WO2024092963A1 - 一种基于肠道菌群的阿尔茨海默病生物标志物及其应用 - Google Patents

一种基于肠道菌群的阿尔茨海默病生物标志物及其应用 Download PDF

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WO2024092963A1
WO2024092963A1 PCT/CN2022/137818 CN2022137818W WO2024092963A1 WO 2024092963 A1 WO2024092963 A1 WO 2024092963A1 CN 2022137818 W CN2022137818 W CN 2022137818W WO 2024092963 A1 WO2024092963 A1 WO 2024092963A1
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alzheimer
disease
biomarker
intestinal flora
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French (fr)
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陈宇
陈艺菁
李寅虎
樊颖颖
陈岳文
杨玉洁
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深圳先进技术研究院
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Definitions

  • the present invention belongs to the technical field of biotechnology and relates to an Alzheimer's disease biomarker based on intestinal flora and an application thereof.
  • AD Alzheimer's disease
  • senile dementia is a progressive degenerative disease of the central nervous system that occurs in the elderly. It is characterized by progressive memory impairment, cognitive decline, and loss of daily living ability, accompanied by neuropsychiatric symptoms such as personality changes, which seriously affect social and living functions. Because the pathogenesis of Alzheimer's disease has not yet been fully clarified, and its early symptoms are relatively hidden, Alzheimer's patients are easily missed or misdiagnosed.
  • diagnosis of AD mainly relies on memory scales, PET, and the level 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.
  • CN114657270A discloses an Alzheimer's disease biomarker based on intestinal flora and its application, which found that Moryella, Hydrogenophage, Spirospira, Rhodozoa and Delftia are related to Alzheimer's disease, and their abundance shows significant differences between Alzheimer's patients and healthy people.
  • ROC curve analysis shows that it has high accuracy, specificity and sensitivity as a detection variable, and Moryella, Hydrogenophage, Spirospira, Rhodozoa and Delftia can be used as detection targets for the diagnosis of Alzheimer's patients.
  • Alzheimer's disease biomarkers Although a variety of Alzheimer's disease biomarkers have been made public, their applicability is limited and they are not suitable for all potential patients. Therefore, in order to further improve the detection rate and accuracy of Alzheimer's disease, it is still necessary to continuously develop new Alzheimer's disease biomarkers to expand Alzheimer's disease detection indicators.
  • the present invention aims to develop novel biomarkers for Alzheimer's disease in order to expand the detection index of Alzheimer's disease and further improve the detection rate and accuracy of Alzheimer's disease, in order to solve the problem that early diagnosis and early warning of Alzheimer's disease require abundant and effective biomarkers.
  • the present invention adopts the following technical solutions:
  • the present invention provides an Alzheimer's disease biomarker based on intestinal flora, wherein the Alzheimer's disease biomarker is an intestinal microorganism, and the intestinal microorganism includes any one or a combination of at least two of the Flavobacteriaceae, Lachnospiraceae, Deferribacteraceae, Olfacteriaceae or Desulfovibrioides.
  • the present invention is based on the Illuminate MiSeq sequencing platform, and deeply analyzes the intestinal flora of individuals with Alzheimer's disease and healthy individuals. It is found that the relative abundance levels of Flavobacteriaceae, Lachnospiraceae and Deferribacteraceae in AD mice are significantly increased compared with normal mice, while the relative abundance of Odoribacteraceae and Desulfovibrionaceae are significantly decreased compared with healthy mice.
  • the relative abundance level is used as a detection indicator for auxiliary judgment or early warning of Alzheimer's disease symptoms. It has the characteristics of high detection accuracy, convenience, speed, safety and non-invasiveness, and has important clinical guidance significance for auxiliary diagnosis and early warning of AD.
  • the Alzheimer's disease biomarker is derived from a biological sample of a subject, and the biological sample includes feces of the subject.
  • the present invention provides the use of a substance for detecting the intestinal flora-based Alzheimer's disease biomarker described in the first aspect in the preparation of a product for diagnosing whether a subject has Alzheimer's disease or predicting the risk of a subject having Alzheimer's disease.
  • the present invention is based on the developed intestinal flora-based Alzheimer's disease biomarker. By detecting the relative abundance of the biomarker, a product for diagnosing whether a subject has Alzheimer's disease or predicting the risk of a subject having Alzheimer's disease can be developed.
  • the present invention provides a product for diagnosing whether a subject has Alzheimer's disease or predicting the risk of a subject having Alzheimer's disease, the product comprising reagents and/or devices for detecting the relative abundance and/or quantity of the intestinal flora-based Alzheimer's disease biomarkers described in the first aspect.
  • the product of the present invention for diagnosing whether a subject has Alzheimer's disease or predicting the risk of a subject having Alzheimer's disease uses relative abundance level as a detection indicator, can effectively characterize Alzheimer's disease samples, and assist in AD diagnosis and early warning.
