Method and apparatus for evaluating risk of alzheimer-type dementia

Abstract

This method for evaluating the risk of Alzheimer-type dementia comprises: a step for extracting DNA from a test sample; a step for identifying bacterial species contained in the test sample on the basis of information on the sequence of the extracted DNA; a step for outputting the abundance of each of bacterial species belonging to a first group and bacterial species belonging to a second group among bacteria contained in the test sample, or outputting the relative abundance ratio between the bacterial species belonging to the first group and the bacterial species belonging to the second group; and an evaluation step for evaluating the abundances or the abundance ratio. The test sample is a fecal sample. The first group includes bacterial species involved in Alzheimer-type dementia. The second group includes bacterial species involved in the suppression of Alzheimer-type dementia.

Description

Method and device for assessing risk of Alzheimer's disease

[0001] The present disclosure relates to a method and an apparatus for assessing the risk of Alzheimer's disease.

[0002] Attempts have been made to evaluate health status and disease risk based on the intestinal microbiota. Patent Document 1 discloses a method for typing intestinal microbiota for disease risk assessment, as one such technique. The typing method in Patent Document 1 includes a measurement step of measuring the abundance ratio of nucleic acids derived from specific bacteria in a test sample, and a classification step of classifying the type of intestinal microbiota of the test sample based on the abundance ratio and a predetermined reference value. Thirty-six specific genera of specific bacteria are presented.

[0003] Patent Literature 2 discloses a method for diagnosing the probability that a subject will develop or have dementia based on the abundance of specific bacteria contained in a sample of the subject's gut microbiota. The diagnostic method of Patent Literature 2 particularly involves comparing the ratio of Firmicutes to Bacteroidetes contained in the sample of the gut microbiota.

[0004] Japanese Patent No. 7193810 Japanese Patent Application Laid-Open No. 2023-508968

[0005] The relationship between the intestinal microbiota and dementia has been studied, for example, as shown in Patent Document 2 mentioned above. However, as dementia is becoming a social issue, there is an ongoing need for new methods for assessing dementia risk.

[0006] In view of this current situation, the present disclosure aims to provide a novel method and system for assessing the risk of Alzheimer's disease.

[0007] A method for assessing the risk of Alzheimer's disease according to the present disclosure includes the steps of: extracting DNA contained in a test sample from the test sample; identifying bacterial species contained in the test sample based on sequence information of the extracted DNA; outputting the abundance of bacterial species belonging to a first group and a second group, or the relative abundance ratio of bacterial species belonging to the first group and the second group, among the bacterial species contained in the test sample; and evaluating the abundance or the abundance ratio. The test sample is a fecal sample. The first group includes bacterial species involved in Alzheimer's disease. The second group includes bacterial species involved in suppressing Alzheimer's disease.

[0008] An apparatus for assessing the risk of Alzheimer's disease according to the present disclosure comprises: a bacterial flora data acquisition unit that extracts DNA contained in a test sample from the test sample and identifies bacterial species contained in the test sample based on sequence information of the extracted DNA; a data derivation unit that outputs, based on the bacterial flora data of the test sample acquired by the bacterial flora data acquisition unit, the abundance of bacterial species belonging to a first group and a second group, or the relative abundance ratio of bacterial species belonging to the first group and the second group, among the bacterial species contained in the test sample; and an evaluation unit that evaluates the risk of Alzheimer's disease based on the results derived by the data derivation unit. The test sample is a fecal sample. The first group includes bacterial species involved in Alzheimer's disease. The second group includes bacterial species involved in suppressing Alzheimer's disease.

[0009] A novel method and system for assessing the risk of Alzheimer's disease is provided.

[0010] Figure 1 shows a graph of the relative abundance of four of the seven bacterial species belonging to Group 1 in healthy individuals (HV), pre-dementia patients (RG), and dementia patients (AD). Figure 2 shows a graph of the relative abundance of three of the seven bacterial species belonging to Group 1 in healthy individuals (HV), pre-dementia patients (RG), and dementia patients (AD). Figure 3 shows a graph of the relative abundance of four of the 17 bacterial species belonging to Group 2 in healthy individuals (HV), pre-dementia patients (RG), and dementia patients (AD). Figure 4 shows a graph of the relative abundance of four of the 17 bacterial species belonging to Group 2 in healthy individuals (HV), pre-dementia patients (RG), and dementia patients (AD). Figure 5 shows a graph of the relative abundance of four of the 17 bacterial species belonging to Group 2 in healthy individuals (HV), pre-dementia patients (RG), and dementia patients (AD). FIG. 6 shows a graph of the relative abundance of five of the 17 bacterial species belonging to Group 2 in healthy subjects (HV), pre-dementia patients (RG), and dementia patients (AD).

