CN115266972A - Combined marker, screening method, diagnostic reagent or kit and application thereof - Google Patents
Combined marker, screening method, diagnostic reagent or kit and application thereof Download PDFInfo
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- G—PHYSICS
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- 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
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- 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
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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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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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- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
Abstract
The invention provides a screening method of macaque spontaneous diabetes biological combined markers, obtains a group of combined markers, and discloses a diagnostic reagent or a kit with one or more diagnostic markers of myristoleic acid, oleic acid and palmitic acid by utilizing the principle of the combined markers. The macaque spontaneous diabetes biomarkers myristic acid, oleic acid and palmitic acid provided by the invention are high in sensitivity and specificity and good in predictability, can be applied to aspects of auxiliary diagnosis of type 2diabetes, auxiliary evaluation of the disease risk and prognosis condition of type 2diabetes, auxiliary research of type 2diabetes and the like, have important guiding significance for early detection and prevention of type 2diabetes, can be popularized, and have important significance in individuation, sensitive identification, screening and genetic consultation.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to screening and application of a blood metabolism marker of a macaque with spontaneous diabetes.
Background
Diabetes is characterized by hyperglycemia and is a chronic metabolic disease caused by long-term combined action of genetic factors and environmental factors. Type 2diabetes (T2DM) accounts for over 90% of the diabetic population and is the leading Type of diabetes. The international diabetes union published data shows: the global prevalence of diabetes in 2019 was 9.3%, which increased to 10.2% in 2030 and 10.9% in 2045. This data continues to grow with the rapid growth of economies. Diabetes not only reduces the quality of life of patients and increases the economic burden of families, but also causes national economic loss, and the medical expenditure of more than 6000 billion RMB in China is related to diabetes every year. Diabetic complications have also become a significant cause of death from non-neoplastic diseases, and thus the prevention and control of diabetes remains a significant challenge. The spontaneous T2DM macaque can simulate the pathogenesis characteristics of human T2DM to the greatest extent, and therefore, the screening and application of the spontaneous T2DM macaque biomarker provides important preconditions for establishing a non-human primate model of spontaneous T2DM and developing human T2DM pathogenesis, pathological mechanism, drug screening and the like.
The pathogenesis of diabetes is very complex, the blood sugar change of early diabetes is not obvious, single blood sugar detection only can carry out preliminary screening on the diabetic macaque, and wrong screening and screen leakage are easy to occur. Therefore, more accurate indexes are needed to judge whether the macaques have diabetes.
The metabolite can reflect the information of apoptosis, energy generation, storage and the like of cells. At present, metabolomics technology has been applied in many fields of T2DM treatment, such as drug treatment of T2DM, bioactive food component treatment of T2DM, weight loss surgery treatment of T2DM, exercise treatment of T2DM, and the like. However, there is no method or product for rapid diagnosis of spontaneous diabetes in macaques using metabonomic methods.
Disclosure of Invention
Aiming at the technical problems that in the prior art, single blood sugar detection only can be used for preliminarily screening diabetic macaques, wrong screening and screen leakage are easy to occur, and the like, the blood samples and excrement of normal macaques and spontaneous diabetic macaques are collected, the samples are analyzed by a metabonomics method of liquid chromatography-mass spectrometry combined analysis, so that differential metabolites of the normal macaques and the spontaneous diabetic macaques are obtained, and further T2DM diagnosis is assisted, and T2DM disease risk or prognosis condition is assisted to be evaluated or T2DM is assisted to be researched. The invention finds out the metabolites related to the spontaneous T2DM of the macaque based on the non-targeted metabolome and the targeted medium-long chain fatty acid metabolome detection technology, and processes and analyzes the metabolites for further application.
In one aspect, the invention provides a diagnostic reagent or kit for macaque idiopathic diabetes, wherein a diagnostic marker of the diagnostic reagent or kit is one or more of myristoleic acid, oleic acid and palmitic acid.