  • the product may be a kit.
  • the present invention provides the use of the intestinal flora-based Alzheimer's disease biomarker described in the first aspect in constructing an early diagnosis model for Alzheimer's disease and/or preparing an early diagnosis device for Alzheimer's disease.
  • the present invention provides an early diagnosis model for Alzheimer's disease, wherein the input variables of the early diagnosis model for Alzheimer's disease include the relative abundance value of the Alzheimer's disease biomarker based on intestinal flora described in the first aspect, and the output variables of the early diagnosis model for Alzheimer's disease include the difference multiple, and the calculation formula of the difference multiple is:
  • the criteria for positive Alzheimer's disease are:
  • the difference multiple of the Flavobacteriaceae is ⁇ 1.31, the difference multiple of the Lachnospiraceae is ⁇ 1.74, the difference multiple of the Deferrobacillaceae is ⁇ 1.65, the difference multiple of the Olfobacteriaceae is ⁇ 0.35, or the difference multiple of the Desulfovibrioaceae is ⁇ 0.63.
  • a model for early diagnosis of Alzheimer's disease was constructed by fully comparing and analyzing the relative abundance of flora in normal stool samples and AD stool samples, and performing rational design.
  • the model uses relative abundance value as input variable and difference multiple as output variable, can output results quickly, and fully characterize samples with abnormal intestinal flora, thereby assisting in the early diagnosis of Alzheimer's disease.
  • the present invention provides an early diagnosis device for Alzheimer's disease, the device comprising the following units:
  • Evaluation units for implementation include:
  • the relative abundance value of the Alzheimer's disease biomarker detected by the analysis unit is input into the Alzheimer's disease early diagnosis model described in the fifth aspect for calculation, the difference multiple is output, and it is determined whether it is Alzheimer's disease positive.
  • the various units cooperate effectively with each other, are simple and efficient, can quickly complete sample processing, detection and obtain difference multiples, and at the same time perform positive assessment of Alzheimer's disease with reasonably designed judgment criteria, which is of great significance for the early diagnosis of Alzheimer's disease.
  • the sample to be tested includes a stool sample.
  • the present invention provides the use of the intestinal flora-based Alzheimer's disease biomarker described in the first aspect as a target in screening drugs for preventing or treating Alzheimer's disease.
  • the biomarkers associated with Alzheimer's disease discovered based on the present invention are used as targets and can be used to screen drugs for preventing or treating Alzheimer's disease.
  • the screening may include determining whether the candidate drug can be used to prevent or treat Alzheimer's disease based on the effects of the candidate drug on the Alzheimer's disease biomarkers before and after use.
  • the present invention has the following beneficial effects:
  • the present invention discovered for the first time that Flavobacteriaceae, Lachnospiraceae, Deferribacteriaceae, Olfacteriaceae and Desulfovibrioides in the intestinal flora are associated with Alzheimer's disease, and their relative abundance is significantly different between healthy individuals and AD individuals.
  • the method can be used to assist in the diagnosis of symptoms of Alzheimer's disease, has the characteristics of high detection accuracy, convenience, speed, safety and non-invasiveness, and has important clinical guiding significance for auxiliary diagnosis and early warning of AD.
  • FIG1 is a graph showing the intestinal flora diversity of AD mice and wild-type mice (WT);
  • FIG2 is a graph showing the relative abundance of Flavobacteriaceae in AD mice and wild-type mice (WT);
  • FIG3 is a graph showing the relative abundance of Lachnospiraceae in AD mice and wild-type mice (WT);
  • FIG4 is a graph showing the relative abundance of Deferribacteriaceae in AD mice and wild-type mice (WT);
  • FIG5 is a graph showing the relative abundance of Olfacteriaceae in AD mice and wild-type mice (WT);
  • FIG6 is a graph showing the relative abundance of Desulfovibrioides in AD mice and wild-type mice (WT).
  • Alzheimer's disease is a neurological disease that occurs in the elderly and pre-elderly, characterized by progressive cognitive dysfunction and behavioral impairment.
  • the main manifestations are memory impairment, aphasia, apraxia, agnosia, visual-spatial ability impairment, abstract thinking and calculation ability impairment, personality and behavioral changes, etc., which can be improved through drug treatment.
  • Biomarker refers to "a characteristic that can be objectively detected and evaluated, and can serve as an indicator of normal biological processes, pathological processes, or pharmacological responses to therapeutic interventions.”
  • nucleic acid markers also known as gene markers, such as DNA
  • protein markers protein markers
  • cytokine markers chemokine markers
  • carbohydrate markers antigen markers
  • antibody markers antibody markers
  • species markers species markers (species/genus markers)
  • functional markers etc.