[0011] [Description of Embodiments of the Present Disclosure] First, embodiments of the present disclosure will be listed and described. A method for assessing the risk of Alzheimer's disease according to the present disclosure includes the steps of extracting DNA contained in a test sample from the test sample, identifying bacterial species contained in the test sample based on sequence information of the extracted DNA, outputting the abundance of bacterial species belonging to a first group and a second group among the bacteria contained in the test sample, or the relative abundance ratio of bacterial species belonging to the first group to bacterial species belonging to the second group, and evaluating the abundance or abundance ratio. The test sample is a fecal sample. The first group includes bacterial species involved in Alzheimer's disease. The second group includes bacterial species involved in suppressing Alzheimer's disease.

[0012] Alzheimer's disease (AD), which accounts for the majority of dementia, is a progressive neurological disorder caused by the accumulation of amyloid beta (Aβ) in the brain and the phosphorylation of Tau protein. Alzheimer's disease develops from a healthy state through mild cognitive impairment (MCI). Recent reports suggest that Aβ accumulation in the brain begins even during a healthy state, and that it takes 20 to 30 years from the onset of Aβ accumulation to the onset of dementia. This state in which Aβ accumulation is observed but cognitive function is healthy is referred to as pre-dementia. Early detection of pre-dementia and inhibition of the progression of Aβ accumulation is believed to delay or prevent the progression to MCI and AD. Therefore, early detection of Aβ accumulation is desirable. Meanwhile, it has been reported that intestinal bacteria of the order Clostridiales control Aβ accumulation in the brain in mice. Furthermore, it has been reported that there are bacterial groups among intestinal bacteria involved in the production or degradation of Aβ. However, there has been no specific species-level knowledge regarding Aβ production and Aβ degradation in human intestinal bacteria.

[0013] Under these circumstances, a novel method for assessing the risk of Alzheimer's disease was investigated. The inventors discovered that there are significant differences in the abundance ratio of specific bacterial species in the intestinal microbiota of healthy individuals, those with pre-dementia, and those with AD. They then found that screening for those with pre-dementia is possible by calculating and evaluating the abundance or abundance ratio of a first group containing bacterial species involved in Alzheimer's disease and a second group containing bacterial species involved in suppressing Alzheimer's disease in the subject's intestinal microbiota, and arrived at the method of the present disclosure.

[0014] The present inventors came up with the idea that the risk of Alzheimer's disease can be assessed by collectively analyzing the intestinal microbiota contained in a sample obtained from a subject, and by dividing the bacteria contained in the intestinal microbiota into a group containing bacterial species involved in Aβ production (group 1) and a group containing bacterial species involved in inhibiting Aβ production (group 2), calculating the amount and abundance ratio of each, and using the amount and relative abundance ratio of each of groups 1 and 2 as indicators. Further investigation revealed that this assessment method can distinguish between AD patients and healthy individuals, as well as pre-dementia groups, and is useful as a method for assessing dementia risk, leading to the completion of the present invention. According to the present disclosure, a novel method for assessing dementia risk is provided.

[0015] In the method for assessing the risk of Alzheimer's disease, the bacterial species involved in Alzheimer's disease may be a bacterial species involved in amyloid beta production, and the bacterial species involved in the suppression of Alzheimer's disease may be a bacterial species involved in the suppression of amyloid beta production.

[0016] In the method for assessing a risk of Alzheimer's disease, the first group may include Blautia obeum, Ruminococcus torques, and Subdoligranalum variabile, and the second group may include Phocaeicola vulgatus 1, Blautia wexlerae, and Clostridium butyricum.