Specifically, the diagnostic marker of the diagnostic reagent or the diagnostic kit comprises three of myristoleate (C14: 1N 5), oleic acid (oleic, C18:1N 9) and palmitic acid (palmitate, C16: 0).
The biomarkers of the invention are derived from stool or ex vivo blood samples.
More preferably, the detection thresholds of the biomarkers of the invention are respectively: the threshold value of the myristic acid is 23.39ug/mL, the threshold value of the oleic acid is 0.15ug/mL, and the threshold value of the palmitic acid is 219.41ug/mL.
The diabetes is macaque spontaneous diabetes.
In a second aspect, the invention also provides the use of one or more of the biomarkers myristoleic acid, oleic acid and palmitic acid in the manufacture of a diabetes aid product, including a product to aid in the diagnosis of type 2diabetes, to aid in the assessment of the risk of developing type 2diabetes or the prognostic status, or to aid in the study of type 2 diabetes.
In a third aspect, the present invention provides the use of one or more of the biomarkers myristoleic acid, oleic acid and palmitic acid in the manufacture of a diabetes detection product.
Specifically, the detection product comprises a detection chip for type 2diabetes, a detection reagent for type 2diabetes or a detection kit for type 2 diabetes.
In a fourth aspect, the present invention also provides a combination marker for assessing idiopathic diabetes in rhesus monkeys, the combination comprising myristoleic acid, oleic acid, and palmitic acid.
In a fifth aspect, the invention provides a screening method for biomarkers of spontaneous diabetes mellitus of macaques, wherein the biomarkers are obtained by a metabonomics method based on liquid chromatography-mass spectrometry analysis.
Specifically, the screening method of the kiwi fruit spontaneous diabetes biomarker comprises the following steps:
1) Primarily screening spontaneous diabetic macaques;
2) Detecting physical and chemical indexes of the spontaneous diabetic macaque;
3) A non-targeted metabolome screening marker;
4) Targeted medium-long chain fatty acid metabolome detection.
Specifically, the preliminary screening of the spontaneous diabetic macaque provided by the invention comprises the following steps:
recording the length of the selected macaque according to the diabetes diagnosis standard and the requirement of not using antibiotics within three months, and simultaneously detecting the Fasting Plasma Glucose (FPG) value of the macaque to be detected; the macaques in the T2DM group have fasting blood glucose values meeting FPG (fast food group) not less than 7mmol/L for 3 times of detection in 1 year, the macaques in the control group have fasting blood glucose values meeting FPG not more than 6.1mmol/L for 3 times of detection in 1 year, the macaques in the T2DM group and the macaques in the control group are respectively numbered and fed in a single cage, and excrement sample and blood sample can be conveniently collected subsequently.
Specifically, the physical and chemical indexes of the spontaneous diabetic macaque provided by the invention are detected as follows:
the screened macaques are fasted and fed for 12 hours without water supply. The method comprises the steps of collecting venous blood of a macaque by using a biochemical tube, noting information such as sampling time, sampling place, sample number and the like on the tube body, and then placing the macaque in a foam box with an ice bag to be transported back to a laboratory. The collected blood samples are immediately subjected to detection of physiological and biochemical indexes of Fasting glucose (FPG), glycosylated hemoglobin A1c (HbA 1 c), fasting insulin (FPI), total Cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL) and Low-density lipoprotein (LDL), and then the BMI value of the experimental macaque is calculated by combining the provided weight data, wherein the specific formula is as follows:
weight (kg)/length (m)2
HOMA-IR is one of the standards for judging T2DM, and the calculation mode is as follows:
(FPI×FPG)/22.5。
specifically, the non-targeted metabolome screening marker comprises the following steps:
s1, collecting manure sample
A sampling person wearing two layers of sterile gloves (the inner layer is latex gloves, and the outer layer is disposable PE gloves) collects a fecal sample in ten minutes after the macaque is fed in a single cage, peels off the outer layer in contact with a fecal receiving plate, puts the fecal sample into a 50mL sterile centrifuge tube for later use, and records information such as collection time, place, sample number and the like on a sampling tube. And when the next excrement sample is collected, replacing the new PE gloves, and repeating the collection process. And finally storing all collected feces samples in a refrigerator at the temperature of-80 ℃.
