Schizophrenia marker microorganisms and their applications

Abstract

The present invention proposes a schizophrenia marker microorganism and its application. The schizophrenia marker microorganism includes a first microorganism set. Therefore, a kit is further proposed, including a reagent suitable for detecting at least one bacterial species in the first microorganism set. The first microorganism set consists of the following bacterial species: Eubacterium sp. CAG: 115, Eubacterium sp. CAG: 252, Firmicutes bacterium CAG: 227, Pseudomonas agarici. The marker microorganism proposed by the present invention has a significant difference in abundance between healthy people and schizophrenia patients, and can be used as a marker for detecting and / or treating schizophrenia.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to schizophrenia marker microorganisms and uses thereof. More specifically, the present application relates to a kit, the use of a reagent in preparing a kit, a pharmaceutical composition for preventing or treating schizophrenia, a method for determining whether an individual suffers from schizophrenia, an apparatus for determining whether an individual suffers from schizophrenia, a device, and a method for screening drugs. Background Art

[0002] Schizophrenia (SCZ) is a devastating psychiatric disorder associated with hallucinations, delusions, and thought disorders that affect perception and social interaction. The etiology of schizophrenia has not yet been elucidated, but it may involve multiple factors, including genetics and the environment. Abnormalities in neurotransmitter systems have been extensively studied, especially focusing on abnormalities in dopamine, glutamate, and γ-aminobutyric acid signaling. There is growing evidence that schizophrenia may be a systemic disease of neuropsychiatric disorders in addition to psychosis. In addition, the importance of inflammation and the gastrointestinal system in schizophrenia has also received attention.

[0003] It is reported that the intestinal microbiota plays an important role in the process of neurogenesis, and the disturbance of the microbiota and microbial products has been shown to affect behavior. A large number of studies have shown that intestinal microorganisms can regulate brain function and behavior through the microbiota-gut-brain axis, and have been reported to affect the host's emotions, memory, cognition and spontaneous activities, and can also regulate neurotransmitters such as 5HT, γ-aminobutyric acid (GABA), norepinephrine (NE), DA and acetylcholine (Ach), thereby participating in the occurrence and development of mental illness, becoming a potential intervention target for mental disorders.

[0004] Recent clinical and preclinical studies have shown that there is a potential link between intestinal flora disorders and the occurrence of SCZ. The intestinal microbiota plays an important role in the metabolism of schizophrenia. Studies have found that the number of Bacteroidetes increases, the number of Bifidobacterium decreases, and there is an imbalance in the proportion of the flora in schizophrenia patients. The number of conditional pathogenic bacteria Bacteroidetes increases significantly, while the number of probiotic Bifidobacterium decreases significantly. The two are in an unbalanced state. Therefore, studying the characteristics of the intestinal microbiota in patients with schizophrenia is of great significance for the detection and treatment of schizophrenia. Summary of the invention

[0005] This application is based on the inventor's discovery and understanding of the following facts and problems:

[0006] The applicant of this application has conducted extensive preliminary research and unexpectedly discovered that some microorganisms can be used as marker microorganisms for detecting schizophrenia, providing a non-invasive method for early detection of schizophrenia; the rational and effective application of marker microorganisms can support the growth of beneficial intestinal bacteria and inhibit potential intestinal pathogenic bacteria, which can treat or alleviate the clinical symptoms of schizophrenia.

[0007] To this end, in the first aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present invention, it includes a reagent suitable for detecting at least one strain in a first microbial set, wherein the first microbial set is composed of the following strains: Eubacterium sp. CAG: 115, Eubacterium sp. CAG: 252, Firmicutes bacterium CAG: 227, Pseudomonas agarici. The kit prepared by the reagent according to the specific embodiment of the present invention can accurately detect at least one strain in the first microbial set, and can accurately distinguish schizophrenia patients from healthy individuals.

[0008] In the second aspect of the present invention, the present invention proposes the use of a reagent in preparing a kit, wherein the reagent is suitable for detecting at least one species in a first microbial set. According to an embodiment of the present invention, the kit is used to diagnose schizophrenia or detect the therapeutic effect of schizophrenia, and the first microbial set is composed of the following species: Eubacterium sp. CAG: 115, Eubacterium sp. CAG: 252, Firmicutesbacterium CAG: 227, Pseudomonas agarici. The kit prepared by the reagent according to a specific embodiment of the present invention can accurately detect at least one species in the first microbial set, and extremely accurately distinguish schizophrenia patients from healthy individuals, thereby effectively diagnosing schizophrenia at an early stage, or detecting changes in schizophrenia during treatment.

[0009] In the third aspect of the present invention, the present invention proposes a pharmaceutical composition for preventing or treating schizophrenia. According to an embodiment of the present invention, at least one strain in the first microbial set is contained, and the first microbial set is composed of the following strains: Eubacterium sp. CAG: 115, Eubacterium sp. CAG: 252, Firmicutes bacterium CAG: 227, Pseudomonas agarici. The strains of the first microbial set in the schizophrenia marker microorganisms according to the embodiment of the present invention can be non-invasively found or assisted in the early detection of schizophrenia, and determine the probability of an individual suffering from schizophrenia or the probability of an individual being in a healthy state; at the same time, increasing the various strains of the first microbial set in the intestinal tract of a high-risk population for schizophrenia or a patient with schizophrenia can reduce the probability of schizophrenia or slow down or cure schizophrenia. Therefore, the pharmaceutical composition comprising at least one strain in the first microbial set can be used to balance the intestinal flora and effectively prevent or treat schizophrenia.