  • nucleic acid markers is not limited to existing genes that can be expressed as biologically active proteins, but also includes any nucleic acid fragments, which can be DNA or RNA, modified DNA or RNA, or unmodified DNA or RNA, and a collection of them.
  • biomarkers refer to intestinal microbial markers, which can also be represented by “intestinal microorganisms” or “intestinal flora”.
  • the microbial markers related to Alzheimer's disease used in the present invention are all from fecal samples after intestinal metabolism of the subject.
  • the biomarkers can be analyzed in batches using high-throughput sequencing for fecal samples of healthy individuals and individuals with Alzheimer's disease. Based on high-throughput sequencing data, healthy individuals and Alzheimer's disease individuals are compared to determine specific nucleic acid sequences associated with the Alzheimer's disease individual group.
  • the specific intestinal microbial nucleic acid sequences associated with Alzheimer's disease individuals are determined by bioinformatics analysis methods. First, the sequencing sequence (reads) is compared with a reference gene set (also referred to as a reference gene set, which can be a newly constructed gene set or a database of any known sequence, for example, a known intestinal microbial community non-redundant gene set).
  • the relative abundance of each gene in the nucleic acid sample from the fecal sample of a healthy individual and an Alzheimer's disease individual group is determined respectively.
  • a corresponding relationship can be established between the sequencing sequence and the gene in the reference gene set, so that for a specific gene in the nucleic acid sample, the number of sequencing sequences corresponding thereto can effectively reflect the relative abundance of the gene.
  • the relative abundance of the gene in the nucleic acid sample can be determined by the comparison results according to conventional statistical analysis.
  • the relative abundance of each gene in the nucleic acid sample from the feces of healthy individuals and Alzheimer's disease individuals is statistically tested. Thus, it can be determined whether there are genes with significant differences in relative abundance between the healthy population and the Alzheimer's disease patient population. If there are genes with significant differences, the gene is regarded as a biomarker of abnormal state, i.e., a nucleic acid marker. For a known or newly constructed reference gene set, it usually contains gene species information and functional annotations.
  • the species information and functional annotations of the gene can be further classified to determine the relative abundance of the species and function of each microorganism in the intestinal flora, and the species markers and functional markers of abnormal state can be further determined.
  • high-throughput sequencing analysis is performed on the highly variable regions of the nucleic acid sequences encoding ribosomal RNA.
  • a set of primers is designed using the conserved regions in the bacterial 16S rDNA to amplify the 16S rRNA V3-V4 region genes of the intestinal flora. Qualified PCR products will be used for library construction and high-throughput sequencing of the Illumina Miseq sequencer.
  • the sequencing data is filtered out through a series of bioinformatics analysis methods such as low quality, reads splicing, OTU clustering, species annotation and diversity analysis, and the analysis results of the sample's flora composition and abundance, species with significant differences between groups, etc. can be obtained.
  • a product is provided, the product is used to include a substance for detecting the above-mentioned biomarker, the product is used to diagnose whether a subject suffers from Alzheimer's disease or predict whether a subject has the risk of Alzheimer's disease.
  • the product includes but is not limited to reagents and/or equipment for detecting the relative abundance and/or quantity of biomarkers in the sample to be tested.
  • the relative abundance information of the biomarker is obtained using a sequencing method, further comprising: isolating a nucleic acid sample from a sample (feces) of the subject, constructing a DNA library based on the obtained nucleic acid sample, sequencing the DNA library to obtain sequencing results, and based on the sequencing results, comparing the sequencing results with a reference gene set to determine the relative abundance information of the biomarker.
  • a model for early diagnosis of Alzheimer's disease wherein the input variables of the model for early diagnosis of Alzheimer's disease include the relative abundance value of the Alzheimer's disease biomarker based on intestinal flora, and the output variables of the model for early diagnosis of Alzheimer's disease include the difference multiple, and the calculation formula of the difference multiple is:
  • the criteria for positive Alzheimer's disease are:
  • the difference multiple of the Flavobacteriaceae is ⁇ 1.31, the difference multiple of the Lachnospiraceae is ⁇ 1.74, the difference multiple of the Deferrobacillaceae is ⁇ 1.65, the difference multiple of the Olfobacteriaceae is ⁇ 0.35, or the difference multiple of the Desulfovibrioaceae is ⁇ 0.63.
  • a device for early diagnosis of Alzheimer's disease comprising the following units:
  • Evaluation units for implementation include:
  • the relative abundance value of the Alzheimer's disease biomarker detected by the analysis unit is input into the Alzheimer's disease early diagnosis model for calculation, the difference multiple is output, and it is determined whether it is Alzheimer's disease positive.
  • the use of the intestinal flora-based Alzheimer's disease biomarker as a target in screening drugs for preventing or treating Alzheimer's disease is provided, and based on the effects of the candidate drug on the Alzheimer's disease biomarker before and after use, it is determined whether the candidate drug can be used to prevent or treat Alzheimer's disease.