[0017] The first group may further include bacteria of the genus Romboutsia, bacteria of the family Lachnospiraceae, bacteria of the genus Terrisporobacter, and bacteria of the genus Bacteroides.The second group may further include bacteria of the genus Agathobacter, bacteria of the genus Bifidobacterium, bacteria of the genus Agathobaculum, bacteria of the genus Faecalibacterium, bacteria of the genus Roseburia, bacteria of the genus Blautia, bacteria of the genus Ruminococcus, bacteria of the genus Phocaeicola, bacteria of the genus Lactobacillus, and bacteria of the genus Bacteroides.

[0018] In the method for assessing the risk of Alzheimer's disease, the test samples may be a first test sample obtained at a first time point and a second test sample obtained at a second time point different from the first time point, both obtained from the same subject. The method for assessing the risk of Alzheimer's disease may include a step of comparing the abundance of the bacterial species belonging to the first group and the bacterial species belonging to the second group, or the relative abundance ratio of the bacterial species belonging to the first group and the bacterial species belonging to the second group, in the first test sample with the abundance of the bacterial species belonging to the first group and the bacterial species belonging to the second group, or the relative abundance ratio of the bacterial species belonging to the first group and the bacterial species belonging to the second group, in the second test sample.

[0019] In the method for assessing the risk of Alzheimer's disease, the step of identifying the bacteria contained in the test sample based on the sequence information of the extracted DNA may be carried out using a 16S rRNA method.

[0020] The device for assessing the risk of Alzheimer's disease according to the present disclosure comprises: a bacterial flora data acquisition unit that extracts DNA contained in a test sample from the test sample and identifies bacterial species contained in the test sample based on sequence information of the extracted DNA; a data derivation unit that outputs, based on the bacterial flora data of the test sample acquired by the bacterial flora data acquisition unit, the abundance of bacterial species belonging to a first group and a second group among the bacteria contained in the test sample, or the relative abundance ratio of bacterial species belonging to the first group and bacterial species belonging to the second group; and an evaluation unit that evaluates the risk of Alzheimer's disease based on the results derived by the data derivation unit. The test sample is a fecal sample. The first group includes bacterial species involved in Alzheimer's disease. The second group includes bacterial species involved in suppressing Alzheimer's disease.

[0021] [Details of the embodiment of the present disclosure] The evaluation method and evaluation device according to the present disclosure will be described in more detail.

[0022] [Evaluation Method] The evaluation method according to the present disclosure includes the steps of extracting DNA contained in a test sample from the test sample, and identifying the bacterial species contained in the test sample based on the sequence information of the extracted DNA.

[0023] The test sample in the evaluation method according to the present disclosure is not particularly limited as long as it is obtained from a subject from which a bacterial flora can be collected. Examples of subjects include humans and non-human mammals (e.g., monkeys, cows, horses, pigs, mice, rats, guinea pigs, hamsters, dogs, cats, rabbits, sheep, goats, etc.). A preferred subject is a human. The subject's attributes are not particularly limited, but may be, for example, male or female subjects aged 30 or over, or 40 or over. In the evaluation method according to the present disclosure, the test sample is a fecal sample obtained from a subject.

[0024] The test sample may be stored until it is subjected to evaluation. The storage conditions are not particularly limited, and may be, for example, frozen storage, freeze-dried storage, refrigerated storage, etc. Specific methods for frozen storage include storage at extremely low temperatures using, for example, dry ice, an ultra-low temperature refrigerator, or liquid nitrogen.

[0025] The specific method for extracting DNA from a test sample is not particularly limited and can be any known method. Specifically, for example, the following procedure is used: A fecal sample, for example, about the size of a grain of rice (approximately 10 mg to 1 g), obtained from a subject is suspended in an extraction solution, such as a Tris / HCl buffer solution. A surfactant, a chelating agent, a digestive enzyme, glass beads, etc. are mixed with the suspended extraction solution, and a disruption treatment is performed. The disrupted solution is centrifuged to obtain a supernatant. Centrifugation and extraction are repeated as necessary to obtain a DNA extract. A commonly available stool collection kit for intestinal flora analysis can also be used for DNA extraction.