[0010] In a fourth aspect of the present invention, the present invention provides a method for determining whether an individual suffers from schizophrenia. According to an embodiment of the present invention, the method comprises: (1) determining the abundance of a marker microorganism in a stool sample of the individual, wherein the marker microorganism comprises at least one species of a first microorganism set and a second microorganism set; (2) comparing the abundance obtained in step (1) with a predetermined threshold value to determine whether the individual suffers from schizophrenia; wherein the first microorganism set comprises the following species: Eubacterium sp. CAG:115, Eubacterium sp. CAG:252, Firmicutes bacterium CAG:227, Pseudomonas agarici; and the second microorganism set comprises the following species: Actinomyces sp. oral taxon180, Akkermansia muciniphila CAG:154, Akkermansia sp. UNK.MGS-1, Alloscardovia omnicolens, Bacillus vallismortis), Cryptobacteriumcurtum, Kytococcus sedentarius, Trueperella pyogenes. The method according to an embodiment of the present invention can determine whether an individual suffers from schizophrenia based on the abundance of the marker microorganisms in the individual's fecal sample. The marker microorganisms are verified by the inventors on a large number of fecal samples of known status, and the abundance of various intestinal microorganisms in the fecal samples of the schizophrenia group and the healthy group is determined by differential comparative analysis.

[0011] In a fifth aspect of the present invention, the present invention provides an apparatus for determining whether an individual suffers from schizophrenia. According to an embodiment of the present invention, the method comprises: an abundance determination unit, used for determining the abundance of marker microorganisms in the stool sample of the individual, wherein the marker microorganisms include at least one species in a first microorganism set and a second microorganism set; a comparison unit, used for comparing the obtained abundance with a predetermined threshold value, so as to determine whether the individual suffers from schizophrenia; wherein the first microorganism set is composed of the following species: Eubacterium sp. CAG:115, Eubacterium sp. CAG:252, Firmicutes bacterium CAG:227, Pseudomonas agarici; and the second microorganism set is composed of the following species: Actinomyces sp. oral taxon 180, Akkermansia muciniphila CAG:154, Akkermansia sp. UNK.MGS-1, Alloscardovia omnicolens, Bacillus vallismortis), Cryptobacterium curtum, Kytococcus sedentarius, Trueperella pyogenes. The marker microorganisms are determined by the inventor through differential comparative analysis of the abundance of various intestinal microorganisms in fecal samples of schizophrenia patients and healthy people and after analysis and verification of a large number of fecal samples with known states. The device according to the embodiment of the present invention can accurately determine whether an individual is a high-risk group for schizophrenia or a schizophrenia patient.

[0012] In a sixth aspect of the present invention, the present invention proposes a device. According to an embodiment of the present invention, it includes: a computer-readable storage medium having a computer program stored thereon, the program being used to execute the method described in the fourth aspect; and one or more processors for executing the program in the computer-readable storage medium. The device according to an embodiment of the present invention can accurately determine whether an individual is a high-risk group for schizophrenia or a schizophrenia patient.

[0013] In the seventh aspect of the present invention, the present invention provides a method for screening drugs. According to an embodiment of the present invention, the drug is used to treat or prevent schizophrenia, and the method comprises: administering a candidate drug to a subject, detecting the abundance of marker microorganisms in the feces of the subject before and after administration, wherein the marker microorganisms include at least one species in a first microbial set and a second microbial set, wherein the candidate drug that satisfies at least one of the following conditions is suitable for treating or preventing schizophrenia: (1) after the administration, the abundance of at least one species in the first microbial set increases; and (2) after the administration, the abundance of at least one species in the second microbial set decreases; wherein the first microbial set consists of the following species: Eubacterium sp. CAG:115, Eubacterium sp. CAG:252, Firmicutes bacterium CAG:227, Pseudomonas agarici; the second microbial set consists of the following species: Actinomyces sp. oral taxon 180, Akkermansia muciniphila) CAG:154, Akkermansia sp. UNK.MGS-1, Alloscardovia omnicolens, Bacillus vallismortis, Cryptobacterium curtum, Kytococcus sedentarius, Trueperella pyogenes. The method according to the embodiment of the present invention can produce or screen out drugs that promote the growth of various species in the first microorganism concentration in the marker microorganism and / or inhibit the growth of various species in the second microorganism concentration in the intestinal marker microorganism, which is of great significance for assisting in alleviating the clinical symptoms of schizophrenia.