S2, treatment and analysis of manure sample
Stool samples stored at-80 ℃ were taken out and placed in a 4 ℃ environment for slow thawing, methanol, acetonitrile and aqueous solution were mixed at a ratio of 2:2:1, uniformly mixing and precooling for later use, adding the unfrozen sample into the mixed solution, carrying out vortex mixing, sequentially carrying out low-temperature ultrasonic treatment for 30min, standing for 10min and centrifuging for 20min (14000g, 4 ℃), and then taking supernatant to carry out chromatography-mass spectrometry. Separating the supernatant by adopting an Agilent 1290Infinity LC ultra-performance liquid chromatography system (UHPLC) HILIC chromatographic column, wherein the separation parameters are a default program: 0-0.5min, the mobile phase composition is 95% acetonitrile; the acetonitrile concentration is linearly reduced to 65 percent in 0.5-7 min; the acetonitrile concentration is linearly reduced to 40 percent within 7-8 min; 8-9min, and maintaining the concentration of acetonitrile at 40%;9-9.1min, acetonitrile concentration is increased from 40% to 95% linearly; 9.1-12min, and the concentration of acetonitrile is maintained at 95%; the separated sample is detected by an AB Triple TOF 6600 mass spectrometer in an electrospray ionization (ESI) mode respectively for positive ions and negative ions to obtain original data.
After the raw data is converted into an mzXML format, XCMS software is used for peak alignment, retention time correction and peak area extraction. And (3) carrying out metabolite structure identification, data preprocessing and data quality evaluation on the data processed by XCMS in sequence, and finally carrying out data analysis. And screening differential metabolites by combining univariate statistical analysis and multidimensional statistical analysis, displaying the difference of the metabolic levels of the experimental group in the control group through cluster analysis, correlation analysis and KEGG channel enrichment analysis, and excavating the biological processes and the regulation and control channels in which the differential metabolites participate.
S3, screening of differential metabolites
14665 metabolites were detected in total between the T2DM group and the control group in the positive and negative ion modes, and the metabolites were significantly different between the T2DM group and the control group in the positive and negative ion modes.
A total of 1564 metabolites were identified between the T2DM group and the control group, of which 116 were differential metabolites.
Specifically, the detection analysis and marker threshold determination of the targeted medium-long chain fatty acid metabolome comprises the following steps:
a1, blood sample collection
A2, treatment and analysis of blood samples
Standard preparation
The 40 mixed standard substance solutions of fatty acid methyl ester are prepared into ten mixed standard substances with the total concentration of 0.5mg/L,1mg/L,5mg/L,10mg/L,25mg/L,50mg/L,100mg/L,250mg/L,500mg/L and 1000mg/L respectively. Adding 25 mu L of n-nonadecanoic acid methyl ester with the concentration of 500ppm as an internal standard into 500 mu L of mixed standard substance, mixing uniformly, and adding into a sample injection bottle. And (3) splitting the sample by using a sample feeding amount of 1 mu L and a split ratio of 10. Then the plasma samples were thawed on ice, 100ul of the sample and 1mL of chloroform methanol solution were added to a 2mL glass centrifuge tube and subjected to the following procedure: performing ultrasonic treatment for 30min, taking supernatant, adding 2mL of 1% sulfuric acid-methanol solution, performing 80 ℃ water bath for 30min, adding 1mL of n-hexane for extraction, adding 5mL of pure water for washing, sucking 500 mu L of supernatant, adding 25 mu L of n-nonadecanoic acid methyl ester for uniform mixing, adding into a sample bottle, and performing GC-MS detection.