  • This embodiment provides an Alzheimer's disease biomarker based on intestinal flora, wherein the Alzheimer's disease biomarker includes any one or a combination of at least two of the family Flavobacteriaceae, Lachnospiraceae, Deferribacteraceae, Olfacteriaceae or Desulfovibrioaceae.
  • the abundance and diversity of intestinal flora were analyzed in fecal samples of AD model male mice (purchased from the Institute of Model Animals of Nanjing University) and wild-type healthy male mice (WT) control group. Fecal samples were collected from AD model mice (10) and wild-type healthy mice (10), and total DNA was isolated using a nucleic acid isolation kit. Primers were designed according to the conserved region, and sequencing adapters were added to the ends of the primers. PCR amplification was performed and the products were purified, quantified and normalized to form sequencing libraries. The constructed libraries were first subjected to library quality inspection, and the qualified libraries were subjected to high-throughput sequencing using the Illumina Miseq/HiSeq2500 system.
  • Reads containing more than 10 low-quality ( ⁇ Q20) bases were filtered from the raw data. Then, the filtered reads were classified and annotated using QIIME2 software (version 2020.11.1): first, high-quality paired-end reads were connected to tags using vsearch; second, amplicon sequence variants (ASVs) of all samples were detected using deblur software; third, sklearn-based classifiers were applied to classify and annotate the Greengenes database (version 13-8-99).
  • the Shannon diversity index (Shannon index) was calculated using the "vegan" package in R to detect the alpha diversity of bacteria. After obtaining the taxonomic spectrum of the bacterial families, the Wilcoxon rank sum test was used to compare the abundance differences of the top 10 bacterial families in the diseased group and the normal control group (p ⁇ 0.05).
  • these biomarkers can all be used as biological markers for Alzheimer's disease detection, and whether the test subject suffers from or is susceptible to Alzheimer's disease (i.e., predicting the risk of suffering from Alzheimer's disease) can be effectively determined by determining whether one, two or more of these markers are present in the intestinal flora of the subject. Therefore, by detecting the content of at least one of these biomarkers in the intestinal flora in the test sample, it can be determined whether the subject suffers from or is susceptible to Alzheimer's disease, and it can also be used to monitor the efficiency of the treatment effect of Alzheimer's patients.
