Use of alzheimer's-disease intestinal flora biomarker combination in preparation of product for detecting ad

Abstract

Provided in the present application is the use of an Alzheimer's-disease intestinal flora biomarker combination in the preparation of a product for detecting AD. The marker combination comprises: any one of or a combination of at least two of Akkermansia muciniphila, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelothrix, Lachnospira, Enterobacter, Corynebacterium, Clostridium, an undefined genus in Erysipelotrichaceae, Brevibacterium or Odoribacter. In the present invention, the abundance level of the intestinal flora biomarker combination is used as a detection index for early auxiliary diagnosis of Alzheimer's disease.

Description

Application of Alzheimer's disease intestinal flora biomarker combination in the preparation of AD detection products Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to the application of a combination of intestinal flora biomarkers for Alzheimer's disease in the preparation of products for detecting AD. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease with an insidious onset. Clinically, it presents with global dementia symptoms, including memory impairment, aphasia, apraxia, agnosia, impaired visual-spatial skills, executive dysfunction, and personality and behavioral changes. The cause remains unknown. Onset before age 65 is termed presenile dementia; onset after age 65 is termed senile dementia.

[0003] Alzheimer's disease has become a major global public health issue. Dementia typically develops after the age of 65. However, with social development, factors such as an accelerated pace of life, high work pressure, and irregular diet and rest have led to an increasingly younger population suffering from Alzheimer's, with many people in their 50s or even their 40s developing the disease. Because the pathogenesis of Alzheimer's disease is not yet fully understood and its early symptoms are relatively subtle, patients with Alzheimer's disease are easily missed or misdiagnosed. Therefore, the search for highly sensitive and accurate biomarkers is crucial for the diagnosis and drug intervention of Alzheimer's disease.

[0004] CN116930510A discloses a combined diagnostic kit and its application for Alzheimer's disease. The kit uses at least four of the following: beta-amyloid protein, phosphorylated tau protein, biomarkers of neurodegeneration or neuronal damage, and biomarkers of neuroimmune disorders, synaptic dysfunction, and blood-brain barrier alterations. The kit also establishes a novel algorithmic model that creatively incorporates AD scale scores and brain function imaging diagnostic results into the algorithmic model for collaborative diagnosis.

[0005] CN117054664A discloses a kit for diagnosing Alzheimer's disease (AD) and its use, specifically disclosing the use of detection reagents for amyloid beta, phosphorylated tau protein, and blood proteins in the preparation of a kit for diagnosing Alzheimer's disease. The kit can use non-cerebrospinal fluids such as blood (whole blood, plasma, serum, etc.), saliva, and tears as samples, enabling early diagnosis and screening of AD. The kit is suitable for use with fully automatic single-molecule immunoassay analyzers and is well-suited for AD screening.

[0006] Currently, the diagnosis of AD mainly relies on memory scales, PET, and the detection of pathological indicators such as A and phosphorylated tau in cerebrospinal fluid and blood. However, the detection results of these diagnostic indicators in clinical practice are still controversial, and there is a lack of effective detection evidence for the early symptoms of AD.

[0007] Recent studies have linked numerous neuropsychiatric disorders, such as Parkinson's disease, depression, and autism, to gut microbial imbalances. Over 80% of patients with Alzheimer's disease (AD) experience gut microbial imbalance, suggesting a close link between gut microbial homeostasis and the pathogenesis of neurodegenerative diseases like AD. Gastrointestinal dysfunction has been reported in AD, and data show that the gut microbiome composition of AD patients differs from that of healthy peers. Increased bacterial toxins and inflammatory cytokine levels have been detected in the blood, suggesting that microbial imbalance in AD may be crucially linked to the development of peripheral immune inflammation. Peripheral immune activation, in turn, may contribute to central nervous system inflammation through the bloodstream. With aging, the permeability of the intestinal mucosal barrier increases, allowing opportunistic pathogens and their products to cross the barrier and invade internal tissues and organs, thereby inducing chronic intestinal and systemic inflammation.