The sample is separated by an Agilent DB-WAX capillary column (30 m multiplied by 0.25mm ID multiplied by 0.25 mu m) gas chromatography system, a QC sample is arranged in a sample queue at intervals of a certain amount of experimental samples for detecting and evaluating the stability and the repeatability of the system, and then the Agilent 7890/5975C gas-mass spectrometer is used for carrying out mass spectrum analysis on the sample.
Chromatographic peak area and retention time were extracted using MSD ChemStation software. Drawing a standard curve and calculating the content of medium-long chain fatty acid in the sample. In the case of differential analysis using the Wilcoxon test, metabolites with a P value <0.05 were considered as differential metabolites and the data results were all expressed as mean. + -. Standard deviation.
A3, determination of the critical value
A diagnostic threshold is determined for the selected marker metabolites.
Compared with the prior art, the invention has the following beneficial effects:
the screening method of the kiwi fruit spontaneous diabetes biomarker can obtain an efficient biological combination marker, and whether the kiwi fruit suffers from diabetes or not can be accurately judged by using the biomarker, so that an important premise is provided for human T2DM pathogenesis, pathological mechanism, drug screening and the like.
In the diagnostic reagent or the kit provided by the invention, the biomarkers of myristoleate (C14: 1N 5), oleic acid (oleate, C18:1N 9) and palmitic acid (palmitate, C16: 0) have high sensitivity and specificity and good predictability, can be applied to the aspects of auxiliary diagnosis of type 2diabetes, auxiliary evaluation of the disease risk and the prognosis condition of type 2diabetes, auxiliary research of type 2diabetes and the like, has important guiding significance on the early detection and prevention of type 2diabetes, can be popularized and has important significance in individuation, sensitive identification, screening and genetic counseling.
One or more of the biomarkers myristoleic acid, oleic acid and palmitic acid provided by the invention have wide application in diabetes auxiliary products for auxiliary diagnosis of type 2diabetes, auxiliary evaluation of the disease risk or prognosis condition of type 2diabetes or auxiliary research of type 2diabetes, detection products such as a detection chip for type 2diabetes, a detection reagent for type 2diabetes or a detection kit for type 2diabetes and the like.
Drawings
FIG. 1 is a volcano plot in positive and negative ion mode;
FIG. 2 is a diagram of PLS-DA in positive and negative ion mode;
FIG. 3 is an OPLS-DA diagram in positive and negative ion mode
Figure 4 is a KEGG signal path.
Detailed Description
The following detailed description of the invention, when taken in conjunction with the examples and the drawings, is intended to illustrate, but not limit the invention.
Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and the products, reagents and materials used in the following examples are all commercially available products.
Example 1: screening method of biomarkers of spontaneous diabetes of macaques
1) Preliminary screening of spontaneous diabetic macaques
Recording the length of the selected macaque according to the diabetes diagnosis standard and the requirement of not using antibiotics within three months, and simultaneously detecting the Fasting Plasma Glucose (FPG) value of the macaque to be detected; the macaques in the T2DM group have fasting blood glucose values meeting FPG (glucose tolerance) of not less than 7mmol/L for 3 times in 1 year, and the macaques in the control group have fasting blood glucose values meeting FPG of not more than 6.1mmol/L for 3 times in 1 year; the 8T 2DM group macaques and the 8 control group macaques meeting the requirements are provided by the Green Hauss Biotechnology Co., ltd, meishan city, sichuan, and the 8T 2DM group macaques and the 8 control group macaques are respectively numbered and fed in a single cage, so that the subsequent collection of excrement samples and blood samples is facilitated.