[0026] The bacterial flora contained in the test sample is analyzed using a DNA extract obtained from the test sample, and the bacterial species contained in the test sample are identified. The bacterial flora contained in the test sample reflects the intestinal bacterial flora of the subject. Methods for analyzing the bacteria contained in the bacterial flora, their abundance, and their abundance ratio include, for example, Gram staining, microscopy, and PCR. However, PCR is preferred due to its advantage of being able to simultaneously analyze the bacteria contained in the bacterial flora and their abundance ratio. Among these, it is preferable to perform bacterial flora analysis using 16S rRNA, which is an amplicon sequence analysis. When performing bacterial flora analysis using 16S rRNA, the genomic region to be analyzed is not particularly limited as long as it can identify the bacteria, but a region centered on the V3 to V4 regions of the 16S rRNA gene is mainly used.

[0027] When analyzing bacteria contained in a bacterial flora and their abundance ratio using the 16S rRNA method, it is preferable to use a next-generation sequencer because it allows for simultaneous analysis of multiple samples. The next-generation sequencer is not particularly limited as long as it can simultaneously determine the base sequences of randomly cleaved DNA fragments, and specific examples include the Illumina MiSeq system. Bacteria constituting the bacterial flora can be identified by referencing the data obtained by the next-generation sequencer and a reference genome database, which is a microbial identification database. The microbial identification database is not particularly limited as long as it can identify bacteria constituting the bacterial flora. The results of amplicon sequence analysis can be statistically processed to determine the abundance and abundance ratio of specific bacterial species in the bacterial flora.

[0028] The method for calculating the abundance ratio of each bacterial species is not particularly limited. For example, a calculation method correcting for sampling error based on a statistical method may be used, or the amount of nucleic acid derived from each bacterium relative to the total amount of nucleic acid in the test sample may be calculated. The calculation may be performed in series with the analysis of the bacterial flora using commercially available software.

[0029] The method according to the present disclosure includes a step of outputting the abundance of bacterial species belonging to a first group and bacterial species belonging to a second group among the bacterial species contained in a test sample, or the relative abundance ratio of bacterial species belonging to the first group and bacterial species belonging to the second group.

[0030] The bacterial species belonging to Group 1 are bacterial species involved in Alzheimer's dementia (involved in the onset of Alzheimer's dementia), more specifically, bacterial species involved in amyloid-β production. The bacterial species involved in amyloid-β production in Group 1 include multiple bacterial species. Specifically, Group 1 may be composed of three or more bacterial species, and preferably five or more, seven or more, or ten or more bacterial species. Specifically, Group 1 includes Blautia obeum, Ruminococcus torques, and Subdoligranalum variabile. Preferably, Group 1 further includes bacteria of the genus Romboutsia, bacteria of the family Lachnospiraceae, bacteria of the genus Terrisporobacter, and bacteria of the genus Bacteroides. More preferably, Group 1 further includes Romboutsia ilealis, Roseburia inulinivorans, Terrisporobacter petrolearius, and Bacteroides stercoris. Typically, the first group may be a group consisting of seven bacterial species: Blautia obeum, Ruminococcus torques, Subdoligranalum variabile, Romboutsia ilealis, Roseburia inulinivorans, Terrisporobacter petrolearius, and Bacteroides stercoris. Previously, bacteria of the order Clostridiales were known to be involved in Aβ accumulation in mice, but no species-level information had been obtained in humans. The method of the present disclosure is characterized by assessing the presence of bacteria containing specific bacterial species as the first group. The first group is an indicator of risk for Alzheimer's disease (a negative factor for Alzheimer's disease).

[0031] The abundance of the bacterial species belonging to the first group may be output as the sum of the abundance of each bacterial species using the species-level taxonomic group estimated in the microbial identification database, or the bacterial species belonging to the first group may be detected collectively and their amounts output. The abundance ratio of the bacterial species belonging to the first group may be output as the abundance ratio of the bacterial species belonging to the first group relative to the entire bacterial flora contained in the test sample.

