WO2023206739A1 - 一种基于粪便的阿尔兹海默症生物标志物及其应用 - Google Patents

一种基于粪便的阿尔兹海默症生物标志物及其应用 Download PDF

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WO2023206739A1
WO2023206739A1 PCT/CN2022/098168 CN2022098168W WO2023206739A1 WO 2023206739 A1 WO2023206739 A1 WO 2023206739A1 CN 2022098168 W CN2022098168 W CN 2022098168W WO 2023206739 A1 WO2023206739 A1 WO 2023206739A1
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alzheimer
disease
sample
early diagnosis
biomarker
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陈宇
陈艺菁
李寅虎
樊颖颖
陈岳文
陈静
柴岳
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中国科学院深圳先进技术研究院
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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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
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    • G—PHYSICS
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • 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
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate

Definitions

  • This application belongs to the field of biotechnology and relates to a stool-based biomarker for Alzheimer's disease and its application.
  • AD Alzheimer's disease
  • mutism a progressive degenerative disease of the central nervous system that occurs in old age and is characterized by progressive memory impairment, cognitive function decline, and loss of daily living abilities. Accompanied by neuropsychiatric symptoms such as personality changes. Since the pathogenesis of Alzheimer's disease has not yet been fully understood, and its early symptoms are relatively secretive, patients with Alzheimer's disease are easily missed or misdiagnosed. Therefore, the search for highly sensitive and accurate biomarkers is important for Alzheimer's disease. The diagnosis and drug intervention of mutism are of great significance.
  • AD mainly relies on the detection of memory scales, PET, and the level detection of pathological indicators such as A ⁇ and phosphorylated tau in cerebrospinal fluid and blood.
  • pathological indicators such as A ⁇ and phosphorylated tau in cerebrospinal fluid and blood.
  • the clinical detection results of these diagnostic indicators are still controversial, and there is still some controversy about the early onset of AD. There is still a lack of effective testing evidence for symptoms.
  • CN106062563A discloses a biomarker and method for early diagnosis of Alzheimer's disease.
  • the AD biomarker is at least four selected from the group consisting of brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1.
  • Biomarkers in (IGF-1), tumor growth factor beta 1 (TGF- ⁇ 1), vascular endothelial growth factor (VEGF), interleukin 18 (IL-18), and monocyte chemoattractant protein-1 (MCP-1) by analyzing its expression level, it can assist in the early diagnosis of AD.
  • the host and intestinal flora produce a large number of metabolites in the process of metabolizing food substances.
  • the diversity and abundance changes of the intestinal flora will have an important impact on the types and concentrations of small molecule metabolites in the body.
  • the composition of the intestinal flora of patients is different from that of healthy peers, and more and more evidence shows that disorders of various metabolic pathways may mediate the pathogenesis and development of AD.
  • the imbalance of bacterial flora in AD may be important in the occurrence of metabolic disorders. Association, and changes in the body's peripheral metabolism may further lead to metabolic disorders in the central nervous system through blood circulation.
  • screening new AD biomarkers can help expand the basis for early diagnosis of AD. It can be combined with other marker detection to improve the accuracy of AD diagnosis and contribute to early warning and pathological analysis of the disease. Prediction and evaluation of types and development stages, etc.
  • This application provides a feces-based Alzheimer's disease biomarker and its application.
  • This application conducts qualitative and quantitative analysis of human feces metabolites based on targeted metabolomics analysis technology, and uses imidazole propionate in feces as Alzheimer's disease biomarker.
  • detecting the level of imidazole propionate in stool can assist in the early diagnosis of Alzheimer's disease, and it is timely, convenient, non-invasive, highly specific and sensitive.
  • the present application provides a stool-based Alzheimer's disease biomarker, which includes Imidazole Propionate.
  • This application conducts qualitative and quantitative analysis of fecal metabolites based on targeted metabolomics analysis technology. It is detected that the level of imidazole propionate in Alzheimer's disease stool samples is significantly higher than that in normal stool samples, and it is used as an Alzheimer's disease biomarker. It can assist in the early diagnosis of Alzheimer's disease by detecting the level of imidazole propionate in feces.