2) Physical and chemical index detection of spontaneous diabetic macaques
The selected 16 macaques were fasted and kept for 12h without water deprivation. The method comprises the steps of collecting venous blood of a macaque by using a biochemical tube, noting information such as sampling time, sampling place, sample number and the like on the tube body, and then placing the macaque in a foam box with an ice bag to be transported back to a laboratory. The collected blood samples are immediately subjected to detection of physiological and biochemical indicators of Fasting Glucose (FGP), glycated hemoglobin A1c (hemoglobin A1 c), fasting insulin (FPI), total Cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and the like, and then combined with body weight data provided by the company to calculate the value of the experimental macaque, wherein the specific formula is as follows:
weight (kg)/length (m)2
HOMA-IR is one of the standards for judging T2DM, and the calculation mode is as follows:
(FPI×FPG)/22.5
the following table shows the physiological and biochemical index values of the control group and the T2DM group:
TABLE 1 physiological and biochemical indices of control and T2DM individuals
*P<0.05，**P<0.01
As shown in table 1, the FPG, FPI and HOMA-IR index of the T2DM group was significantly increased (P < 0.01) compared to the control group; there was no significant change in HbA1c, TG, TC, HDL, LDL and BMI indicators (P < 0.05).
3) Non-targeted metabolome screening markers
S1, collecting a fecal sample
A sampling person wearing two layers of sterile gloves (the inner layer is latex gloves, and the outer layer is disposable PE gloves) collects a fecal sample in ten minutes after the macaque is fed in a single cage, peels off the outer layer in contact with a fecal receiving plate, puts the fecal sample into a 50mL sterile centrifuge tube for later use, and records information such as collection time, place, sample number and the like on a sampling tube. And when the next excrement sample is collected, replacing the new PE glove, and repeating the collection process. And finally storing all the collected feces in a refrigerator at the temperature of-80 ℃.
S2, treatment and analysis of the manure sample
Stool samples stored at-80 ℃ were removed and placed in a 4 ℃ environment for slow thawing, methanol, acetonitrile and aqueous solution were mixed at a ratio of 2:2:1, uniformly mixing and precooling for later use, adding the unfrozen sample into the mixed solution, carrying out vortex mixing, sequentially carrying out low-temperature ultrasonic treatment for 30min, standing for 10min and centrifuging for 20min (14000g, 4 ℃), and then taking supernatant to carry out chromatography-mass spectrometry. Separating the supernatant by adopting an Agilent 1290Infinity LC ultra-performance liquid chromatography system (UHPLC) HILIC chromatographic column, wherein the separation parameters are a default program: 0-0.5min, the mobile phase composition is 95% acetonitrile; the acetonitrile concentration is linearly reduced to 65 percent in 0.5-7 min; the acetonitrile concentration is linearly reduced to 40 percent within 7-8 min; 8-9min, and maintaining the concentration of acetonitrile at 40%;9-9.1min, acetonitrile concentration is increased from 40% to 95% linearly; 9.1-12min, and the concentration of acetonitrile is maintained at 95%; the separated sample is detected by an AB Triple TOF 6600 mass spectrometer in an electrospray ionization (ESI) mode respectively for positive ions and negative ions to obtain original data.
After the raw data is converted into an mzXML format, XCMS software is used for peak alignment, retention time correction and peak area extraction. And (3) sequentially carrying out metabolite structure identification, data preprocessing and data quality evaluation on the data processed by XCMS, and finally carrying out data analysis. And screening different metabolites by combining univariate statistical analysis and multidimensional statistical analysis, displaying the difference of the metabolic levels of the experimental group in the control group by clustering analysis, correlation analysis and KEGG (Kegg open-ended Key genetic code) path enrichment analysis, and mining the biological processes and the regulation and control paths in which the different metabolites participate. Univariate statistical Analysis mainly included Fold difference Analysis (FC Analysis) and T-test, metabolites with FC >1.5 or FC <0.67, p value woven-straw 0.05 were considered differential metabolites and visualized using volcano plots. Multidimensional statistical Analysis obtains Variable Importance for the project (VIP) mainly by Partial Least Squares discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares discriminant Analysis (OPLS-DA), the VIP value is used to evaluate the influence strength of each metabolite on the discriminant Analysis, and metabolites with significant contribution in model interpretation are usually screened with the standard of VIP >1. PLS-DA is a supervised statistical method for establishing a metabolite and sample phenotype relation model by using partial least squares regression, and OPLS-DA is an analysis method for reducing noise and improving the analysis capability and effectiveness of the model on the basis of PLS-DA. FC >1.5 or FC <0.67, P value <0.05 and VIP >1 were used as significant differential metabolic screening criteria in this study. KEGG pathway enrichment analysis of differential metabolites was performed using Metabioanalyst (https:// www. MetaboAnalyst. Ca/home. Xhtml) online software, and significance levels of metabolite enrichment in each pathway were calculated by Fisher's exact test, with metabolic pathways with Pvalue <0.05 being significant enrichment pathways.