[0014] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0016] Figure 1 is a schematic diagram of an experimental analysis process for screening schizophrenia marker microorganisms according to an embodiment of the present invention; and

[0017] Figure 2: is a schematic diagram of the AUC evaluation results of the comprehensive indicator of marker microorganisms according to an embodiment of the present invention, wherein Specificity represents specificity, that is, predicted to be positive and actually positive, true positive, the ordinate Sensitivity represents sensitivity, that is, true negative, and CI (Confidence interval) represents confidence interval:

[0018] 2-A is the AUC value and confidence interval result diagram under the ROC curve of 122 sample data in the first phase;

[0019] 2-B is the AUC value and confidence interval result graph under the ROC curve of the second phase sample data. DETAILED DESCRIPTION

[0020] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0021] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0022] The term "optionally" is used for descriptive purposes only and cannot be understood as indicating or implying relative importance. Thus, a feature defined as "optionally" may explicitly or implicitly include or exclude the feature.

[0023] Biomarkers are cellular / biochemical or molecular changes that can be detected from biological media, including various body fluids, tissues, cells, feces, hair, exhaled breath, etc.

[0024] The abundance of a microorganism refers to the abundance of that microorganism in a certain microbial population, such as the abundance of that microorganism in an intestinal microbial population, which can be expressed as the content of that microorganism in the population.

[0025] According to one embodiment of the present invention, a kit is provided, comprising a reagent suitable for detecting at least one bacterial species in a first microbial set, wherein the first microbial set consists of the following bacterial species: Eubacterium sp. CAG: 115, Eubacterium sp. CAG: 252, Firmicutes bacterium CAG: 227, and Pseudomonas agarici.

[0026] According to a specific embodiment of the present invention, the kit further includes a reagent suitable for detecting at least one bacterial species in a second microbial set, and the second microbial set is composed of the following bacterial species: Actinomyces sp. oraltaxon 180, Akkermansia muciniphila CAG: 154, Akkermansia sp. UNK.MGS-1, Alloscardovia omnicolens, Bacillus vallismortis, Cryptobacterium curtum, Kytococcus sedentarius, and Trueperella pyogenes.

[0027] According to a specific embodiment of the present invention, the kit includes reagents suitable for detecting all of the bacterial species in the first microbial collection.

[0028] According to a specific embodiment of the present invention, the kit comprises reagents suitable for detecting all of the bacterial species in the second microbial collection.

[0029] According to a specific embodiment of the present invention, the marker microorganisms are determined by the inventors through comparative analysis and verification of the difference in the abundance of microorganisms in fecal samples of a large number of individuals with schizophrenia and a large number of healthy control individuals, and the marker microorganisms associated with schizophrenia in the intestinal microorganisms are clarified. The kit containing reagents for detecting the marker microorganisms can determine the probability of an individual suffering from schizophrenia or the probability of being in a healthy state, which can be used for non-invasive early detection or auxiliary detection of schizophrenia.

[0030] According to a specific embodiment of the present invention, the reagent suitable for detecting the first microbial set or the second microbial set is not particularly limited, and any reagent that can detect the microbial species is included in the scope of the present invention, such as reagents that detect the microbial species through morphological characteristics, physiological and biochemical reaction characteristics, ecological characteristics, serological reactions, sensitivity to bacteriophages, molecular biology, etc., specifically, antibodies, enzymes, nucleic acid molecules, etc.

[0031] In this article, the morphological characteristics of microorganisms refer to: the size, shape, arrangement, etc. of microorganisms observed under a microscope, cell structure, Gram staining reaction, whether they can move, the location and number of flagella, the presence or absence of spores and capsules, the size and position of spores, the shape and structure of the reproductive organs of actinomycetes and fungi, the number, shape, size, color and surface characteristics of spores, etc.

[0032] In this article, the physiological and biochemical reaction characteristics of microorganisms refer to: the ability of microorganisms to utilize substances, the specificity of metabolic products, such as whether H2S, indole, CO2, alcohol, organic acid are produced, whether nitrates can be reduced, whether milk can be coagulated or frozen, etc., the growth environment (temperature, humidity, concentration of gases such as oxygen and carbon dioxide, pH, whether it is resistant to hyperosmosis, whether it is halophilic, etc.), the relationship with other organisms (such as symbiosis, parasitism, host range and pathogenicity), etc.

[0033] Herein, the microbial serological reaction refers to: using the highly sensitive and specific reaction between antigen and antibody to identify similar bacterial species, or to type the same microorganisms, such as using antiserum made from known bacterial species, types or strains to identify the microorganism by whether a specific serological reaction occurs with the microorganism to be identified.

[0034] In this article, molecular biology methods for detecting microorganisms mainly include: using PCR technology, high-throughput sequencing and other methods.

[0035] According to the use of the reagent provided by the present invention in preparing a kit, the reagent is suitable for detecting at least one species in a first microbial set, and the kit is used to diagnose schizophrenia or detect the therapeutic effect of schizophrenia, and the first microbial set consists of the following species: Eubacterium sp. CAG: 115, Eubacterium sp. CAG: 252, Firmicutes bacterium CAG: 227, and Pseudomonas agarici.

[0036] According to a specific embodiment of the present invention, the marker microorganisms are determined by the inventors through comparative analysis and verification of the abundance of microorganisms in fecal samples of a large number of individuals with schizophrenia and a large number of healthy control individuals, and the microbial markers related to schizophrenia in the intestinal microorganisms are clarified. The reagents for detecting the marker microorganisms can determine the probability of an individual suffering from schizophrenia or being in a healthy state, and can be used for non-invasive early detection or auxiliary detection of schizophrenia.