  • this application provides the application of the Alzheimer's disease biomarker as described in the first aspect in constructing an early diagnosis model of Alzheimer's disease and/or preparing an early diagnosis device for Alzheimer's disease.
  • this application provides an Alzheimer's disease early diagnosis model.
  • the input variables of the Alzheimer's disease early diagnosis model include the mass spectrum peak intensity of the Alzheimer's disease biomarker described in the first aspect. value.
  • the output variables of the Alzheimer's disease early diagnosis model include differential expression folds, and the calculation formula of the differential expression folds is as shown in equation (1):
  • the criterion for determining Alzheimer's disease positivity is that the differential expression fold is ⁇ 1.66.
  • an early diagnosis model for Alzheimer's disease was constructed through full comparative analysis of the mass spectrum peak intensity values of Alzheimer's disease biomarkers in normal stool samples and AD stool samples, and rational design.
  • the model uses the mass spectrum peak intensity values of Alzheimer's disease biomarkers as input variables and differential expression folds as output variables. It can quickly output results and fully characterize samples with abnormal levels of Alzheimer's disease biomarkers. Thus assisting in the early diagnosis of Alzheimer’s disease.
  • this application provides an early diagnosis device for Alzheimer's disease, which device includes the following units:
  • the sample preparation unit is used to perform the following steps:
  • the detection unit is used to perform the following steps:
  • the analysis unit is used to perform the following steps:
  • each unit cooperates effectively, is simple and efficient, and can quickly complete sample processing, detection and obtain differential expression multiples. At the same time, it can conduct Alzheimer's disease positivity with reasonably designed judgment criteria. Assessment is of great significance for the early diagnosis of Alzheimer’s disease.
  • the sample to be tested includes a stool sample.
  • the preparation method of the sample solution to be tested includes adding the sample to be tested into a methanol-acetonitrile aqueous solution, centrifuging and collecting the supernatant to obtain the sample solution to be tested.
  • the preparation method of the sample solution to be tested includes the following steps:
  • 12000 ⁇ 16000 ⁇ g for example, it can be 12200 ⁇ g, 12400 ⁇ g, 12600 ⁇ g, 12800 ⁇ g, 13200 ⁇ g, 12600 ⁇ g, 15000 ⁇ g or 15800 ⁇ g
  • Centrifuge for 15-25min for example, it can be 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min or 24min), take the supernatant and vacuum dry it to obtain a pretreated sample;
  • the volume ratio of methanol, acetonitrile and water in the methanol/acetonitrile/water solution is (1 ⁇ 2):(1 ⁇ 2):1, including but not limited to 1.2:2:1, 1.2:1:1, 2 :2:1, 1.4:1.5:1, 1.6:1.2:1, 1.8:2:1, 1.9:1.8:1 or 1.1:1.4:1.
  • the volume ratio of acetonitrile and water in the acetonitrile aqueous solution is (1-2):1, including but not limited to 1.1:1, 1.2:1, 1.3:1, 1.5:1, 1.6:1, 1.7:1 , 1.8:1 or 1.9:1.
  • the liquid chromatograph includes an ultra-high performance liquid chromatograph.
  • the ultra-high performance liquid chromatograph includes Agilent 1290 Infinity LC ultra-high performance liquid chromatograph.
  • the mass spectrometer includes a triple quadrupole mass spectrometer.
  • the triple quadrupole mass spectrometer includes an AB 5500/6500 Q-trap mass spectrometer (AB SCIEX).
  • the data processing includes:
  • the Alzheimer's disease early diagnosis device includes the following units:
  • the sample preparation unit is used to perform the following steps:
  • the detection unit is used to perform the following steps:
  • the analysis unit is used to perform the following steps:
  • the peak intensity value of the detected Alzheimer's disease biomarker mass spectrum is input into the Alzheimer's disease early diagnosis model according to any one of claims 3 to 5 for data analysis, and the differential expression fold corresponding to the sample is output, and Determine whether it is positive for Alzheimer's disease.