  • the present invention discovered for the first time that Flavobacteriaceae, Lachnospiraceae, Deferribacteriaceae, Olfacteriaceae and Desulfovibrioides in the intestinal flora are associated with Alzheimer's disease, and their relative abundance is significantly different between healthy individuals and AD individuals. It can be used to assist in the diagnosis or early warning of symptoms of Alzheimer's disease, has the characteristics of high detection accuracy, convenience, speed, safety and non-invasiveness, and has important clinical guidance significance for assisting the diagnosis and early warning of AD.
  • the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented.
  • Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

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Abstract

本发明公开了一种基于肠道菌群的阿尔茨海默病生物标志物及其应用。所述阿尔茨海默病生物标志物为肠道微生物，所述肠道微生物包括黄杆菌科、毛螺菌科、脱铁杆菌科、嗅杆菌科或脱硫弧菌科中的任意一种或至少两种组合。本发明首次发现了肠道菌群中黄杆菌科、毛螺菌科、脱铁杆菌科、嗅杆菌科和脱硫弧菌科与阿尔茨海默病相关，其相对丰度在健康个体和AD个体中存在显著差异，可用于阿尔茨海默病的症状辅助判断。

Description

一种基于肠道菌群的阿尔茨海默病生物标志物及其应用 技术领域
本发明属于生物技术技术领域，涉及一种基于肠道菌群的阿尔茨海默病生物标志物及其应用。
背景技术
阿尔茨海默病(Alzheimer disease，AD)，又称老年痴呆症，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，严重影响社交与生活功能。由于阿尔茨海默病发病机制尚未完全明确，加上其早期症状比较隐秘，阿尔茨海默病患者容易被漏诊或错诊。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。
寻找高灵敏度、高准确度的生物标志物，对于阿尔茨海默病的诊断和药物干预具有重要意义，如CN106062563A公开了一种用于阿尔茨海默病的早期诊断的生物标志物及方法，所述AD生物标志物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)，通过分析其表达水平，能够辅助AD的早期诊断。
最近研究发现多种神经精神疾病如帕金森、抑郁症、自闭症等与肠道菌群失衡有关，并且80％以上AD患者存在肠道菌群失衡的现象，提示肠道菌群稳态与AD等神经退行性疾病的发病进程密切相关。如CN114657270A公开了一种基于肠道菌群的阿尔茨海默病生物标志物及其应用，其发现了Moryella、氢噬胞菌属、草螺菌属、红游动菌属和代尔夫特菌属与阿尔茨海默病相关，其丰度在阿尔茨海默病患者和健康人群中呈现显著性差异，ROC曲线分析其作为检 测变量具有较高的准确性、特异性和敏感性，可以将Moryella、氢噬胞菌属、草螺菌属、红游动菌属和代尔夫特菌属作为检测靶标应用于阿尔茨海默病患者的诊断。
综上所述，虽然目前已经公开了多种阿尔茨海默病生物标志物，但适用性均有限，并非适用于所有潜在患者，因此，为进一步提高阿尔茨海默病检出率及检出准确性，仍需不断开发新型阿尔茨海默病生物标志物，以扩充阿尔茨海默病检测指标。
发明内容
本发明针对阿尔茨海默病的早期诊断、预警需要丰富的有效生物标志物的问题，开发新型阿尔茨海默病生物标志物，以扩充阿尔茨海默病检测指标，进一步提高阿尔茨海默病检出率及检出准确性。
为达上述目的，本发明采用以下技术方案：
第一方面，本发明提供一种基于肠道菌群的阿尔茨海默病生物标志物，所述阿尔茨海默病生物标志物为肠道微生物，所述肠道微生物包括黄杆菌科、毛螺菌科、脱铁杆菌科、嗅杆菌科或脱硫弧菌科中的任意一种或至少两种组合。