S3, screening of differential metabolites
In the positive and negative ion mode, 14665 metabolites were detected in total between the T2DM group and the control group, and it was shown from the volcano plot that there were a large number of significantly different metabolites between the T2DM group and the control group for subsequent identification (fig. 1). The PLS-DA score maps and OPLS-DA score maps (fig. 2 and 3) of the T2DM group and the control group show that the metabolites of the T2DM group and the control group are significantly different in the positive and negative ion modes.
A total of 1564 metabolites were identified between the T2DM group and the control group, of which 116 were differential metabolites. 68 differential metabolites were identified in positive ion mode (VIP >1, P < -0.1), with 38 species of significantly different metabolites (VIP >1, P < -0.05); 48 different metabolites were identified in negative ion mode, with 26 of the significantly different metabolites (VIP >1, P-tres 0.05). Wherein the Fold difference of L-propionylcarnitine is highest among all the different metabolites (Fold change = 16.19). To further analyze the function of the differential metabolites, we performed KEGG pathway enrichment analysis on them and found that a total of 1 signaling pathway (P < 0.05) was enriched for tryptophan metabolism in the T2DM group (fig. 4).
TABLE 2 differential analysis of acyl carnitine metabolites between groups
*P<0.05，**P<0.01
As can be seen from table 2, the levels of the fatty acid metabolites L-propionylcarnitine (L-propionylcarnitine), L-hexanoylcarnitine (Hexanoyl-L-Carnitine), (r) -butyrylcarnitine ((r) -butyrylcarnitine), carnitine (Carnitine) and isovalerylcarnitine (Isovaleryl-L-Carnitine) were significantly increased in the T2DM group. It is known that fatty acid metabolites such as L-propionyl carnitine, L-caproyl carnitine, (r) -butyryl carnitine, carnitine and isovaleryl carnitine are significantly increased in the T2DM group of individuals, and an increase in fatty acid acyl carnitine indicates that mitochondrial beta-oxidation is hindered, which may lead to accumulation of free fatty acids. Therefore, the medium-long chain free fatty acid of the blood of two groups of macaques is subjected to targeted quantitative detection.
4) Targeted medium and long chain fatty acid metabolome assays
A1, blood sample collection
The 16 experimental macaques are fed in a single cage for 12 hours without water supply. The intravenous blood of the macaque is collected by using a heparin anticoagulant blood collection tube, information such as sampling time, sampling place, sample number and the like is noted on the tube body, then the macaque is placed in a foam box with an ice bag and is transported back to a laboratory, and the collected blood sample is stored in a refrigerator at the temperature of 80 ℃ below zero.