  • the detection of Alzheimer's disease biomarker levels in stool samples can be used as a diagnostic basis, combined with other test results, to assist in the early diagnosis of Alzheimer's disease, and is expected to improve Alzheimer's disease. Diagnostic accuracy, but does not alone serve as a diagnostic indicator that can 100% diagnose Alzheimer's disease.
  • this application provides the application of the Alzheimer's disease biomarker described in the first aspect in screening drugs for treating and/or preventing Alzheimer's disease.
  • Alzheimer's disease biomarker described in the first aspect as a target to screen drugs for treating and/or preventing Alzheimer's disease.
  • This application detects for the first time that the level of imidazole propionate in Alzheimer's disease stool samples is significantly higher than that in normal stool samples, using it as an Alzheimer's disease biomarker, and provides an early diagnosis model and device for Alzheimer's disease, through Detecting the level of imidazole propionate in feces can assist in the early diagnosis of Alzheimer's disease, facilitate non-invasive and rapid detection, and is timely, convenient, highly specific and sensitive.
  • Figure 1 shows the levels of imidazole propionate in fecal samples of AD model mice and wild-type mice.
  • This example conducts qualitative and quantitative analysis of metabolites in fecal samples of 9-month-old AD model mice (APP/PS1 transgenic mice, provided by the Model Animal Institute of Nanjing University) and wild-type (WT) mice.
  • AD model mice and 9 AD model mice cultured under the same conditions were collected (mice were housed in a specific pathogen-free environment, with a 12-hour light/dark cycle, the temperature was maintained at 24°C, and sterile drinking water and standard feed were provided).
  • ultra-high performance liquid chromatography-mass spectrometry was used to detect ⁇ -murinecholic acid, taurine- ⁇ -murinecholic acid, ursolic acid, 7-dehydrocholic acid, and Deoxycholic acid, glycolithocholic acid-3-sulfate, isodeoxycholic acid, hyodeoxycholic acid, and omega-murinecholic acid levels by:
  • the Agilent 1290 Infinity LC ultra-high performance liquid chromatography system (UHPLC) is used for separation through HILIC and C18 chromatographic columns in sequence.
  • the HILIC column temperature is 25°C; the flow rate is 0.3mL/min; the injection volume is 2 ⁇ L; the mobile phase composition includes : Phase A: Mix 90% water with 10% acetonitrile by volume and add ammonium formate with a final concentration of 2mM (mmol/L).
  • Phase B Add 0.4% (volume percentage) formic acid to methanol; the gradient elution procedure is as follows: 0 ⁇ 1.0min, 85% B phase; 1.0 ⁇ 3min, B phase linearly changes from 85% to 80%; 3 ⁇ 4min, 80% B phase; 4 ⁇ 6min, B phase linearly changes from 80% to 70%; 6 ⁇ 10min, B changes linearly from 70% to 50%; 10 ⁇ 12.5min, B phase maintains 50%; 12.5 ⁇ 12.6min, B phase linearly changes from 50% to 85%; 12.6 ⁇ 18min, B phase maintains 85% ;
  • phase B mix according to volume ratio 99.5% acetonitrile and 0.5% ammonia
  • the gradient elution program is as follows: 0 to 5 minutes, phase B changes linearly from 5% to 60%; 5 to 11 minutes, phase B changes linearly from 60% to 100%; 11 to 13 minutes, phase B Maintained at 100%; 13-13.1min, phase B changes linearly from 100% to 5%; 13.1-16min, phase B maintained at 5%; throughout the analysis process, the sample is placed in the 4°C autosampler; in the sample queue Insert isotope standards (from Cambridge Isotope Laboratories) to monitor and evaluate the stability of the system and the reliability of experimental data;
  • AB 6500 QTRAP mass spectrometer (AB SCIEX) to perform mass spectrometry analysis of the sample after ultra-high performance liquid chromatography separation in step (4).
  • the ESI source conditions are as follows: sheath gas temperature, 350°C; dry gas temperature, 350°C; sheath gas flow, 11L/min; dry gas flow, 10L/min; capillary voltage, 4000V or-3500V in positive or negative modes, respectively; nozzle voltage, 500V; and nebulizer pressure, 30psi, monitored in MRM mode, using MultiQuant software Peak extraction was performed on the original MRM data, and the ratio of the peak area of each substance to the corresponding isotope standard internal standard peak area was obtained as the normalized peak intensity value of these metabolites, and the average peak intensity was plotted.