[0032] The bacterial species belonging to the second group are bacterial species involved in the suppression of Alzheimer's disease, more specifically, bacterial species involved in the suppression of amyloid beta production. The bacterial species belonging to the second group are multiple bacterial species. Specifically, the second group may be composed of three or more bacterial species, and preferably five or more, seven or more, ten or more, fifteen or more, or seventeen or more bacterial species. Specifically, the second group may include Phocaeicola vulgatus 1, Blautia wexlerae, and Clostridium butyricum. The second group preferably further includes bacteria of the genus Agathobacter, Bifidobacterium, Agathobaculum, Faecalibacterium, Roseburia, Blautia, Ruminococcus, Phocaeicola, Lactobacillus, and Bacteroides. In addition, the second group further includes Agathobacter rectalis, Bifidobacterium breve, Bifidobacterium longum, Agathobaculum butyriciproducens, Faecalibacterium prausnitzii, Roseburia intestinalis, Blautia hansenii / hominis, Blautia faecis, Ruminococcus gnavus, Roseburia hominis, Phocaeicola vulgatus 4, Lactobacillus acidophilus, Bacteroides uniformis, and Bacteroides caccae.Typically, the second group may be a group consisting of 17 bacterial species: Phocaeicola vulgatus 1, Blautia wexlerae, Clostridium butyricum, Agathobacter rectalis, Bifidobacterium breve, Bifidobacterium longum, Agathobaculum butyriciproducens, Faecalibacterium prausnitzii, Roseburia intestinalis, Blautia hansenii / hominis, Blautia faecis, Ruminococcus gnavus, Roseburia hominis, Phocaeicola vulgatus 4, Lactobacillus acidophilus, Bacteroides uniformis, and Bacteroides caccae. The method according to the present disclosure is characterized in that the presence of bacteria including a specific bacterial species is evaluated as the second group. The second group is an index for evaluating risk reduction (positive factors related to Alzheimer's dementia) in terms of Alzheimer's dementia risk.

[0033] The abundance of the bacterial species belonging to the second group may be output as the sum of the abundance of each bacterial species using the species-level taxonomic group estimated in the microbial identification database, or the bacterial species belonging to the second group may be detected collectively and their amounts output. The abundance ratio of the bacterial species belonging to the second group may be output as the abundance ratio of the bacterial species belonging to the second group relative to the entire bacterial flora contained in the test sample.

[0034] The method according to the present disclosure includes an evaluation step of evaluating the abundance or abundance ratio of bacterial species belonging to Group 1 and Group 2. Typically, dementia risk is assessed based on the relative abundance ratio of bacterial species belonging to Group 1 and bacterial species belonging to Group 2. The risk of Alzheimer's disease is classified into three stages, for example. The classification may be, for example, three stages: healthy, pre-AD dementia, and MCI or AD dementia. The risk of Alzheimer's disease may also be the likelihood of developing Alzheimer's disease or the likelihood of transitioning to MCI. Furthermore, the risk may be the likelihood of changes (deterioration) in health status (physical characteristics such as weight and height, diet, exercise, sleep, etc.), cognitive function, mental function, or motor function, which are preliminary indicators of the onset of Alzheimer's disease and / or MCI.

[0035] Specifically, for example, if the abundance of bacterial species belonging to Group 1 is minimal and the abundance of bacterial species belonging to Group 2 is abundant, the subject is evaluated as healthy. If the abundance of bacterial species belonging to Group 1 is on the rise and the abundance of bacterial species belonging to Group 2 is on the decline, the subject is evaluated as having pre-Alzheimer's disease. If the abundance of bacterial species belonging to Group 1 is abundant and the abundance of bacterial species belonging to Group 2 is minimal, the subject is evaluated as having MCI or Alzheimer's disease. Objective and / or empirical criteria may be applied as evaluation criteria. Furthermore, the evaluation criteria may be set using AI.

[0036] Furthermore, the method according to the present disclosure may involve obtaining test samples from the same subject at multiple time points and evaluating changes in the presence of bacterial species belonging to the first and second groups in the intestinal microbiota. That is, the test samples are a first test sample obtained at a first time point and a second test sample obtained at a second time point different from the first time point. The interval between the first and second time points may be any time interval, such as one month or more, six months or more, one year or more, or three years or more. The multiple time points may be two or three or more points. For example, two or more points may be spaced apart by six months, one year, or the like. Obtaining test samples at multiple time points allows for a more accurate assessment of Alzheimer's disease risk.

[0037] The abundance and abundance ratio of each of the bacterial species belonging to the first group and the bacterial species belonging to the second group may be analyzed for multiple samples taken at multiple time points at the time the samples are obtained, or the samples from multiple time points may be obtained and stored and then analyzed simultaneously.