[0037] According to some specific embodiments of the present invention, the reagent is further suitable for detecting at least one bacterial species in a second microbial set, and the second microbial set is composed of the following bacterial species: Actinomyces sp. oral taxon180, Akkermansia muciniphila CAG:154, Akkermansia sp. UNK.MGS-1, Alloscardovia omnicolens, Bacillus vallismortis, Cryptobacterium curtum, Kytococcus sedentarius, and Trueperella pyogenes.

[0038] According to a specific embodiment of the present invention, the reagents suitable for detecting the first microbial set or the second microbial set are not particularly limited, and the reagents that can detect the microbial species are included in the scope of the present invention, such as reagents that detect the microbial species through morphological characteristics, physiological and biochemical reaction characteristics, ecological characteristics, serological reactions, sensitivity to bacteriophages, molecular biology, etc., specifically, antibodies, enzymes, and nucleic acid molecules.

[0039] In this article, the morphological characteristics of microorganisms refer to: the size, shape, arrangement, etc. of microorganisms observed under a microscope, cell structure, Gram staining reaction, whether they can move, the location and number of flagella, the presence or absence of spores and capsules, the size and position of spores, the shape and structure of the reproductive organs of actinomycetes and fungi, the number, shape, size, color and surface characteristics of spores, etc.

[0040] In this article, the physiological and biochemical reaction characteristics of microorganisms refer to: the ability of microorganisms to utilize substances, the specificity of metabolic products, such as whether H2S, indole, CO2, alcohol, organic acid are produced, whether nitrates can be reduced, whether milk can be coagulated or frozen, etc., the growth environment (suitable growth temperature, humidity, concentration of gases such as oxygen and carbon dioxide, pH, whether it is resistant to hyperosmosis, whether it is halophilic, etc.), the relationship with other organisms (such as symbiosis, parasitism, host range and pathogenicity), etc.

[0041] Herein, the microbial serological reaction refers to: using the highly sensitive and specific reaction between antigen and antibody to identify similar bacterial species, or to type the same microorganisms, such as using antiserum made from known bacterial species, types or strains to identify the microorganism by whether a specific serological reaction occurs with the microorganism to be identified.

[0042] In this article, molecular biology methods for detecting microorganisms mainly include: using PCR technology, high-throughput sequencing and other methods.

[0043] A pharmaceutical composition for preventing or treating schizophrenia provided by the present invention contains at least one species of a first microbial collection, wherein the first microbial collection consists of the following species: Eubacterium sp. CAG: 115, Eubacterium sp. CAG: 252, Firmicutes bacterium CAG: 227, and Pseudomonas agarici.

[0044] The above-mentioned marker microorganisms are determined by the inventor through differential comparative analysis of the abundance of various intestinal microorganisms in fecal samples of schizophrenia disease groups and healthy groups, as well as verification of a large number of fecal samples of known status. The species of the first microbial collection in the marker microorganisms are significantly enriched in the healthy group compared with the schizophrenia patient group. The significant enrichment means that compared with the abundance in the schizophrenia patient group, the abundance of the above-mentioned species in the healthy group is statistically significantly higher or significantly and substantially higher than the abundance in the schizophrenia patient group; substances that can increase the abundance of this part of the species can be used to treat schizophrenia or be beneficial to schizophrenia patients. The substances that can increase their abundance are not limited to drugs for treating schizophrenia and functional foods that are beneficial to the balance of intestinal flora. The marker microorganisms provided in this embodiment can be used to prepare drugs for treating schizophrenia, and the drugs can effectively treat or relieve schizophrenia.

[0045] A method for determining whether an individual suffers from schizophrenia according to the present invention comprises steps (1) and (2).

[0046] (1) Determining the abundance of marker microorganisms in a stool sample of the individual.

[0047] The marker microorganisms include at least one species in the first microorganism set and the second microorganism set. The first microorganism set is composed of the following species: Eubacterium sp. CAG: 115, Eubacterium sp. CAG: 252, Firmicutes bacterium CAG: 227, Pseudomonas agarici; the second microorganism set is composed of the following species: Streptococcus parasanguinis, Streptococcus salivarius, Eubacterium recale, Coprococcus comes, Lachnospiraceae bacterium 5163FAA and Roseburia hominis.

[0048] According to some specific embodiments of the present invention, step (1) further includes: obtaining nucleic acid sequencing data from the stool sample of the individual; comparing the sequencing data with a reference genome; and determining the abundance of the marker microorganism based on the result of the comparison.

[0049] According to a specific embodiment of the present invention, in step (1), the abundance of the marker microorganism is determined according to the following formula:

[0050] Ab(S)=Ab(U S )+Ab(M S ), wherein S represents the number of the marker microorganism, Ab(S) represents the abundance of the marker microorganism S, and Ab(U S )=U S / l S , U S is the number of reads in the sequencing data that are uniquely aligned with the reference genome of the marker microorganism S, l S is the total length of the reference genome of the marker microorganism S, M S is the number of reads in the sequencing data that are non-uniquely aligned with the reference genome of the marker microorganism S, i represents the number of the non-uniquely aligned reads, Co i is the abundance coefficient corresponding to the i-th read segment, Co i,s represents the abundance coefficient of the non-uniquely aligned read i for the marker microorganism S, N is the total number of microorganisms that the non-uniquely aligned read i can be aligned with, and j represents the number of the microorganism that the non-uniquely aligned read i can be aligned with.