  • Imidazole propionate produced from histidine, is an important microbial metabolite that promotes the development of insulin resistance. and eventually leads to type 2 diabetes (T2DM). These results suggest that changes in fecal metabolite levels may reflect abnormalities in AD-related metabolic pathways, which is of great significance for early clinical diagnosis.
  • this application detects for the first time that the level of imidazole propionate in Alzheimer's disease stool samples is significantly higher than that in normal stool samples. It uses fecal imidazole propionate as a biomarker for Alzheimer's disease and provides information on Alzheimer's disease. Early diagnosis models and devices for Alzheimer's disease can assist in the early diagnosis of Alzheimer's disease by detecting the level of imidazole propionate in feces, contribute to non-invasive and rapid detection, and are timely, convenient, highly specific and sensitive.

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Abstract

一种基于粪便的阿尔兹海默症生物标志物及其应用。阿尔兹海默症生物标志物包括丙酸咪唑。首次检测到阿尔兹海默症粪便样本中丙酸咪唑水平显著高于正常粪便样本，将粪便样本中丙酸咪唑作为阿尔兹海默症生物标志物，通过检测粪便中丙酸咪唑水平能够辅助阿尔兹海默症早期诊断，有助于无创快速检测，且具备及时、方便、高特异性及高灵敏度的特点。

Description

一种基于粪便的阿尔兹海默症生物标志物及其应用 技术领域
本申请属于生物技术领域，涉及一种基于粪便的阿尔兹海默症生物标志物及其应用。
背景技术
阿尔兹海默症(Alzheimer disease，AD)，是一种发生于老年期的进行性发展的中枢神经系统退行性变性疾病，以渐进性记忆障碍及认知功能下降和日常生活能力丧失为特征，伴随人格改变等神经精神症状。由于阿尔兹海默症发病机制尚未完全明确，加上其早期症状比较隐秘，阿尔兹海默症患者容易被漏诊或错诊，因此寻找高灵敏度、高准确度的生物标志物，对于阿尔兹海默症的诊断和药物干预具有重要意义。
目前对于AD的诊断主要依靠记忆量表、PET以及脑脊液、血液中Aβ、磷酸化tau等病理指标的水平检测，然而这些诊断指标在临床中的检测结果仍存在一定争议，而且对于AD发病早期的症状尚缺乏有效的检测证据。
CN106062563A公开了一种用于阿尔兹海默症的早期诊断的生物标志物及方法，所述AD生物标志物是至少四种选自脑源性神经营养因子(BDNF)、胰岛素样生长因子-1(IGF-1)、肿瘤生长因子β1(TGF-β1)、血管内皮生长因子(VEGF)、白介素18(IL-18)和单核细胞趋化蛋白-1(MCP-1)中的生物标志物，通过分析其表达水平，能够辅助AD的早期诊断。