[0038] The method for assessing the risk of Alzheimer's disease according to the present disclosure may combine the above-described assessment method based on the abundance and abundance ratio of bacterial species in Groups 1 and 2 with another assessment index. The assessment index that can be combined with the assessment method according to the present disclosure may be, for example, one or more selected from gender, age, height, weight, blood pressure, lifestyle habits, eyesight, hearing, etc. It may also be test results such as cognitive function tests, mental function tests, and motor function tests. It may also be diagnostic results such as MRI imaging diagnosis. It may also be surgical history, medical history, medication history, the presence or absence of diseases currently being treated, etc.

[0039] [Evaluation Device] The evaluation device according to the present disclosure is suitable for carrying out the above-described evaluation method. The evaluation device according to the present disclosure includes a bacterial flora data acquisition unit that extracts DNA contained in a test sample from the test sample and estimates the bacterial species contained in the test sample based on sequence information of the extracted DNA. The data acquisition unit can include a pre-processing device such as an automatic DNA separator, a PCR device, a sequencer, or other device. It can also include a control unit for executing processing by these devices. Mechanical processing performed in response to commands from the control unit can be combined with manual operation by a human operator.

[0040] The evaluation device according to the present disclosure includes a data derivation unit that outputs, based on the bacterial flora data of the test sample acquired by the bacterial flora data acquisition unit, the abundance of each of the bacterial species belonging to the first group and the bacterial species belonging to the second group among the bacteria contained in the test sample, or the relative abundance ratio of the bacterial species belonging to the first group to the bacterial species belonging to the second group. The data derivation unit includes software, and may also include a control unit that causes the software to perform statistical processing.

[0041] The evaluation device according to the present disclosure includes an evaluation unit that evaluates the risk of Alzheimer's disease based on the results derived by the data derivation unit. The evaluation unit may perform the evaluation based on data provided by the data derivation unit, or may perform the evaluation by combining the data provided by the data derivation unit with information obtained from a database that associates the state of the intestinal microbiota with the risk of dementia.

[0042] [Use of Evaluation Method and Evaluation Results] Information on dementia risk obtained by the evaluation method according to the present disclosure can be used as information for indicating the risk of Alzheimer's disease to a subject.

[0043] Furthermore, information on dementia risk obtained by the evaluation method according to the present disclosure can be used as information for evaluating the anticognitive effects of foods and supplements. When used as information for evaluating the anticognitive effects of foods and supplements, for example, multiple subjects (e.g., 100, 300, 500, etc.) are asked to ingest the food or supplement to be evaluated a certain number of times (or for a certain period of time, at certain intervals, and in a certain amount). The anticognitive effects of foods and supplements can be evaluated by analyzing the change in the abundance ratio of bacterial species belonging to Group 1 and bacterial species belonging to Group 2 before and after ingestion. Furthermore, the anticognitive effects can be evaluated by analyzing the increase or decrease in the amount of bacterial species belonging to Group 1 before and after ingestion, and the increase or decrease in the amount of bacterial species belonging to Group 2 before and after ingestion. The evaluation method according to the present disclosure can be used not only to evaluate the anticognitive effects of foods and supplements, but also to develop novel foods and supplements with anticognitive effects.

[0044] [Example] An example of the evaluation method according to the present disclosure is shown below. [Preparation and Analysis of Test Samples] Fecal samples were obtained from healthy individuals (HV), pre-dementia group (RG), and dementia (AD) subjects. Healthy individuals (HV) and dementia (AD) were diagnosed using cognitive function tests, brain imaging tests, etc. For the pre-dementia group (RG), candidates aged 40 years or older were subjected to cognitive function tests and were determined to belong to the pre-dementia group in advance. The number of samples was 8 healthy individuals (HV), 5 pre-dementia group (RG), and 10 dementia (AD) samples.

[0045] 1. Amplicon sequence analysis (1) DNA was extracted from each fecal sample. DNA extraction was performed using a pretreatment method according to the method of Takahashi et al. (Reference 1). The crude DNA was purified using an automated DNA separator (GENE PREP STAR PI-480, manufactured by Kurabo Industries, Ltd.).

[0046] (2) PCR amplification was performed according to the method of Takahashi et al. (Reference 1) using a primer set (341f - R806) (References 2, 3) that amplifies the V3-V4 region of the bacterial 16S rRNA gene. In this case, an index sequence unique to each sample (Reference 4) was inserted into each primer.

