic performance of the combination containing multiple selected biomarkers (metabolites).

[0097] Example 1

[0098] The present embodiment discloses a reagent combination for detecting depression, which comprises calibrators, quality control samples, extraction solutions, diluents, internal standards of 3-hydroxyanthranilic acid, kynurenine and quinolinic acid, mobile phase additives. 13 C6, quinolinic acid 13 C3 15 N.

[0099] The blood sample of the testee is detected by the above-mentioned reagent combination, the plasma in the blood sample is extracted, and the contents of 3-hydroxyanthranilic acid, kynurenine and quinolinic acid are detected by liquid chromatography tandem mass spectrometry. The experimental results, the detection accuracy for depression and the difference data are shown in Table 6-1.

[0100] Examples 2-36

[0101] The specific reagent composition, test instrument and test process of examples 2-36 are the same as those of example 1, and the only difference is that the selected biomarkers are shown in Table 4-1 as follows.

[0102] The experimental results, the detection accuracy for depression and the difference data are shown in Table 6-1.

[0103] Comparative examples 1-8

[0104] The specific reagent kit composition, test instrument and test process of comparative examples 1-8 are the same as those of example 1, and the only difference is that the selected biomarkers are shown in Table 4-2 as follows.

[0105] The experimental results, the detection accuracy for depression and the difference data are shown in Table 6-2.

[0106] Table 4-1 selected biomarkers in each example

[0107]

[0108]

[0109] Table 4-2 selected biomarkers in each comparative example

[0110]

[0111] Table 5-1 Model formula of examples

[0112]

[0113]

[0114]

[0115] Table 5-2 Model formula of comparative examples

[0116]

[0117] Table 6-1 Experimental test results of each example

[0118]

[0119]

[0120] wherein: *p<0.05 **p<0.01

[0121] Table 6-2 Experimental test results of each comparative example

[0122]

[0123] wherein: *p<0.05 **p<0.01

[0124] Figures 14-55 The ROC curve figures of various combinations of the 13 preferred biomarkers of the present application for diagnosing major depressive disorder and healthy controls. Each figure is matched with a part of the examples according to the combination of the detected compounds (biomarkers) therein, for example Figure 14 corresponding to example 1, Figure 20 corresponding to example 22. The above figures are not strictly sorted according to the examples, and the number exceeds the number of examples, which is intended to better demonstrate the advantages and benefits of the present application.

[0125] The above figures are combined with the Figures 14-55As can be seen from Examples 1-36 and Comparative Examples 1-8, when the above data is for three components, although the part sensitivity and / or specificity values are lower than 0.900, the overall performance can still meet the general detection requirements. When the preferred components are more than three, the specificity is generally more than 0.930, and even reaches 1.000; the sensitivity is at least 0.900, thus indicating that from the ten or so biomarkers selected by the inventor, the more selected, the specificity and sensitivity of the final result are not necessarily better, but at least will not be significantly reduced to below 0.900, and can all meet the purposes of the present application and be used for clinical practical application.

[0126] In addition, as can be seen from Comparative Examples 1-8, when the candidate biomarkers are less than 3, the fitted results cannot be used for diagnosis of major depressive disorder.

[0127] Based on the above data, it can be seen that the preferred biomarker combination provided by the present application for assisting in predicting and diagnosing depression has high sensitivity and specificity, and can accurately determine whether the testee has major depressive disorder and bipolar depression.

[0128] In addition, the above data is collected from two city-level hospitals with a certain geographical distance in Jiangsu, and by verifying the data of two different hospitals, it is proved that the method of the present application has good reproducibility, high accuracy of clinical diagnosis and the like, and can fully perform the clinical diagnosis work of general hospitals for severe patients, and has good practicability.