宿主和肠道菌群在代谢食物物质的过程中产生大量的代谢物，肠道菌群的多样性以及丰度的改变会对机体中小分子代谢物的种类和浓度产生重要影响，有数据显示AD患者肠道菌群组成与健康同龄人不同，且越来越多证据表明各种代谢途径的紊乱可能介导AD的病理发生和发展，AD中的菌群失衡可能与机体代谢紊乱的发生有重要关联，而机体外周代谢改变又可能通过血液循环进一步导致中枢神经系统代谢紊乱。
综上所述，筛选新的AD生物标志物，有助于扩充AD早期诊断的判断依据，可与其他标志物检测相互结合，提高AD诊断的准确性，有助于疾病的早期预警、病理分型以及发展阶段的预测评估等。
发明内容
本申请提供一种基于粪便的阿尔兹海默症生物标志物及其应用，本申请基于靶向代谢组学分析技术对人粪便代谢物进行定性定量分析，将粪便中丙酸咪唑作为阿尔兹海默症标志物，通过检测粪便中丙酸咪唑水平能够辅助阿尔兹海默症早期诊断，且具备及时、方便、无创、高特异性及高灵敏度的特点。
第一方面，本申请提供一种基于粪便的阿尔兹海默症生物标志物，所述阿尔兹海默症生物标志物包括丙酸咪唑(Imidazole Propionate)。
本申请基于靶向代谢组学分析技术对粪便代谢物进行定性定量分析，检测到阿尔兹海默症粪便样本中丙酸咪唑水平显著高于正常粪便样本，将其作为阿尔兹海默症生物标志物，通过检测粪便中丙酸咪唑水平能够辅助阿尔兹海默症早期诊断。
第二方面，本申请提供如第一方面所述的阿尔兹海默症生物标志物在构建阿尔兹海默症早期诊断模型和/或制备阿尔兹海默症早期诊断装置中的应用。
第三方面，本申请提供一种阿尔兹海默症早期诊断模型，所述阿尔兹海默症早期诊断模型的输入变量包括第一方面所述的阿尔兹海默症生物标志物的质谱峰强度值。
优选地，所述阿尔兹海默症早期诊断模型的输出变量包括差异表达倍数，所述差异表达倍数的计算公式如方程式(1)所示：
Figure PCTCN2022098168-appb-000001
优选地，阿尔兹海默症阳性的判断标准为所述差异表达倍数≥1.66。
本申请中，通过对正常粪便样本和AD粪便样本中阿尔兹海默症生物标志物的质谱峰强度值进行充分对比分析，并进行理性设计，构建了一种阿尔兹海默症早期诊断模型，所述模型以阿尔兹海默症生物标志物的质谱峰强度值为输入变量，以差异表达倍数为输出变量，能够快速输出结果，且充分表征阿尔兹海默症生物标志物水平异常的样本，从而辅助阿尔兹海默症早期诊断。
第四方面，本申请提供一种阿尔兹海默症早期诊断装置，所述装置包括如下单元：
样本配制单元，用于执行包括以下步骤：
用于将待测样本配制成可用于液相色谱仪分离的待测样本溶液；
检测单元，用于执行包括以下步骤：
利用所述液相色谱仪分离所述待测样本溶液，利用质谱仪对分离后样本进行检测，进行数据处理，测定样本中第一方面所述的阿尔兹海默症生物标志物的质谱峰强度值；以及
分析单元，用于执行包括以下步骤：
将检测到的阿尔兹海默症生物标志物质谱的峰强度值输入第三方面所述的阿尔兹海默症早期诊断模型进行数据分析，输出样本对应的差异表达倍数，并判断是否为阿尔兹海默症阳性。
本申请的阿尔兹海默症早期诊断装置中，各单元间有效配合，简单高效，能够快速完成样本处理、检测及获得差异表达倍数，同时以经过合理设计的判断标准进行阿尔兹海默症阳性评估，对于阿尔兹海默症早期诊断具有重要意义。
优选地，所述待测样本包括粪便样本。
优选地，所述待测样本溶液的配制方法包括将待测样本加入甲醇-乙腈水溶液中，离心并收集上清液，得到所述待测样本溶液。
优选地，所述待测样本溶液的配制方法包括以下步骤：
(1)取待测样本加入预冷甲醇/乙腈/水溶液中，混合并超声25～35min(例如可以是26min、27min、28min、29min或32min)，置于-20～-15℃(例如可以是-19℃、-18℃、-16℃或-17℃)静置5～15min(例如可以是6min、7min、8min、9min、10min、12min或14min)，于0～4℃(例如可以是1℃、2℃或3℃)、12000～16000×g(例如可以是12200×g、12400×g、12600×g、12800×g、13200×g、12600×g、15000×g或15800×g)离心15～25min(例如可以是16min、17min、18min、19min、20min、21min、22min、23min或24min)，取上清进行真空干燥，得到预处理样本；