[0047] (3) For sequencing, paired-end sequencing was performed using the Miseq system (Illumina) and MiSeq Reagent Kit v 3 (600 Cycle) (Illumina) at 2 × 301 bp cycles.

[0048] 2. Analysis of Amplicon Sequencing Data: (1) After paired-end sequencing, primer sequences were removed from the obtained reads using Cutadapt version 1.18 (Reference 5) software with default settings. (2) Paired-end sequences were joined using fastq-join (Reference 6) software with default settings. (3) Reads with 99% or more of the base sequence satisfying a QV (Quality Value) of 20 or higher were selected using FASTX-Toolkit version 0.0.14 (Reference 7) software, and chimeric sequences were removed using usearch61 (References 8, 9) software. (4) After chimeric sequences were removed, the taxonomic group assignment of the base sequences was estimated using Metagenome@KIN version 2.2.1 software with the RDP version 2.13 (Reference 10) database and the NGS-DB-BA 16.04 microbial identification database (Reference 11) (TechnoSuruga Lab Co., Ltd.). The RDP estimated the taxonomic group with a confidence of 0.8 or higher, and the microbial identification database estimated the taxonomic group with a homology rate of 97% or higher.

[0049] 3. Data Analysis Using Statistical Analysis Software R (1) Using the species-level taxonomic groups estimated in the microbial identification database, the proportions of bacterial species associated with amyloid-β production (Table 1) and those associated with amyloid-β production inhibition (Table 2) were extracted. (2) Using the extracted proportions of each bacterial species, box-and-whisker plots were plotted using the statistical analysis software R version 3.1.01 (Reference 12) based on the grouping information. (3) To confirm whether there were significant differences in the proportions of each bacterial species between groups, a Mann-Whitney U test (Reference 15) was performed using the vegan version 2.5.7 (References 13, 14) package. Statistics and p-values ​​were calculated using the statistical test. The false positive rate (FDR) correction for p-values ​​was performed using the Bonferroni method (Reference 16).

[0050]

[0051]

[0052] [Results] Figures 1 and 2 show graphs of the relative abundance of seven bacterial species belonging to Group 1 in dementia (AD), healthy subjects (HV), and pre-dementia groups (RG). As shown in Figures 1 and 2, the relative abundance of each of the seven bacterial species in AD was higher than that in HV and RG. Furthermore, a tendency for differences in relative abundance was observed among AD, HV, and RG. In particular, the three bacterial species Blautia obeum, Ruminococcus torques, and Subdoligranalum variabile tended to be more abundant in AD. It was confirmed that a group of bacterial species including Blautia obeum, Ruminococcus torques, and Subdoligranalum variabile could be considered a group involved in Alzheimer's dementia and could be used as a parameter for assessing the risk of developing Alzheimer's dementia. Furthermore, it was confirmed that a group of bacterial species including the seven bacterial species shown in Table 1 is a group involved in Alzheimer's dementia and can be used as a parameter for assessing the risk of developing Alzheimer's dementia.

[0053] Figures 3 to 6 show graphs of the relative abundance of each of the 17 bacterial species belonging to Group 2 in dementia (AD), healthy subjects (HV), and pre-dementia groups (RG). As shown in Figures 3 to 6, the relative abundance of all 17 bacterial species in HV was higher than that in RG and AD. Furthermore, a tendency for differences in relative abundance was observed between AD, HV, and RG. In particular, the three bacterial species Phocaeicola vulgatus 1, Blautia wexlerae, and Clostridium butyricum tended to have a higher relative abundance in HV. It was confirmed that a group of bacterial species including Phocaeicola vulgatus 1, Blautia wexlerae, and Clostridium butyricum, which are involved in the suppression of Alzheimer's dementia, can be used as a parameter for assessing the risk of developing Alzheimer's dementia. Furthermore, it was confirmed that a group of bacterial species including the 17 bacterial species shown in Table 2 is a group involved in suppressing Alzheimer's disease and can be used as a parameter for assessing the risk of developing Alzheimer's disease.