[0008] Therefore, studying the relationship between intestinal flora and the onset of AD and screening AD early diagnostic biomarkers based on intestinal flora are expected to improve the accuracy of AD diagnosis, help with early warning of the disease, pathological typing, and predictive evaluation of the development stage.

[0009] Summary of the Invention

[0010] This application provides the application of a combination of gut microbiota biomarkers for Alzheimer's disease in the preparation of products for detecting AD. This application provides a highly sensitive and accurate combination of Alzheimer's disease biomarkers based on gut microbiota and its application for the early auxiliary diagnosis of Alzheimer's disease. This application uses a combination of 14 gut microbiota markers and their abundance levels as detection indicators for auxiliary judgment 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 of AD-related indicators.

[0011] In a first aspect, the present application provides an Alzheimer's disease intestinal flora biomarker combination or its detection reagent for the preparation of a product for detecting Alzheimer's disease, wherein the marker combination includes: Akkermansia, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelatoclostridium, Lachnospiraceae NK4A136 group, Enterobacter, Oscillibacter, Clostridia UCG-014, Erysipelotrichaceae undefined, Curtobacterium or Odoribacter, or a combination of at least two of the following:

[0012] Preferably, the combination of Alzheimer's disease intestinal flora biomarkers includes: Akkermansia, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelatoclostridium, Lachnospiraceae NK4A136 group, Enterobacter, Oscillibacter, Clostridia UCG-014, Erysipelotrichaceae undefined, Curtobacterium and Odoribacter.

[0013] This application detected the relative abundance information of the intestinal flora in the Alzheimer's disease group and the normal control group. The results showed that the relative abundance levels of Akkermansia, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelatoclostridium and Lachnospiraceae NK4A136 group in the Alzheimer's disease group were significantly increased compared with the normal control group. The relative abundance levels of Enterobacter, Oscillibacter, Clostridia UCG-014, Erysipelotrichaceae undefined, Curtobacterium and Odoribacter in the Alzheimer's disease group were significantly decreased compared with the normal control group. Therefore, this application is based on the above-mentioned combination of intestinal flora markers and uses its abundance level as a detection indicator to develop a method for auxiliary judgment of symptoms of Alzheimer's disease. This detection method has the characteristics of high detection accuracy, convenience, speed, safety and non-invasiveness, and has important clinical guidance significance for auxiliary diagnosis of AD-related indicators.

[0014] In the present application, the nucleotide sequence of the 16S V3-V4 region of the Erysipelotrichaceae undefined is as follows:

[0015] SEQ ID No. 1:

[0016] Preferably, the product for detecting Alzheimer's disease uses the relative abundance level of the Alzheimer's disease intestinal flora biomarker combination as an evaluation standard for assisting in judging AD symptoms; the relative abundance level of the marker combination is detected using a method comprising the following steps:

[0017] (1) Designing specific primers based on the bacteria in the Alzheimer's disease intestinal flora biomarker combination, using the DNA of the sample to be tested as a template, performing PCR amplification, and purifying, quantifying and normalizing the products to form a sequencing library;

[0018] (2) Perform high-throughput sequencing on the sequencing library, perform quality control, read segment splicing, OTU clustering, species annotation and diversity analysis on the sequencing data to obtain the expression abundance of the bacterial community.

[0019] Preferably, in step (1), the specific primers are a set of primers designed for the conserved region in the 16S rDNA of the intestinal flora.

[0020] Preferably, in step (2), the expression abundance of the bacterial population is obtained by high-throughput sequencing analysis of the hypervariable region of the nucleic acid sequence encoding ribosomal RNA based on the Illuminate MiSeq sequencing platform.

[0021] In this application, the relative abundance information of the intestinal flora is obtained by high-throughput sequencing analysis of the hypervariable region of the nucleic acid sequence encoding ribosomal RNA based on the Illuminate MiSeq sequencing platform. A set of primers was designed using the conserved region in the bacterial 16S rDNA to amplify the 16S rRNA V3-V4 region gene 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 subjected to a series of bioinformatics analysis methods such as filtering low quality, read segment 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.