(2)将所述预处理样本加入80～120μL乙腈水溶液中复溶，涡旋，于0～4℃、12000～16000×g(例如可以是12200×g、12400×g、12600×g、12800×g、13200×g、12600×g、15000×g或15800×g)离心10～20min(例如可以是11min、12min、13min、14min、15min、16min、17min、18min或19min)，取上清液，得到所述待测样本溶液。
优选地，所述甲醇/乙腈/水溶液中甲醇、乙腈和水的体积比为(1～2):(1～2):1 包括但不限于1.2:2:1、1.2:1:1、2:2:1、1.4:1.5:1、1.6:1.2:1、1.8:2:1、1.9:1.8:1或1.1:1.4:1。
优选地，所述乙腈水溶液中乙腈和水的体积比为(1～2):1，包括但不限于1.1:1、1.2:1、1.3:1、1.5:1、1.6:1、1.7:1、1.8:1或1.9:1。
优选地，所述液相色谱仪包括超高效液相色谱仪。
优选地，所述超高效液相色谱仪包括Agilent 1290Infinity LC超高效液相色谱仪。
优选地，所述质谱仪包括三重四级杆质谱仪。
优选地，所述三重四级杆质谱仪包括AB 5500/6500Q-trap质谱仪(AB SCIEX)。
优选地，所述数据处理包括：
使用MultiQuant软件对MRM原始数据进行峰提取，计算阿尔兹海默症生物标志物的峰面积和内标峰面积的比值，作为质谱峰强度值。
作为优选的技术方案，所述阿尔兹海默症早期诊断装置包括如下单元：
样本配制单元，用于执行包括以下步骤：
将待测样本配制成可用于液相色谱仪分离的待测样本溶液；
检测单元，用于执行包括以下步骤：
利用所述液相色谱仪分离所述待测样本溶液，利用质谱仪对分离后样本进行检测，使用MultiQuant软件对MRM原始数据进行峰提取，计算阿尔兹海默症生物标志物的峰面积和内标峰面积的比值，作为质谱峰强度值；以及
分析单元，用于执行包括以下步骤：
将检测到的阿尔兹海默症生物标志物质谱的峰强度值输入权利要求3-5任一项所述的阿尔兹海默症早期诊断模型进行数据分析，输出样本对应的差异表达倍数，并判断是否为阿尔兹海默症阳性。
本申请中，对粪便样本中阿尔兹海默症生物标志物水平进行检测，可以作为一种诊断依据，与其他检测结果结合，辅助阿尔兹海默症早期诊断，预期可以提高阿尔兹海默症诊断的准确性，但并不单独作为能够100％诊断阿尔兹海默症的诊断指标。
第五方面，本申请提供第一方面所述的阿尔兹海默症生物标志物在筛选治疗和/或预防阿尔兹海默症的药物中的应用。
即以第一方面所述的阿尔兹海默症生物标志物作为靶点筛选治疗和/或预防阿尔兹海默症的药物。
与现有技术相比，本申请具有以下有益效果：
本申请首次检测到阿尔兹海默症粪便样本中丙酸咪唑水平显著高于正常粪便样本，将其作为阿尔兹海默症生物标志物，并提供阿尔兹海默症早期诊断模型和装置，通过检测粪便中丙酸咪唑水平能够辅助阿尔兹海默症早期诊断，有助于无创快速检测，且具备及时、方便、高特异性及高灵敏度的特点。
附图说明
图1为AD模型小鼠和野生型小鼠的粪便样本中丙酸咪唑水平图。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
本申请实施例中实验仪器和试剂包括：
AB 5500/6500 Q-trap质谱仪(AB SCIEX)；
Agilent 1290 Infinity LC超高压液相色谱仪(Agilent)；
低温高速离心机(Eppendorf 5430R)；
色谱柱：Waters，ACQUITY UPLC BEH Amide 1.7μm，2.1mm×100mm column；Waters，ACQUITY UPLC BEH C18 1.7μm，2.1mm×100mm column；
乙腈(Merck，1499230-935)；
乙酸铵(Sigma，70221)；