[0129] The above-described specific embodiments further illustrate the purposes, technical solutions and beneficial effects of the present application, and it should be understood that the above-described is only a specific embodiment of the present application, and is not used to limit the present application, and any modification, equivalent replacement, improvement and the like made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A detection system for major depressive disorder, characterized in that, include: A reagent kit for detecting major depressive disorder, extraction reagents for the target compound as a biomarker, and detection equipment; The reagent combination includes calibrators, quality control samples, and extraction solutions for the compound to be detected as a biomarker; The compound to be detected is selected from any of the following combinations: 3-Hydroxy-anaminobenzoic acid, kynurenic acid, quinolinic acid; 2-Pyridinecarboxylic acid, 3-hydroxy-o-aminobenzoic acid, quinoline acid; 5-Hydroxytryptophan, 2-pyridinecarboxylic acid, 3-hydroxy-an-aminobenzoic acid; 3-Hydroxykynurenine, 2-pyridinecarboxylic acid, 5-hydroxytryptophan; 2-Pyridinecarboxylic acid, 3-hydroxy-an-aminobenzoic acid, 3-hydroxykynurenine; Quinolinic acid, 3-hydroxykynurenine, 2-pyridinecarboxylic acid; 5-Hydroxytryptophan, 2-pyridinecarboxylic acid, 3-hydroxy-an-aminobenzoic acid, kynurenine; 2-Pyridinecarboxylic acid, 3-hydroxy-an-aminobenzoic acid, 3-hydroxykynurenine, homocysteine; 5-Hydroxytryptophan, homocysteine, 2-pyridinecarboxylic acid, 3-hydroxy-an-aminobenzoic acid; 3-Hydroxy-anaminobenzoic acid, kynurenine, quinolinic acid, 2-pyridinecarboxylic acid, 5-hydroxytryptophan; 5-Hydroxytryptophan, 2-pyridinecarboxylic acid, 3-hydroxy-an-aminobenzoic acid, kynurenine, 3-hydroxykynurenine, quinolinic acid; 3-Hydroxy-anaminobenzoic acid, kynurenine, quinolinic acid, 2-pyridinecarboxylic acid, 5-hydroxytryptophan, homocysteine, 3-hydroxykynurenine; 3,4-Dihydroxyphenylacetic acid, 3-hydroxy-o-aminobenzoic acid, quinoline acid; γ-aminobutyric acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxy-o-aminobenzoic acid; Homocysteine, quinolinic acid, 3-hydroxy-an-aminobenzoic acid; Homocysteine, quinolinic acid, 3-hydroxy-2-aminobenzoic acid, 5-hydroxy-2-aminobenzoic acid; Kyreninine, 2-pyridinecarboxylic acid, 3-hydroxy-an-aminobenzoic acid, 3,4-dihydroxyphenylacetic acid, 5-hydroxytryptophan; Quinolinic acid, 3-hydroxykynurenine, 2-pyridinecarboxylic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxy-o-aminobenzoic acid; 3,4-Dihydroxyphenylacetic acid, 3-hydroxy-o-aminobenzoic acid, quinolinic acid, 2-pyridinecarboxylic acid, kynurenine, 5-hydroxytryptophan; γ-aminobutyric acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxy-o-aminobenzoic acid, 3-hydroxykynurenine, homocysteine, 2-pyridinecarboxylic acid; 3-Hydroxyanaminobenzoic acid, kynurenine, quinolinic acid, 2-pyridinecarboxylic acid, 5-hydroxytryptophan, homocysteine, 5-hydroxyanaminobenzoic acid; 3-Hydroxy-anaminobenzoic acid, kynurenine, quinolinic acid, 2-pyridinecarboxylic acid, 5-hydroxytryptophan, 5-hydroxy-anaminobenzoic acid, homocysteine, 3-hydroxykynurenine; 5-Hydroxyanaminobenzoic acid, 3,4-dihydroxyphenylacetic acid, homocysteine, 3-hydroxyanaminobenzoic acid, 2-pyridinecarboxylic acid, quinolinic acid, kynurenine, 5-hydroxytryptophan, 3-hydroxykynurenine; 3-Hydroxy-o-aminobenzoic acid, dopamine, 5-Hydroxy-o-aminobenzoic acid; Homocysteine, 3-hydroxy-an-aminobenzoic acid, kynurenine; Kyrenine, 2-pyridinecarboxylic acid, norepinephrine; 3-Hydroxy-o-aminobenzoic acid, dopamine, 2-pyridinecarboxylic acid; Methionine, 3,4-dihydroxyphenylacetic acid, 2-pyridinecarboxylic acid; 3-Hydroxy-anaminobenzoic acid, 3,4-dihydroxyphenylacetic acid, methionine; 5-Hydroxytryptophan, 2-pyridinecarboxylic acid, 3-hydroxy-an-aminobenzoic acid, kynurenine, dopamine; Norepinephrine, 2-pyridinecarboxylic acid, γ-aminobutyric acid, 3-hydroxy-an-aminobenzoic acid, homocysteine; 3-Hydroxy-anaminobenzoic acid, 3,4-dihydroxyphenylacetic acid, methionine, 2-pyridinecarboxylic acid, homocysteine, 5-hydroxytryptophan; Methionine, 3,4-dihydroxyphenylacetic acid, 2-pyridinecarboxylic acid, 3-hydroxy-an-aminobenzoic acid, kynurenine, 5-hydroxytryptophan; Kynurinine, quinolinic acid, 5-hydroxytryptophan, homocysteine, 5-hydroxy-an-aminobenzoic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxykynurinine, dopamine, norepinephrine, methionine; Kynurinine, quinolinic acid, 5-hydroxytryptophan, homocysteine, 5-hydroxy-an-aminobenzoic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxykynurinine, dopamine, norepinephrine, methionine, 2-pyridinecarboxylic acid; 3-Hydroxyanaminobenzoic acid, kynurenine, quinolinic acid, 2-pyridinecarboxylic acid, 5-hydroxytryptophan, homocysteine, 5-hydroxyanaminobenzoic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxykynurenine, dopamine, norepinephrine, methionine; The detection device is used to detect the sample of the test subject after it has been treated with the extraction reagent, and to obtain the content of the compound to be detected; The sample from the test subject was blood.

2. The detection system for major depressive disorder according to claim 1, characterized in that, The reagent kit for detecting major depressive disorder also includes an isotopic internal standard for mass spectrometry detection; and / or The reagent combination used for the detection of major depressive disorder is a kit.

3. The detection system for major depressive disorder according to claim 1, characterized in that, The detection equipment is a chromatographic, high-performance liquid chromatography, mass spectrometry, or chromatographic-mass spectrometry device; and / or The detection system also includes a data processing module, which is part of the detection device or a separate computing device independent of the detection device. The data processing module receives the detection results from the detection device and assesses the risk of major depressive disorder in the test subject based on a fitting formula obtained by fitting a multivariate linear equation or power exponential equation to the concentration of the detected biomarkers in advance. The fitting formula is as follows: ； or, ; in, p This is the probability of developing severe depression. These are the fitting correction coefficients. The concentration detected for each biomarker.

4. A method for manufacturing a reagent combination for detecting major depressive disorder, characterized in that, Includes the following steps: Obtain all or part of the reagents in the reagent kit for detecting major depressive disorder as described in any one of claims 1-2 by self-manufacturing or purchasing, and combine them into a reagent kit for detecting major depressive disorder.

5. The application of a reagent combination for detecting biomarkers in the preparation of a kit for detecting major depressive disorder, characterized in that, The biomarker is the compound to be detected as described in claim 1.

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