[0022] In a second aspect, the present application provides a device for early auxiliary diagnosis of Alzheimer's disease, the device comprising:

[0023] PCR amplification unit: used to perform PCR amplification using the DNA of the sample to be tested as a template, and to purify, quantify and normalize the product to form a sequencing library; the primers used in the PCR amplification are specific primers designed based on the bacteria in the Alzheimer's disease intestinal flora biomarker combination described in the first aspect;

[0024] High-throughput sequencing unit: used to perform high-throughput sequencing on the sequencing library, perform quality control, read segment splicing, OTU clustering, species annotation and diversity analysis on the sequencing data, and obtain the expression abundance of the bacterial community; and

[0025] Result analysis unit: used to determine whether the sample to be tested is from a patient with Alzheimer's disease based on the bacterial community composition and expression abundance results.

[0026] Preferably, in the PCR amplification unit, the specific primers are a set of primers designed for the conserved region in the 16S rDNA of the intestinal flora.

[0027] Preferably, in the high-throughput sequencing unit, the expression abundance of the bacterial community is obtained by high-throughput sequencing analysis of the hypervariable region of the nucleic acid sequence encoding ribosomal RNA based on the Illuminate MiSeq sequencing platform.

[0028] Preferably, in the result analysis unit, the criteria for judging whether the sample to be tested is derived from a patient with Alzheimer's disease are: the relative abundance level of any one or a combination of at least two of the genus Akkermansia, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelatoclostridium and Lachnospiraceae NK4A136 group is significantly higher than that of the normal control group, and the relative abundance level of Enterobacter, Oscillibacter, Clostridia UCG-014, Erysipelotrichaceae undefined genus is significantly higher than that of the normal control group. The relative abundance levels of any one or a combination of at least two of the genera undefined, Curtobacterium, and Odoribacter were significantly lower than those in the normal control group.

[0029] In a third aspect, the present application provides the use of the Alzheimer's disease intestinal flora biomarker combination described in the first aspect in screening drugs for treating and / or preventing Alzheimer's disease.

[0030] In a fourth aspect, the present application provides the use of the device for early auxiliary diagnosis of Alzheimer's disease described in the second aspect in the preparation of medical equipment for diagnosing Alzheimer's disease.

[0031] In a fifth aspect, the present application provides the use of the device for early auxiliary diagnosis of Alzheimer's disease described in the second aspect in screening therapeutic drugs and / or preventive drugs for Alzheimer's disease.

[0032] In a sixth aspect, the present application provides a reagent for screening or assisting in screening for Alzheimer's disease, the reagent comprising a reagent for detecting a combination of Alzheimer's disease intestinal flora biomarkers in the intestinal flora;

[0033] Among them, the Alzheimer's disease intestinal flora biomarker combination includes: Akkermansia muciniphila, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelatoclostridium, Lachnospiraceae NK4A136 group, Enterobacter, Oscillibacter, Clostridia UCG-014, Erysipelotrichaceae undefined, Curtobacterium or Odoribacter, or a combination of at least two of them.

[0034] Preferably, the combination of Alzheimer's disease intestinal flora biomarkers includes: Akkermansia, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelatoclostridium, Lachnospiraceae NK4A136 group, Enterobacter, Oscillibacter, Clostridia UCG-014, Erysipelotrichaceae undefined, Curtobacterium and Odoribacter.

[0035] Compared with the prior art, this application has the following beneficial effects:

[0036] This application uses the abundance level of the Alzheimer's disease intestinal flora biomarker combination as a detection indicator for auxiliary judgment 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 of AD-related indicators. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 shows the differences in microbial diversity between AD mice and the WT control group. Specific Embodiments

[0038] The technical solution of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present application and should not be regarded as specific limitations on the present application.

[0039] For those technical or conditions not specified in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be purchased through regular channels.