甲醇(Fisher，A456-4)；
氨水(Sigma，221228)；
甲酸铵(Sigma，70221)；
甲酸(Sigma，00940)；
同位素标准品(Cambridge Isotope Laboratories)。
实施例1
本实施例对9月龄AD模型小鼠(APP/PS1转基因小鼠，由南京大学模式动物研究所提供)与野生型(WT)小鼠的粪便样本进行代谢物定性定量分析。
分别采集在相同条件下培养的(小鼠饲养在无特定病原体的环境中，12小时光照/黑暗循环，温度保持在24℃，提供灭菌饮用水和标准饲料)10只AD模型小鼠和9只野生型小鼠的粪便，采用超高效液相色谱-质谱联用分别检测粪便样本中的β-鼠胆酸、牛磺-β-鼠胆酸、熊果酸、7-脱氢胆酸、脱氧胆酸、甘氨石胆酸-3-硫酸盐、异脱氧胆酸、猪去氧胆酸和ω-鼠胆酸水平，具体方法包括：
(1)取粪便样本加入预冷甲醇/乙腈/水溶液(体积比为2:2:1)，涡旋混合，低温超声30min，置于-20℃静置10min，于4℃、14000×g离心20min，取上清进行真空干燥，得到预处理样本；
(2)将所述预处理样本加入100μL乙腈水溶液(体积比为乙腈:水＝1:1)中复溶，涡旋，于4℃、14000×g离心15min，取上清液，以备进样分析；
(3)采用Agilent 1290 Infinity LC超高效液相色谱系统(UHPLC)依次经HILIC和C18色谱柱进行分离，HILIC色谱柱柱温25℃；流速0.3mL/min；进样量2μL；流动相组成包括：A相：按体积比将90％水混合10％乙腈并加入终浓度为2mM(mmol/L)的甲酸铵，B相：甲醇中加入0.4％(体积百分比)甲酸；梯度洗脱程序如下：0～1.0min，85％B相；1.0～3min，B相从85％线性变化至80％；3～4min，80％B相；4～6min，B相从80％线性变化至70％；6～10min，B从70％线性变化至50％；10～12.5min，B相维持50％；12.5～12.6min，B相从50％线性变化至85％；12.6～18min，B相维持在85％；
C18色谱柱柱温40℃，流速0.4mL/min，进样量2μL；流动相组成A相：水中加入终浓度为5mM的乙酸铵和0.2％(体积百分比)氨水，B相：按体积比混合99.5％乙腈和0.5％氨水；梯度洗脱程序如下：0～5min，B相从5％线性变化至60％；5～11min，B相从60％线性变化至100％；11～13min，B相维持在100％；13～13.1min，B相从100％线性变化至5％；13.1～16min，B相维持在5％；整个分析过程中样本置于4℃自动进样器中；样本队列中插入同位素标准品(来自于Cambridge Isotope Laboratories)，用于监测和评价系统的稳定性及实验数据的可靠性；
(5)采用AB 6500 QTRAP质谱仪(AB SCIEX)对步骤(4)超高效液相 色谱系分离后的样本质谱分析，ESI源条件如下sheath gas temperature，350℃；dry gas temperature，350℃；sheath gas flow，11L/min；dry gas flow，10L/min；capillary voltage，4000V or-3500V in positive or negative modes，respectively；nozzle voltage，500V；and nebulizer pressure，30psi，采用MRM模式监测，使用MultiQuant软件对MRM原始数据进行峰提取，得到各物质的峰面积和相对应的同位素标准品内标峰面积的比值，作为这些代谢物归一化后的峰强度值，以峰强度平均值作图，结果如图1所示，随后，基于各个样本归一化后的代谢物峰强度值，使用OPLS-DA分析去检测不同组别代谢物组成的差异，并筛选变量重要性(VIP)大于1的代谢物，然后，使用秩和检验对不同组别间的代谢物进行差异分析，获得p值小于0.05的差异代谢物，最后将筛选VIP>1和p<0.05的组间差异代谢物作为阿尔兹海默症生物标志物。