A2, treatment and analysis of blood samples
Standard preparation
The 40 mixed standard substance solutions of fatty acid methyl ester are prepared into ten mixed standard substances with total concentrations of 0.5mg/L,1mg/L,5mg/L,10mg/L,25mg/L,50mg/L,100mg/L,250mg/L,500mg/L and 1000mg/L respectively. Adding 25 mu L of n-nonadecanoic acid methyl ester with the concentration of 500ppm as an internal standard into 500 mu L of mixed standard substance, mixing uniformly, and adding into a sample injection bottle. And (4) splitting and injecting samples according to the standard of a sample injection amount of 1 mu L and a splitting ratio of 10, and detecting by GC-MS. Then the plasma samples were thawed on ice, 100ul of the sample and 1mL of chloroform methanol solution were added to a 2mL glass centrifuge tube and subjected to the following procedure: performing ultrasonic treatment for 30min, taking supernatant, adding 2mL of 1% sulfuric acid-methanol solution, performing 80 ℃ water bath for 30min, adding 1mL of n-hexane for extraction, adding 5mL of pure water for washing, sucking 500 mu L of supernatant, adding 25 mu L of n-nonadecanoic acid methyl ester, uniformly mixing, adding into a sample injection bottle, and performing GC-MS detection (sample injection amount is 1 mu L, split-flow ratio is 10, split-flow sample injection is performed.
The sample is separated by an Agilent DB-WAX capillary column (30 m multiplied by 0.25mm ID multiplied by 0.25 mu m) gas chromatography system, a QC sample is arranged in a sample queue at intervals of a certain amount of experimental samples for detecting and evaluating the stability and the repeatability of the system, and then the Agilent 7890/5975C gas-mass spectrometer is used for carrying out mass spectrum analysis on the sample.
Chromatographic peak area and retention time were extracted using MSD ChemStation software. Drawing a standard curve and calculating the content of medium-long chain fatty acid in the sample. In the case of differential analysis using the Wilcoxon test, metabolites with a P value <0.05 were considered as differential metabolites and the data results were all expressed as mean. + -. Standard deviation.
The difference between the different types of fatty acids between the two groups
TABLE 3 analysis of the differences between the different types of fatty acids between the two groups
*P<0.05，**P<0.01
As shown in table 3, the content of Saturated Fatty Acids (SFA) in macaques in T2DM group was significantly higher than that in control group (P < 0.05), while the content of monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), omega-3 polyunsaturated fatty acids (N3) and omega-6 polyunsaturated fatty acids (N6) was not significantly changed between the two groups (P > 0.05).
Differences between the two groups in medium-long chain fatty acids
TABLE 4 Difference of medium-and long-chain fatty acids
*P<0.05，**P<0.01
The specific medium-long chain fatty acid differential analysis result shows that the content of myristoleic acid, oleic acid and palmitic acid in the T2DM group is obviously increased compared with that in the control group (P < 0.05).
A3, determination of the critical value
Determining a diagnostic threshold for the selected marker metabolites as follows: and taking the lowest value of the contents of myristoleic acid, oleic acid and palmitic acid in all the T2DM macaque individuals as the lowest threshold of the metabolite, and if the three metabolites of the macaque individual to be detected exceed the threshold, judging the macaque individual as the T2DM individual. The threshold value of the myristic acid is 23.39ug/mL, the threshold value of the oleic acid is 0.15ug/mL, and the threshold value of the palmitic acid is 219.41ug/mL.
Example 2 diagnostic reagent for Actinidia chinensis diabetes mellitus
This example provides six diagnostic reagents, and the diagnostic markers of the diagnostic reagents are myristoleic acid, oleic acid, palmitic acid, myristoleic acid and oleic acid, myristoleic acid and palmitic acid, oleic acid and palmitic acid, respectively.
In addition, the present embodiment provides another set of diagnostic reagents, wherein the diagnostic markers include three of myristoleate acid (C14: 1N 5), oleic acid (oleate, C18:1N 9), and palmitic acid (palmitate, C16: 0).
The biomarkers of this example were derived from stool.
The detection thresholds for the biomarkers of this example were respectively: the threshold value of the myristoleic acid is 23.39ug/mL, the threshold value of the oleic acid is 0.15ug/mL, and the threshold value of the palmitic acid is 219.41ug/mL.
The diabetes of this example was rhesus monkey idiopathic diabetes.