[0040] Example 1

[0041] Fecal samples were collected from diseased individuals and normal healthy control donors, and total DNA was isolated using a nucleic acid isolation kit. Primers were designed based on the conserved regions, sequencing adapters were added to the ends of the primers, PCR amplification was performed, and the products were purified, quantified, and homogenized to form a sequencing library. The constructed library was first subjected to library quality inspection, and the library with qualified quality inspection was 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 QIIME2 software (version 2021.11.0) was used to classify and annotate the filtered reads: First, the high-quality paired-end reads were connected to the tags using vsearch; second, the amplicon sequence variants (ASVs) of all samples were detected using the deblur software; third, a classifier based on sklearn was applied to compare and classify and annotate against the SILVA database (version 13-8-99). The Shannon index was calculated using the "vegan" package in R to detect the α-diversity of bacteria. After obtaining the genus classification spectrum, the ANCOM-BC bias correction method was used to compare the abundance differences at the genus level between the diseased group and the normal control group (p<0.05).

[0042] Using the above technical methods, in this example, the intestinal flora abundance and diversity of fecal samples of AD model male mice and their littermate wild-type healthy male mouse (WT) control group (10 each, all purchased from the Model Animal Research Institute of Nanjing University) were analyzed.

[0043] Figure 1 shows that there are differences in the flora diversity between AD mice and the WT control group. Both the Shannon and Chao1 indices show that the intestinal flora of 9-month-old AD mice has higher diversity, but the difference is not significant (P>0.05, Figure 1).

[0044] Further analysis of the differences in bacterial abundance levels showed that the relative abundance levels of Akkermansia, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelatoclostridium and Lachnospiraceae NK4A136 group in AD mice were significantly increased compared with normal mice, while the relative abundance levels of Enterobacter, Oscillibacter, Clostridia UCG-014, Erysipelotrichaceae undefined, Curtobacterium and Odoribacter in AD mice were significantly decreased compared with normal mice.

[0045] Example 2

[0046] This embodiment provides a device for early auxiliary diagnosis of Alzheimer's disease, comprising:

[0047] PCR amplification unit: Used to amplify the 16S rRNA V3-V4 region of the intestinal flora using the DNA of the sample to be tested as a template and a set of primers designed from the conserved regions of bacterial 16S rDNA. The products are then purified, quantified, and normalized to form a sequencing library. The primers used in the PCR amplification are specific primers designed based on the bacteria in the Alzheimer's disease intestinal flora biomarker panel.

[0048] The Alzheimer's disease intestinal flora biomarker combination includes: Akkermansia, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelatoclostridium, Lachnospiraceae NK4A136 group, Enterobacter, Oscillibacter, Clostridia UCG-014, Erysipelotrichaceae undefined, Curtobacterium and Odoribacter combination;

[0049] High-throughput sequencing unit: used for high-throughput sequencing of sequencing libraries, quality control, read segment splicing, OTU clustering, species annotation and diversity analysis of sequencing data, and obtaining the expression abundance of bacterial communities;

[0050] Result analysis unit: used to judge whether the sample to be tested is from a patient with Alzheimer's disease based on the results of microbial composition and expression abundance; the criteria for judging whether the sample to be tested is from a patient with Alzheimer's disease are: the relative abundance level of the combination of Akkermansia, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelatoclostridium and Lachnospiraceae NK4A136 group is significantly higher than that of the normal control group, and the relative abundance level of Enterobacter, Oscillibacter, Clostridia UCG-014, Erysipelotrichaceae undefined genus is significantly higher than that of the normal control group. The relative abundance levels of the combination of undefined), Curtobacterium and Odoribacter were significantly lower than those in the normal control group.

[0051] In summary, this application provides a biomarker combination to assist in the diagnosis of Alzheimer's disease, using its abundance level as a detection indicator for assisting in the diagnosis 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 assisting in the diagnosis of AD-related indicators.

[0052] The applicant declares that the above is only a specific implementation method of the present application, but the protection scope of the present application is not limited thereto. Technical personnel in the relevant technical field should understand that any changes or substitutions that can be easily thought of by technical personnel in the relevant technical field within the technical scope disclosed in this application fall within the protection scope and disclosure scope of this application.

Claims

1. Use of the Alzheimer's disease gut microbiota biomarker combination or its detection reagent in the preparation of a product for detecting Alzheimer's disease, wherein, The biomarker combination includes any one or a combination of at least two of the following: Akkermansia, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelatoclostridium, Lachnospiraceae NK4A136 group, Enterobacter, Oscillibacter, Clostridia UCG-014, Erysipelotrichaceae undefined, Curtobacterium, or Odoribacter.