[0051] The alignment can be performed using known alignment software, such as SOAP, BWA and TeraMap. During the alignment process, the alignment parameters are generally set to allow a maximum of s base mismatches for one or a pair of reads, such as setting s≤2. If more than s bases in the reads are mismatched, the reads are deemed to be unable to be aligned to the assembly fragment. The alignment results obtained include the alignment of each read with the reference genome of each species, including whether the read can be aligned to the reference genome of one or some species, whether it is only aligned to one species or to the reference genomes of multiple species, the position of the reference genome of the species, and the unique position or multiple positions of the reference genome of the species.

[0052] The reference genome of the strain / microorganism refers to a predetermined sequence of the microbial species, which can be any reference template of the biological category to which the sample to be tested belongs or is contained, for example, the target is the microorganism in the sample to be tested, and the reference sequence can select the reference genome of various microorganisms in the NCBI database and / or the DACC intestinal reference genome disclosed by the HMP and MetaHIT projects. Furthermore, a resource library containing more reference sequences can also be pre-configured, for example, based on the status of the individual from which the sample to be tested is derived, the region and other factors, select or measure and assemble a closer sequence as a reference sequence. According to one embodiment of the present invention, the reference genomes of various microorganisms are obtained from a public database. Usually, there are multiple versions of the reference genome of a microorganism, that is, there are multiple public reference genomes of a microorganism.

[0053] Reads are aligned with the reference genome of the species, and the aligned reads can be divided into two parts: a) Unique reads (U): reads that are aligned to the reference genome of a species only; these reads are called unique reads. That is, if the reference genomes aligned to the reads are all from the same species, these reads are defined as unique reads; b) Multiple reads (M): reads that are aligned to the reference genomes of more than one species are defined as multiple reads. That is, if the reference genomes aligned to the reads are from at least two species, these reads are defined as multiple reads.

[0054] (2) Abundance comparison to determine whether an individual suffers from schizophrenia.

[0055] According to one embodiment of the present invention, the abundance obtained in step (1) is compared with a predetermined threshold value to determine whether the individual suffers from schizophrenia.

[0056] According to some specific embodiments of the present invention, the threshold is pre-set, including a threshold for abundance of schizophrenia and a threshold for abundance of non-schizophrenia. The abundance of the marker microorganism in the sample of the individual to be tested is compared with the threshold to determine the state of the individual to be tested. The threshold can be a value or a range of values. For example, based on the mean abundance of the marker microorganism in individuals with known disease or health conditions, the threshold corresponding to the microorganism can be set to a 95% confidence interval of the mean abundance.

[0057] The confidence interval is an estimated interval of a population parameter constructed from a sample statistic. In statistics, a confidence interval of a probability sample is an interval estimate of a population parameter of the sample. The confidence interval shows the degree to which the true value of the parameter has a certain probability of falling around the measurement result. The confidence interval gives the degree of credibility of the measured value of the measured parameter, that is, the "certain probability" required above, and this probability is called the confidence level.

[0058] According to some specific embodiments of the present invention, when the abundance of the marker microorganism determined in step (1) reaches the abundance threshold for suffering from schizophrenia but does not reach the abundance threshold for not suffering from schizophrenia, it is determined that the individual suffers from schizophrenia; when the abundance of the marker microorganism determined in (1) reaches the abundance threshold for not suffering from schizophrenia but does not reach the abundance threshold for suffering from schizophrenia, it is determined that the individual does not suffer from schizophrenia.

[0059] It should be noted that, depending on the purpose or requirements, there may be different requirements for the reliability of determining the individual status results, and those skilled in the art may select different significance levels or thresholds.

[0060] The method is based on detecting the abundance of various species of marker microorganisms in the fecal samples of individuals, comparing the abundance of various species determined by the detection with their thresholds, and determining the probability that the individual is a schizophrenia individual or a healthy individual based on the comparison results. This provides a non-invasive auxiliary detection or auxiliary intervention treatment method for the early detection of schizophrenia.

[0061] All or part of the steps of the method for determining whether an individual suffers from schizophrenia using marker microorganisms in any of the above embodiments can be implemented using a device / system comprising detachable corresponding unit functional modules, or the method can be programmed and stored in a machine-readable medium and implemented by running the machine-readable medium.

[0062] According to the present invention, a device for determining whether an individual suffers from schizophrenia is provided, the device comprising: an abundance determination unit, used to determine the abundance of marker microorganisms in a stool sample of the individual; a comparison unit, used to compare the obtained abundance with a predetermined threshold value, so as to determine whether the individual suffers from schizophrenia; wherein the first microbial set is composed of the following species: Eubacterium sp. CAG:115, Eubacterium sp. CAG:252, Firmicutes bacterium CAG:227, Pseudomonas agarici; the second microbial set is composed of the following species: Actinomyces sp. oral taxon180, Akkermansia muciniphila CAG:154, Akkermansia sp. UNK.MGS-1, Alloscardovia omnicolens, Bacillus vallismortis), Cryptobacterium curtum, Kytococcus sedentarius, Trueperella pyogenes. The above description of the technical features and advantages of the method for determining whether an individual suffers from schizophrenia using marker microorganisms in any embodiment of the present invention is also applicable to the device of this aspect of the present invention, and will not be repeated here.