本发明基于Illuminate MiSeq测序平台，深入分析阿尔茨海默病个体和健康个体肠道菌群，发现黄杆菌科(Flavobacteriaceae)，毛螺菌科(Lachnospiraceae)和脱铁杆菌科(Deferribacteraceae)在AD小鼠中的相对丰度水平与正常小鼠相比显著上升，而嗅杆菌科(Odoribacteraceae)和脱硫弧菌科(Desulfovibrionaceae)的相对丰度与健康小鼠相比显著下降，以相对丰度水平作为检测指标，用于阿尔茨海默病的症状辅助判断或预警，具有检测精确度高、方便快捷以及安全无创的特点，对辅助诊断及预警AD具有重要的临床指导意义。
优选地，所述阿尔茨海默病生物标志物来源于受试者的生物样本，所述生物样本包括受试者的粪便。
第二方面，本发明提供检测第一方面所述的基于肠道菌群的阿尔茨海默病 生物标志物的物质在制备诊断受试者是否患有阿尔茨海默病或预测受试者是否患有阿尔茨海默病的风险的产品中的应用。
本发明基于开发的基于肠道菌群的阿尔茨海默病生物标志物，利用检测其相对丰度的物质即可开发诊断受试者是否患有阿尔茨海默病或预测受试者是否患有阿尔茨海默病的风险的产品。
第三方面，本发明提供一种诊断受试者是否患有阿尔茨海默病或预测受试者是否患有阿尔茨海默病的风险的产品，所述产品包括检测第一方面所述的基于肠道菌群的阿尔茨海默病生物标志物的相对丰度和/或数量的试剂和/或设备。
本发明的诊断受试者是否患有阿尔茨海默病或预测受试者是否患有阿尔茨海默病的风险的产品，以相对丰度水平作为检测指标，可有效表征阿尔茨海默病样本，辅助AD诊断及预警。
本发明中，所述产品可以为试剂盒。
第四方面，本发明提供第一方面所述的基于肠道菌群的阿尔茨海默病生物标志物在构建阿尔茨海默病早期诊断模型和/或制备阿尔茨海默病早期诊断装置中的应用。
第五方面，本发明提供一种阿尔茨海默病早期诊断模型，所述阿尔茨海默病早期诊断模型的输入变量包括第一方面所述的基于肠道菌群的阿尔茨海默病生物标志物的相对丰度值，所述阿尔茨海默病早期诊断模型的输出变量包括差异倍数，所述差异倍数的计算公式为：
Figure PCTCN2022137818-appb-000001
阿尔茨海默病阳性的判断标准为：
所述黄杆菌科的差异倍数≥1.31、所述毛螺菌科的差异倍数≥1.74、所述脱铁杆菌科的差异倍数≥1.65、所述嗅杆菌科的差异倍数≤0.35或所述脱硫弧菌科的差异倍数≤0.63。
本发明中，通过对正常粪便样本和AD粪便样本中菌群相对丰度进行充分 对比分析，并进行理性设计，构建了一种阿尔茨海默病早期诊断模型，所述模型以相对丰度值为输入变量，以差异倍数为输出变量，能够快速输出结果，且充分表征肠道菌群异常异常的样本，从而辅助阿尔茨海默症早期诊断。
第六方面，本发明提供一种阿尔茨海默病早期诊断装置，所述装置包括如下单元：
分析单元，用于执行包括：
检测受试者待测样品中第一方面所述的基于肠道菌群的阿尔茨海默病生物标志物的相对丰度值；
评估单元，用于执行包括：
将分析单元检测到的阿尔茨海默病生物标志物的相对丰度值输入第五方面所述的阿尔茨海默病早期诊断模型中进行计算，输出差异倍数，并判断是否为阿尔茨海默病阳性。
本发明的阿尔茨海默病早期诊断装置中，各单元间有效配合，简单高效，能够快速完成样本处理、检测及获得差异倍数，同时以经过合理设计的判断标准进行阿尔茨海默病阳性评估，对于阿尔兹海默症早期诊断具有重要意义。
优选地，所述待测样品包括粪便样品。
第七方面，本发明提供第一方面所述的基于肠道菌群的阿尔茨海默病生物标志物作为靶点在筛选预防或治疗阿尔茨海默病的药物中的应用。
基于本发明发现的与阿尔茨海默病相关的生物标志物为靶点，可用于筛选预防或治疗阿尔茨海默病的药物，所述筛选可包括基于候选药物使用前和使用后对所述阿尔茨海默病生物标志物的影响，从而确定候选药物是否可以用于预防或治疗阿尔茨海默病。
与现有技术相比，本发明具有以下有益效果：
本发明首次发现了肠道菌群中黄杆菌科、毛螺菌科、脱铁杆菌科、嗅杆菌科和脱硫弧菌科与阿尔茨海默病相关，其相对丰度在健康个体和AD个体中存在显著差异，用于阿尔茨海默病的症状辅助判断，具有检测精确度高、方便快 捷以及安全无创的特点，对辅助诊断及预警AD具有重要的临床指导意义。
附图说明
图1为AD小鼠和野生型小鼠(WT)的肠道菌群多样性结果图；
图2为AD小鼠和野生型小鼠(WT)的黄杆菌科相对丰度结果图；
图3为AD小鼠和野生型小鼠(WT)的毛螺菌科相对丰度结果图；
图4为AD小鼠和野生型小鼠(WT)的脱铁杆菌科相对丰度结果图；
图5为AD小鼠和野生型小鼠(WT)的嗅杆菌科相对丰富度结果图；
图6为AD小鼠和野生型小鼠(WT)的脱硫弧菌科相对丰度结果图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。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发明，而非对本发明的限定。
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。
本发明所用术语具有相关领域普通技术人员通常理解的含义。然而，为了更好地理解本发明，对一些定义和相关术语的解释如下：
“阿尔茨海默病”，是发生于老年和老年前期、以进行性认知功能障碍和行为损害为特征的神经系统疾病，主要表现为记忆障碍、失语、失用、失认、视空间能力损害、抽象思维和计算能力损害、人格和行为改变等，可通过药物治疗改善。