[0054] As shown in Figures 1 to 6, the abundance ratio of bacteria in Group 1, which includes bacterial species associated with Alzheimer's disease, and the abundance ratio of bacteria in Group 2, which includes bacterial species associated with the suppression of Alzheimer's disease, differed between healthy individuals (HV), pre-dementia groups (RG), and dementia (AD). In particular, the balance of gut bacterial species in the pre-dementia group (RG), even with normal cognitive function, was found to be different from that of healthy individuals (HV) and closer to the balance of gut bacterial species in AD dementia patients, suggesting that screening for subjects in the pre-dementia group is possible. Furthermore, it was confirmed that screening for healthy individuals, pre-dementia groups, and dementia can be performed based on the balance between the abundance ratio of bacterial species in Group 1 and Group 2.

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[0056] It should be understood that the embodiments disclosed herein are illustrative in all respects and are not limiting in any respect. The scope of the present invention is defined not by the above description but by the claims, and it is intended to include all modifications within the meaning and scope of the claims.

Claims

1. A step of extracting DNA contained in a test sample from the test sample, a step of identifying bacterial species contained in the test sample based on the sequence information of the extracted DNA, and among the bacterial species contained in the test sample, a step of outputting the abundance of bacterial species belonging to the first group and bacterial species belonging to the second group, or the relative abundance ratio of the bacterial species belonging to the first group and the bacterial species belonging to the second group, and an evaluation step of evaluating the abundance or the abundance ratio, wherein the test sample is a fecal sample, the first group includes bacterial species involved in Alzheimer's disease, and the second group includes bacterial species involved in suppressing Alzheimer's disease. A method for evaluating the risk of Alzheimer's disease.

2. The bacterial species involved in Alzheimer's disease are bacterial species involved in amyloid-β production, and the bacterial species involved in suppressing Alzheimer's disease are bacterial species involved in suppressing amyloid-β production. The method for evaluating the risk of Alzheimer's disease according to claim 1.

3. The first group includes Blautia obeum, Ruminococcus torques, and Subdoligranalum variabile, and the second group includes Phocaeicola vulgatus 1, Blautia wexlerae, and Clostridium butyricum. The method for evaluating the risk of Alzheimer's disease according to claim 1 or claim 2.

4. The first group further includes bacteria of the genus Romboutsia, bacteria of the family Lachnospiraceae, bacteria of the genus Terrisporobacter, and bacteria of the genus Bacteroides, and the second group further includes bacteria of the genus Agathobacter, bacteria of the genus Bifidobacterium, bacteria of the genus Agathobaculum, bacteria of the genus Faecalibacterium, bacteria of the genus Roseburia, bacteria of the genus Blautia, bacteria of the genus Ruminococcus, bacteria of the genus Phocaeicola, bacteria of the genus Lactobacillus, and bacteria of the genus Bacteroides. The method for evaluating the risk of Alzheimer's disease according to claim 3.

5. A method for evaluating the risk of Alzheimer's disease according to claim 1 or claim 2, comprising a step of comparing, as the test sample, each of a first test sample at a first time point and a second test sample at a second time point different from the first time point, both obtained from the same subject, with the abundance of bacterial species belonging to the first group and the second group regarding the first test sample, or the relative abundance ratio between the bacterial species belonging to the first group and the bacterial species belonging to the second group, and the abundance of bacterial species belonging to the first group and the second group regarding the second test sample, or the relative abundance ratio between the bacterial species belonging to the first group and the bacterial species belonging to the second group.

6. The method for evaluating the risk of Alzheimer's disease according to claim 1 or claim 2, wherein the step of identifying bacteria contained in the test sample based on the sequence information of the extracted DNA is performed using the 16S rRNA method.

7. An apparatus for evaluating the risk of Alzheimer's disease, comprising: a microbiota data acquisition unit that extracts DNA contained in a test sample from the test sample and identifies bacterial species contained in the test sample based on the sequence information of the extracted DNA; a data derivation unit that outputs the abundance of bacterial species belonging to a first group and a second group, or the relative abundance ratio between the bacterial species belonging to the first group and the bacterial species belonging to the second group, among the bacterial species contained in the test sample, based on the microbiota data of the test sample acquired by the microbiota data acquisition unit; and an evaluation unit that evaluates the risk of Alzheimer's disease based on the result derived by the data derivation unit, wherein the test sample is a fecal sample, the first group includes bacterial species involved in Alzheimer's disease, and the second group includes bacterial species involved in suppressing Alzheimer's disease.

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