[0063] According to an embodiment of the present invention, the abundance determination unit is suitable for determining the abundance through the following steps: obtaining nucleic acid sequencing data in the stool sample of the individual; comparing the sequencing data with a reference genome; and determining the abundance of the marker microorganism based on the result of the comparison.

[0064] The alignment can be performed using known alignment software, such as SOAP, BWA, and TeraMap. During the alignment process, the alignment parameters are generally set to allow a maximum of s base mismatches for one or a pair of reads, such as setting s≤2. If more than s bases in the reads are mismatched, the reads are deemed to be unable to be aligned to (aligned on) the assembly fragment. The alignment results obtained include the alignment of each read with the reference genome of each species, including whether the read can be aligned to the reference genome of one or some species, whether it is only aligned to one species or to the reference genome of multiple species, the position of the reference genome of the species, and the unique position or multiple positions of the reference genome of the species. According to one embodiment of the present invention, SOAPalign 2.21 is used for alignment, and the parameters are set to -r 2-m 100-x 1000.

[0065] The reference genome of the microorganism refers to a predetermined sequence of the microorganism species, which can be any reference template of the biological category to which the sample to be tested belongs or is contained, for example, the target is the microorganism in the sample to be tested, and the reference sequence can select the reference genome of various microorganisms in the NCBI database and / or the DACC intestinal genome disclosed by the HMP and MetaHIT projects. Furthermore, a resource library containing more reference sequences can also be pre-configured, for example, based on the state of the individual from which the sample to be tested is derived, the region and other factors, select or measure and assemble a closer sequence as a reference sequence. According to one embodiment of the present invention, the reference genomes of various microorganisms are obtained from a public database. Usually, there are multiple versions of the reference genome of a microorganism, that is, there are multiple public reference genomes of a microorganism.

[0066] Reads are aligned with the reference genome of the species, and the aligned reads can be divided into two parts: a) Unique reads (U): reads that are aligned to the genome of only one species; these reads are called unique reads. That is, if the reference genomes aligned to the reads are all from the same species, these reads are defined as unique reads. b) Multiple reads (M): reads that are aligned to the reference genomes of more than one species are defined as multiple reads. That is, if the reference genomes aligned to the reads are from at least two species, these reads are defined as multiple reads.

[0067] According to one embodiment of the present invention, the abundance of the marker microorganism is determined according to the following formula:

[0068] Ab(S)=Ab(U S )+Ab(MS ), wherein S represents the number of the marker microorganism, Ab(S) represents the abundance of the marker microorganism S, Ab(U S )=U S / l S , U S is the number of reads in the sequencing data that are uniquely aligned with the reference genome of the marker microorganism S, l S is the total length of the reference genome of the marker microorganism S, M S is the number of reads in the sequencing data that are non-uniquely aligned with the reference genome of the marker microorganism S, i represents the number of the non-uniquely aligned reads, Co i is the abundance coefficient corresponding to the i-th read segment, Co i,s represents the abundance coefficient of the non-uniquely matched read i for the marker microorganism S, N is the total number of microorganisms that the non-uniquely matched read i can match, and j represents the number of the microorganism that the non-uniquely matched read i can match. The above description of the technical features and advantages of the method for determining whether an individual suffers from schizophrenia using marker microorganisms in any embodiment of the present invention is also applicable to the device of this aspect of the present invention, and will not be repeated here.

[0069] According to another embodiment of the present invention, a device is provided, comprising: a computer-readable storage medium having a computer program stored thereon, wherein the program is used to execute the aforementioned method for determining whether an individual suffers from schizophrenia; and one or more processors, for executing the program in the computer-readable storage medium.

[0070] According to another embodiment of the present invention, there is provided a method for screening drugs for treating or preventing schizophrenia, the method comprising: administering a candidate drug to a subject, detecting the abundance of marker microorganisms in the feces of the subject before and after administration, the marker microorganisms comprising at least one species of a first microbial set and a second microbial set, wherein the candidate drug that satisfies at least one of the following conditions is suitable for treating or preventing schizophrenia: (1) after the administration, the abundance of at least one species of the first microbial set increases; and (2) after the administration, the abundance of at least one species of the second microbial set decreases; wherein the first microbial set consists of the following species: Eubacterium sp. CAG:115, Eubacterium sp. CAG:252, Firmicutes bacterium CAG:227, Pseudomonas agarici; and the second microbial set consists of the following species: Actinomyces sp. oral taxon 180. Akkermansia muciniphila CAG:154. Akkermansia sp. UNK.MGS-1. Alloscardovia omnicolens. Bacillus vallismortis. Cryptobacterium curtum. Kytococcus sedentarius. Trueperella pyogenes.