“生物标志物”，是指“一种可客观检测和评价的特性，可作为正常生物学过程、病理过程或治疗干预药理学反应的指示因子”。例如，核酸标志物(也可以称为基因标志物，例如DNA)，蛋白质标志物，细胞因子标记物，趋化因子标记物，碳水化合物标志物，抗原标志物，抗体标志物，物种标志物(种/属的标 记)和功能标志物(KO/OG标记)等。其中，核酸标志物的含义并不局限于现有可以表达为具有生物活性的蛋白质的基因，还包括任何核酸片段，可以为DNA，也可以为RNA，可以是经过修饰的DNA或者RNA，也可以是未经修饰的DNA或者RNA，以及由它们组成的集合。在本发明中，“生物标志物”指肠道微生物标志物，也可用“肠道微生物”、“肠道菌群”表示，本发明中使用的与阿尔茨海默病相关的微生物标志物均来自经受试者肠道代谢后的粪便样本。
所述的生物标志物，可以运用高通量测序，批量分析健康个体和阿尔茨海默病个体的粪便样本。基于高通量测序数据，对健康个体与阿尔茨海默病个体群进行比对，从而确定与阿尔茨海默病个体群相关的特异性核酸序列。通过生物信息学的分析方法，确定与阿尔茨海默病个体相关的特异性肠道微生物核酸序列。首先，将测序序列(reads)与参照基因集(也称为参考基因集，可以为新构建的基因集或任何已知序列的数据库，例如，采用已知的肠道微生物群落非冗余基因集)进行比对。接下来，基于比对结果，分别确定来自健康个体和阿尔茨海默病个体群粪便样品的核酸样本中各基因的相对丰度。通过将测序序列与参照基因集进行比对，可以将测序序列与参照基因集中的基因建立对应关系，从而针对核酸样本中的特定基因，与其相对应的测序序列的数目可以有效地反映该基因的相对丰度。由此，可以通过比对结果，按照常规的统计分析，确定在核酸样本中基因的相对丰度。最后，在确定核酸样本中各基因的相对丰度后，对来自健康个体和阿尔茨海默病个体群粪便的核酸样本中各基因的相对丰度进行统计检验，由此，可以判断在健康人群和阿尔茨海默病患者人群中是否存在相对丰度有显著差异的基因，如果存在基因是显著差异的，则该基因被当作是异常状态的生物标志物，即核酸标志物。对于已知或新构建的参照基因集，其通常包含基因物种信息和功能注释，由此，在确定基因相对丰度的基础上，可以进一步通过将基因的物种信息和功能注释进行分类，从而确定肠道菌群中各微生物的物种相对丰度和功能相对丰度，也就可以进一步确定异常状态的物种标志物和功能标志物。
例如一具体实施例中可采用如下方法：
基于Illuminate MiSeq测序平台，针对编码核糖体RNA的核酸序列的高变区进行高通量测序分析。利用细菌16S rDNA中的保守区域设计一套引物对肠道菌群的16S rRNA V3-V4区域基因进行扩增，合格的PCR产物将用于Illumina Miseq测序仪的文库构建与高通量测序。测序数据经过过滤低质量、reads拼接、OTU聚类、物种注释及多样性分析等一系列生物信息分析的方法，可以获得样品的菌群组成及丰度、组间显著差异的物种等分析结果。
本发明一具体实施方式中，提供一种产品，所述产品用于包括用于检测上述生物标志物的物质，所述产品用于诊断受试者是否患有阿尔茨海默病或者预测受试者是否患有阿尔茨海默病的风险。所述产品包括但不限于检测待测样品中生物标志物的相对丰度和/或数量的试剂和/或设备。所述生物标志物的相对丰度信息是利用测序方法得到的，进一步包括：从受试者的样本(粪便)中分离得到核酸样本，基于所获得的所述核酸样本，构建DNA文库，对所述DNA文库进行测序，以便获得测序结果，以及基于所述测序结果，将测序结果与参考基因集进行比对，以确定所述生物标志物的相对丰度信息。
本发明又一具体实施方式中，提供一种阿尔茨海默病早期诊断模型，所述阿尔茨海默病早期诊断模型的输入变量包括所述的基于肠道菌群的阿尔茨海默病生物标志物的相对丰度值，所述阿尔茨海默病早期诊断模型的输出变量包括差异倍数，所述差异倍数的计算公式为：
Figure PCTCN2022137818-appb-000002
阿尔茨海默病阳性的判断标准为：
所述黄杆菌科的差异倍数≥1.31、所述毛螺菌科的差异倍数≥1.74、所述脱铁杆菌科的差异倍数≥1.65、所述嗅杆菌科的差异倍数≤0.35或所述脱硫弧菌科的差异倍数≤0.63。
本发明又一具体实施方式中，提供一种阿尔茨海默病早期诊断装置，所述 装置包括如下单元：
分析单元，用于执行包括：
检测受试者待测样品中所述的基于肠道菌群的阿尔茨海默病生物标志物的相对丰度值；
评估单元，用于执行包括：
将分析单元检测到的阿尔茨海默病生物标志物的相对丰度值输入所述阿尔茨海默病早期诊断模型中进行计算，输出差异倍数，并判断是否为阿尔茨海默病阳性。
本发明又一具体实施方式中，提供所述基于肠道菌群的阿尔茨海默病生物标志物作为靶点在筛选预防或治疗阿尔茨海默病的药物中的应用，基于候选药物使用前和使用后对所述阿尔茨海默病生物标志物的影响，从而确定候选药物是否可以用于预防或治疗阿尔茨海默病。
实施例1
本实施例提供基于肠道菌群的阿尔茨海默病生物标志物，所述阿尔茨海默病生物标志物包括黄杆菌科、毛螺菌科、脱铁杆菌科、嗅杆菌科或脱硫弧菌科中的任意一种或至少两种组合。