结果如图1及表1所示，AD模型小鼠粪便样本中丙酸咪唑水平显著高于野生型小鼠，表明可将粪便中丙酸咪唑作为阿尔兹海默症生物标志物，通过检测粪便中丙酸咪唑水平能够辅助阿尔兹海默症早期诊断。
表1
Figure PCTCN2022098168-appb-000002
注：**为p<0.01。
实施例2
本实施例对实施例中阿尔兹海默症生物标志物丙酸咪唑进行分析，由组氨酸产生的丙酸咪唑(imidazole propionate)，是一种重要的微生物代谢物，会促进胰岛素抵抗的发展并最终导致2型糖尿病(T2DM)，这些结果提示粪便代谢物水平变化可能反映AD相关代谢通路的异常，对临床早期诊断具有重要意义。
综上所述，本申请首次检测到阿尔兹海默症粪便样本中丙酸咪唑水平显著高于正常粪便样本，将粪便中丙酸咪唑作为阿尔兹海默症生物标志物，并提供阿尔兹海默症早期诊断模型和装置，通过检测粪便中丙酸咪唑水平能够辅助阿尔兹海默症早期诊断，有助于无创快速检测，且具备及时、方便、高特异性及高灵敏度的特点。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。 所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (10)

  1. 一种基于粪便的阿尔兹海默症生物标志物，其包括丙酸咪唑。
  2. 如权利要求1所述的基于粪便的阿尔兹海默症生物标志物在构建阿尔兹海默症早期诊断模型和/或制备阿尔兹海默症早期诊断装置中的应用。
  3. 一种阿尔兹海默症早期诊断模型，其输入变量包括权利要求1所述的阿尔兹海默症生物标志物的质谱峰强度值；
    所述阿尔兹海默症早期诊断模型的输出变量包括差异表达倍数。
  4. 根据权利要求3所述的阿尔兹海默症早期诊断模型，其中，所述差异表达倍数的计算公式如方程式(1)所示：
    Figure PCTCN2022098168-appb-100001
  5. 根据权利要求4所述的阿尔兹海默症早期诊断模型，其中，阿尔兹海默症阳性的判断标准为所述差异表达倍数≥1.66。
  6. 一种阿尔兹海默症早期诊断装置，其包括如下单元：
    样本配制单元，用于执行包括以下步骤：
    用于将待测样本配制成可用于液相色谱仪分离的待测样本溶液；
    检测单元，用于执行包括以下步骤：
    利用所述液相色谱仪分离所述待测样本溶液，利用质谱仪对分离后样本进行检测，进行数据处理，测定样本中权利要求1所述的阿尔兹海默症生物标志物的质谱峰强度值；以及
    分析单元，用于执行包括以下步骤：
    将检测到的阿尔兹海默症生物标志物质谱的峰强度值输入权利要求3-5任一项所述的阿尔兹海默症早期诊断模型进行数据分析，输出样本对应的差异表达倍数，并判断是否为阿尔兹海默症阳性。
  7. 根据权利要求6所述的装置，其中，所述待测样本包括粪便样本。
  8. 根据权利要求6或7所述的装置，其中，所述数据处理包括：
    使用MultiQuant软件对MRM原始数据进行峰提取，计算阿尔兹海默症生物标志物的峰面积和内标峰面积的比值，作为质谱峰强度值。
  9. 根据权利要求6-8任一项所述的装置，其中，所述装置包括如下单元：
    样本配制单元，用于执行包括以下步骤：
    将待测样本配制成可用于液相色谱仪分离的待测样本溶液；
    检测单元，用于执行包括以下步骤：
    利用所述液相色谱仪分离所述待测样本溶液，利用质谱仪对分离后样本进行检测，使用MultiQuant软件对MRM原始数据进行峰提取，计算阿尔兹海默症生物标志物的峰面积和内标峰面积的比值，作为质谱峰强度值；以及
    分析单元，用于执行包括以下步骤：
    将检测到的阿尔兹海默症生物标志物的质谱峰强度值输入权利要求3-5任一项所述的阿尔兹海默症早期诊断模型进行数据分析，输出样本对应的差异表达倍数，并判断是否为阿尔兹海默症阳性。
  10. 权利要求1所述的基于粪便的阿尔兹海默症生物标志物在筛选治疗和/或预防阿尔兹海默症的药物中的应用。
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