2. The use according to claim 1, wherein, The product for detecting Alzheimer's disease uses the relative abundance level of the gut microbiota biomarker combination for Alzheimer's disease as an evaluation criterion for assisting in judging AD symptoms; Among them, the relative abundance level of the biomarker combination is detected by a method including the following steps: (1) Design specific primers according to the bacteria in the gut microbiota biomarker combination for Alzheimer's disease, use the DNA of the test sample as a template, perform PCR amplification, and purify, quantify, and homogenize its product to form a sequencing library; (2) Perform high-throughput sequencing on the sequencing library, perform quality control, read segment splicing, OTU clustering, species annotation, and diversity analysis on the sequencing data to obtain the expression abundance of the microbiota.

3. The use according to claim 2, wherein, In step (1), the specific primers are a set of primers designed for the conserved region in the 16S rDNA of the gut microbiota; Preferably, in step (2), the expression abundance of the microbiota is obtained by performing high-throughput sequencing analysis on the hypervariable region of the nucleic acid sequence encoding ribosomal RNA based on the Illumina MiSeq sequencing platform.

4. A device for early auxiliary diagnosis of Alzheimer's disease, comprising: PCR amplification unit: used to use the DNA of the test sample as a template, perform PCR amplification, and purify, quantify, and homogenize its product to form a sequencing library; The primers in the PCR amplification are specific primers designed according to the bacteria in the gut microbiota biomarker combination for Alzheimer's disease described in claim 1; High-throughput sequencing unit: used to perform high-throughput sequencing on the sequencing library, perform quality control, read segment splicing, OTU clustering, species annotation, and diversity analysis on the sequencing data to obtain the expression abundance of the microbiota; and Result analysis unit: used to judge whether the test sample is from a patient with Alzheimer's disease according to the microbiota composition and expression abundance results.

5. The device for early auxiliary diagnosis of Alzheimer's disease according to claim 4, wherein, In the PCR amplification unit, the specific primers are a set of primers designed for the conserved region in the 16S rDNA of the gut microbiota; Preferably, in the high-throughput sequencing unit, the expression abundance of the flora is obtained by performing high-throughput sequencing analysis on the hypervariable region of the nucleic acid sequence encoding ribosomal RNA based on the Illumina MiSeq sequencing platform.

6. The device for early auxiliary diagnosis of Alzheimer's disease according to claim 4 or 5, wherein, In the result analysis unit, the criteria for determining that the test sample is from a patient with Alzheimer's disease are as follows: the relative abundance level of any one or a combination of at least two of Akkermansia, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelatoclostridium, and Lachnospiraceae NK4A136 group is significantly higher than that of the normal control group, and the relative abundance level of any one or a combination of at least two of Enterobacter, Oscillibacter, Clostridia UCG-014, Erysipelotrichaceae undefined, Curtobacterium, and Odoribacter is significantly lower than that of the normal control group.

7. Use of the Alzheimer's disease gut microbiota biomarker combination according to claim 1 in screening for drugs for treating and / or preventing Alzheimer's disease.

8. Use of the device for early auxiliary diagnosis of Alzheimer's disease according to any one of claims 4 - 6 in the preparation of Alzheimer's disease diagnostic medical equipment.

9. Use of the device for early auxiliary diagnosis of Alzheimer's disease according to any one of claims 4 - 6 in screening for drugs for treating and / or preventing Alzheimer's disease.

10. A reagent for screening or auxiliary screening of Alzheimer's disease, comprising a reagent for detecting the Alzheimer's disease gut microbiota biomarker combination in gut microbiota; wherein, The Alzheimer's disease gut microbiota biomarker combination includes: Akkermansia, Turicibacter, Dubosiella, Pantoea, Alistipes, Lactobacillus, Erysipelatoclostridium, Lachnospiraceae NK4A136 group, Enterobacter, Oscillibacter, Clostridia UCG-014, Erysipelotrichaceae undefined, Curtobacterium, or Odoribacter, either alone or in combination of at least two.

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