[0071] The method for producing or screening drugs for treating schizophrenia according to this aspect of the present invention can obtain drugs that can support the growth of beneficial intestinal bacteria and / or inhibit potential intestinal pathogenic bacteria by rationally and effectively applying determined schizophrenia biomarkers for screening, which is of great significance for assisting in alleviating the clinical symptoms of schizophrenia.

[0072] The following examples are described in detail. The reagents, sequences, software and instruments not specifically mentioned in the following examples are all conventional commercially available products.

[0073] Example 1 Identification of biomarkers

[0074] In this embodiment, the inventors studied fecal samples from 64 schizophrenia patients and 58 healthy controls to obtain the characteristics of intestinal flora microbial communities and functional components. In general, the inventors downloaded about 334Gb of high-quality sequencing data. Metagenomic analysis showed that 27 microbial species were closely related to schizophrenia, of which 6 bacteria were enriched in the intestinal microorganisms of healthy people and 21 bacteria were enriched in the intestinal microorganisms of schizophrenia patients.

[0075] 1. Acquisition of sequencing data:

[0076] Samples from schizophrenia patients and healthy people were from the First Affiliated Hospital of Chongqing Medical University. A total of 64 Chinese schizophrenia patients and 58 healthy people's stool samples were collected in the experiment. The fresh stool sample of each individual was divided into 200 mg / portion, a total of 5 portions, and immediately frozen in a -80℃ refrigerator.

[0077] Total DNA was extracted from all fecal samples using the phenol-chloroform DNA extraction method.

[0078] 2. Library construction and sequencing, as well as reference data download

[0079] DNA library construction was carried out according to the operating instructions of the instrument manufacturer (Illumina). The library was sequenced by PE150 bp. The Illumina HiSeq2000 (Illumina, San Diego, CA) platform sequenced the libraries of 122 samples. Each sample generated an average of 11.21 Gb (sd.±2.1 Gb) of high-quality sequencing results, with a total of 1368 Gb of sequencing data.

[0080] Reference Figure 1 The experimental process shown is for identifying biomarkers related to schizophrenia, wherein the omitted steps or details are well known to those skilled in the art, and several important steps are introduced as described below.

[0081] 3. Analysis of microbial species abundance

[0082] 3.1 Sequence Optimization Statistics

[0083] 1) First, the first phase of sequencing was performed. This phase of sequencing collected data from 122 samples. After obtaining the sequencing data of the first phase of 122 samples, it was filtered and quality controlled according to the following standards: a) remove reads with more than 5 N bases; b) remove reads with low-quality bases (Q20) greater than 50%; c) remove tail low-quality (Q20) and N bases. The reads sequence with missing pairs was considered as a single read for assembly.

[0084] 2) The downloaded healthy person data are processed in the same manner using the method described in 1).

[0085] 2.2 Species abundance analysis

[0086] SOAPalign 2.21 is used to match paired-end clean reads for redundant genomes. Here, the so-called redundant genomes come from the reference genomes of bacteria published in various databases, and the alignment parameters are -r 2-m 200-x1000. The alignment results of reads and redundant genomes can be divided into two parts: a) Unique reads (U): reads are aligned to the genome of only one species; these reads are defined as unique reads. That is, if these genomes come from the same species, the inventors define these reads as unique reads. b) Multiple reads (M): If reads are aligned to the genomes of two or more species, they are defined as multiple reads. That is, if the aligned genomes come from different species, the inventors define these reads as multiple reads.

[0087] For species S, its abundance is Ab(S), which is related to the unique U reads and shared M reads. The abundance is calculated as follows:

[0088] Ab(S)=Ab(U S )+Ab(M S ),

[0089] Wherein, S represents the number of the marker microorganism,

[0090] Ab(S) represents the abundance of the marker microorganism S,

[0091] Ab(U S )=U S / l S ,

[0092] U S is the number of reads in the sequencing data that are uniquely aligned with the reference genome of the marker microorganism S,

[0093] l S is the total length of the reference genome of the marker microorganism S,

[0094]

[0095] M S is the number of reads in the sequencing data that are non-uniquely aligned to the reference genome of the marker microorganism S,

[0096] Co i is the abundance coefficient corresponding to the i-th read segment,

[0097]

[0098] i represents the number of the non-uniquely aligned read segment,

[0099] Co i,s Indicates that for the marker microorganism S,

[0100] The abundance coefficient of the non-uniquely aligned read segment i,

[0101] N is the total number of microorganisms that the non-uniquely aligned read i can align to,

[0102] j represents the number of the microorganism that the non-uniquely aligned read segment i can be aligned with.

[0103] The species abundance values ​​calculated in each sample were divided by the total abundance of each sample to obtain the normalized species abundance table.

[0104] 3.3 Screening of microbial species markers

[0105] In order to obtain intestinal microbial species markers closely related to schizophrenia, the inventors used the intestinal microbial species abundance data of two groups of schizophrenia patients (SCZ) (64 cases) and healthy people (HC) (58 cases) to conduct a study on the correlation with the disease at the species level. Based on the species abundance table obtained in step 3.2, the inventors set the following criteria: (1) The median of the species abundance in the schizophrenia patient group or the healthy person group must be greater than 0; (2) The Wilcoxon rank sum test combined with Benjamini Hochberg's multiple test was used to test and obtain the difference p value between each species and schizophrenia disease; (3) A strict threshold p_values<0.01 was used to screen using the above parameters. The inventors obtained 27 intestinal microbial species closely related to schizophrenia, of which 21 species were enriched in the intestines of schizophrenia patients and 6 species were enriched in healthy people. The 27 microbial species markers are shown in Table 1.