Example 3 diagnostic kit for idiopathic diabetes of Kiwi
The present example provides six diagnostic kits, and the diagnostic markers of the diagnostic kit are myristoleic acid, oleic acid, palmitic acid, myristoleic acid and oleic acid, myristoleic acid and palmitic acid, oleic acid and palmitic acid, respectively.
In addition, this embodiment also provides another set of diagnostic kits, and the diagnostic markers include three of myristoleate (C14: 1N 5), oleic acid (oleic, C18:1N 9), and palmitic acid (palmitate, C16: 0).
The biomarkers of this example were derived from stool.
The detection thresholds for the biomarkers of this example were respectively: the threshold value of the myristic acid is 23.39ug/mL, the threshold value of the oleic acid is 0.15ug/mL, and the threshold value of the palmitic acid is 219.41ug/mL.
The diabetes of this example was rhesus monkey idiopathic diabetes.
Example 4 diabetes-aid product
The present embodiment provides a diabetes-assisting product, in which the biomarkers are myristoleic acid, oleic acid, and palmitic acid, and the diabetes-assisting product includes a product for assisting in diagnosing type 2diabetes, assisting in evaluating the disease risk or prognosis status of type 2diabetes, or assisting in researching type 2 diabetes.
Example 5 diabetes detection products
This example provides a diabetes detection product in which the biomarkers are myristoleic acid, oleic acid, and palmitic acid.
The detection product of the embodiment comprises a detection chip for type 2diabetes, a detection reagent for type 2diabetes or a detection kit for type 2 diabetes.
Example 6 evaluation of combination markers for idiopathic diabetes in macaques
The combined markers for evaluating spontaneous diabetes of macaques of this example include myristoleic acid, oleic acid, and palmitic acid, and the biomarkers of this example are derived from blood samples.
The detection thresholds for the biomarkers of this example were: the threshold value of the myristic acid is 23.39ug/mL, the threshold value of the oleic acid is 0.15ug/mL, and the threshold value of the palmitic acid is 219.41ug/mL.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The diagnostic reagent or the kit for the spontaneous diabetes of the macaques is characterized in that a diagnostic marker of the diagnostic reagent or the kit is one or more of myristoleic acid, oleic acid and palmitic acid.
2. The diagnostic reagent or kit as claimed in claim 1, wherein the diagnostic marker of the diagnostic reagent or kit comprises three of myristoleic acid, oleic acid and palmitic acid.
3. The diagnostic reagent or kit of claim 1, wherein the biomarker is derived from stool.
4. The diagnostic reagent or kit of claim 1, wherein the detection thresholds for the biomarkers are: the threshold value of the myristic acid is 23.39ug/mL, the threshold value of the oleic acid is 0.15ug/mL, and the threshold value of the palmitic acid is 219.41ug/mL.
5. The diagnostic reagent or kit of any one of claims 1 to 4, wherein the diabetes is cynomolgus monkey idiopathic diabetes.
6. The screening method of the biomarkers of the spontaneous diabetes of the macaques is characterized in that the biomarkers are obtained by a metabonomics method based on liquid chromatography-mass spectrometry analysis.
7. Use of one or more of the biomarkers myrtenoic acid, oleic acid and palmitic acid in the manufacture of a diabetes aid product, including a product that aids in the diagnosis of type 2diabetes, aids in the assessment of risk or prognostic status of type 2diabetes, or aids in the study of type 2 diabetes.
8. Use of one or more of the biomarkers myristoleic acid, oleic acid and palmitic acid in the manufacture of a diabetes detection product.
9. The use of the biomarker of claim 8 in the preparation of a diabetes detection product, wherein the detection product comprises a type 2diabetes detection chip, a type 2diabetes detection reagent, or a type 2diabetes detection kit.
10. A combination marker for assessing idiopathic diabetes in a cynomolgus monkey, wherein the combination marker comprises: the composition comprises myristoleic acid, oleic acid, and palmitic acid.
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