对AD模型雄性小鼠(购自南京大学模式动物研究所)与野生型健康雄性小鼠(WT)对照组的粪便样品进行肠道菌群丰度与多样性分析。从AD模型小鼠(10只)和野生型健康小鼠(10只)中采集粪便样品，使用核酸分离试剂盒进行总DNA分离。根据保守区设计得到引物，在引物末端加上测序接头，进行PCR扩增并对其产物进行纯化、定量和均一化形成测序文库，建好的文库先进行文库质检，质检合格的文库用Illumina Miseq/HiSeq2500系统进行高通量测序。从原始数据中过滤那些包含超过10个低质量(<Q20)碱基的reads。然后，使用QIIME2软件(版本2020.11.1)对过滤后的reads进行分类注释：首先，使用vsearch将高质量配对末端reads连接到标签中；其次，使用deblur软件检测所有样本的扩增子序列变体(ASV)；第三，应用基于sklearn的分类器，比 对Greengenes数据库(版本13-8-99)进行分类注释。使用R中的“vegan”包计算香农多样性指数(Shannon指数)来检测细菌的α多样性。在获得菌科分类谱后，使用Wilcoxon秩和检验来比较患病组和正常对照组丰度排名前10位的菌科的丰度差异(p<0.05)。
Shannon指数显示9月龄健康小鼠与AD小鼠鼠菌群多样性存在显著差异(P＝0.035，图1)。如图2～图6所示，进一步对菌群丰度水平进行差异分析发现黄杆菌科(Flavobacteriaceae)、毛螺菌科(Lachnospiraceae)和脱铁杆菌科(Deferribacteraceae)在AD小鼠中的相对丰度水平与健康小鼠相比显著上升，而嗅杆菌科(Odoribacteraceae)和脱硫弧菌科(Desulfovibrionaceae)的相对丰度与健康小鼠相比显著下降。
表明这些生物标志物均可以作为阿尔茨海默病检测的生物学标记物，可以通过确定对象肠道菌群中是否存在这些标志物中的一种或者两种或者多种，从而有效地确定检测对象是否患有或者易感阿尔茨海默病(即预测患有阿尔茨海默病的风险)。由此，通过对检测样本中这些生物标志物的至少一种在肠道菌群中的含量进行检测，来确定对象是否患有或者易感阿尔茨海默病，同时可以用来监控阿尔茨海默病患者的治疗效果的效率。
综上所述，本发明首次发现了肠道菌群中黄杆菌科、毛螺菌科、脱铁杆菌科、嗅杆菌科和脱硫弧菌科与阿尔茨海默病相关，其相对丰度在健康个体和AD个体中存在显著差异，用于阿尔茨海默病的症状辅助判断或预警，具有检测精确度高、方便快捷以及安全无创的特点，对辅助诊断及预警AD具有重要的临床指导意义。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明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，对本发明的任何改进，对本发明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，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 一种基于肠道菌群的阿尔茨海默病生物标志物，其特征在于，所述阿尔茨海默病生物标志物为肠道微生物，所述肠道微生物包括黄杆菌科、毛螺菌科、脱铁杆菌科、嗅杆菌科或脱硫弧菌科中的任意一种或至少两种组合。
  2. 根据权利要求1所述基于肠道菌群的阿尔茨海默病生物标志物，其特征在于，所述阿尔茨海默病生物标志物来源于受试者的生物样本，所述生物样本包括受试者的粪便。
  3. 检测权利要求1或2所述的基于肠道菌群的阿尔茨海默病生物标志物的物质在制备诊断受试者是否患有阿尔茨海默病或预测受试者是否患有阿尔茨海默病的风险的产品中的应用。
  4. 一种诊断受试者是否患有阿尔茨海默病或预测受试者是否患有阿尔茨海默病的风险的产品，其特征在于，所述产品包括检测权利要求1或2所述的基于肠道菌群的阿尔茨海默病生物标志物的相对丰度和/或数量的试剂和/或设备。
  5. 权利要求1或2所述的基于肠道菌群的阿尔茨海默病生物标志物在构建阿尔茨海默病早期诊断模型和/或制备阿尔茨海默病早期诊断装置中的应用。
  6. 一种阿尔茨海默病早期诊断模型，其特征在于，所述阿尔茨海默病早期诊断模型的输入变量包括权利要求1或2所述的基于肠道菌群的阿尔茨海默病生物标志物的相对丰度值；
    所述阿尔茨海默病早期诊断模型的输出变量包括差异倍数，所述差异倍数的计算公式为：
    Figure PCTCN2022137818-appb-100001
    阿尔茨海默病阳性的判断标准为：
    所述黄杆菌科的差异倍数≥1.31、所述毛螺菌科的差异倍数≥1.74、所述脱铁杆菌科的差异倍数≥1.65、所述嗅杆菌科的差异倍数≤0.35或所述脱硫弧菌科的差异倍数≤0.63。
  7. 一种阿尔茨海默病早期诊断装置，其特征在于，所述装置包括如下单元： 分析单元，用于执行包括：
    检测受试者的待测样品中权利要求1或2所述的基于肠道菌群的阿尔茨海默病生物标志物的相对丰度值；
    评估单元，用于执行包括：
    将分析单元检测到的阿尔茨海默病生物标志物的相对丰度值输入权利要求6所述的阿尔茨海默病早期诊断模型中进行计算，输出差异倍数，并判断是否为阿尔茨海默病阳性。
  8. 根据权利要求7所述的装置，其特征在于，所述待测样品包括粪便样品。
  9. 权利要求1或2所述的基于肠道菌群的阿尔茨海默病生物标志物作为靶点在筛选预防或治疗阿尔茨海默病的药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述筛选包括基于候选药物使用前和使用后对所述阿尔茨海默病生物标志物的影响，从而确定候选药物是否可以用于预防或治疗阿尔茨海默病。
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