[0106] Table 1:

[0107]

[0108]

[0109] Example 2 Validation of microbial species markers

[0110] In order to verify the findings in Example 1, the inventors further analyzed the abundance of the 27 bacterial genera in the stool samples of 23 healthy people and 26 schizophrenia patients in the verification group, and selected the 27 microbial species markers according to the enrichment of each species in the healthy group and the disease group. The DNA extraction, sequencing and species abundance analysis of the verification group were carried out with reference to Example 1.

[0111] The validation results are as follows: of the above 6 species enriched in healthy people, 4 were validated with high quality in the validation set (p_values<0.01). The mean and P value results of the validation of the microbial species markers enriched in healthy people are shown in Table 2.

[0112] Table 2:

[0113]

[0114] Among the 21 species enriched in schizophrenia patients mentioned above, 8 of them were verified with high quality in the validation set (p value < 0.01). The p value and q value of the two microbial markers enriched in schizophrenia patients in the validation set data are shown in Table 3.

[0115] Certain Bacillus metabolites inhibit the production of norepinephrine and epinephrine, which may have a significant impact on the sympathetic nervous system and adrenal glands to secrete neurotransmitters. This change seems to be the main factor leading to schizophrenia. Trueperella pyogenes is an important conditional pathogen.

[0116]

[0117] The inventors believe that the four microbial species markers enriched from healthy people can be used as reverse indicators of schizophrenia, or as components of the microbial flora of microbial preparations developed for the treatment of schizophrenia, or as recovery indicators for detecting schizophrenia and monitoring the progress of schizophrenia treatment; the eight microbial species markers enriched in schizophrenia patients can be used as positive indicators of schizophrenia, especially for non-invasive detection and diagnosis of schizophrenia.

[0118] The inventors used these 12 microbial species markers to construct a comprehensive index to estimate the area under the ROC (Receiver-operating characteristic) curve AUC. The larger the AUC, the higher the diagnostic ability. The evaluation comprehensive score corresponds to its diagnostic ability for schizophrenia. By evaluating 122 samples from the first phase and 49 samples from the second phase, the specific situation is as follows Figure 2As shown in Figure 2, they all showed good diagnostic ability, with AUC = 91.1% obtained in the first phase. Figure 2 As shown in a, the confidence interval is 86.2%-96%; the AUC obtained in the second phase is 88.6%. Figure 2 As shown in b, the confidence interval is 74.9%-97.8%.

[0119] Example 3 Detection of individual status

[0120] In this embodiment, the inventors used 60 stool samples to detect the individual status of the sample sources.

[0121] The abundance of Actinomyces sp. oralaxon 180, Akkermansia muciniphila CAG: 154, Alloscardovia omnicolens, Bacillus vallismortis, Cryptobacterium curtum, Kytococcus sedentarius, and Trueperella pyogenes shown in Table 3 in each stool sample was determined by referring to the method of Example 2, and it was determined whether the abundance of these 7 strains in each sample fell within the 95% confidence interval of their abundance in the disease control group or the healthy control group. The state of the individual corresponding to the sample whose abundance of these 7 species fell within the corresponding interval of the disease group was determined to be a schizophrenia patient, and the state of the individual corresponding to the sample whose abundance of the 7 species fell within the corresponding interval of the healthy group was determined to be a non-schizophrenia patient.

[0122] The results show that the method described in this embodiment can be used to judge the individual status of 56 samples, and the judgment of the status of the individuals corresponding to 50 samples out of the 56 samples is consistent with the recorded status of the individuals from which the samples are derived.

[0123] In addition, the inventors found that the combined detection of species in Table 2 and Table 3, such as detecting that the species markers in Table 3 are enriched while the species markers in Table 2 are not enriched, can more accurately determine and discover schizophrenia patients or susceptible populations.

[0124] In the scheme of using markers to treat schizophrenia, the inventors found that the growth of species markers in 3 was inhibited or eliminated, while the species markers in Table 2 were enriched, and the treatment effect was excellent.

[0125] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0126] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of a reagent in preparing a kit, wherein the reagent is suitable for detecting strains in a first microbial collection, and the kit is used to diagnose schizophrenia or detect the therapeutic effect of schizophrenia, and the first microbial collection consists of the following strains: Eubacterium sp. CAG: 115, Eubacterium sp. CAG: 252, Firmicutes bacterium CAG: 227, Pseudomonas agarici, The reagent is further suitable for detecting bacterial species in a second microbial set, which is composed of the following bacterial species: Actinomyces sp. oral taxon 180, Akkermansia muciniphila CAG:154, Akkermansia sp. UNK.MGS-1, Alloscardovia omnicolens, Bacillus vallismortis, Cryptobacterium curtum, Kytococcus sedentarius, and Trueperella pyogenes.

2. A device for determining whether an individual suffers from schizophrenia, characterized in that include: an abundance determination unit, configured to determine the abundance of marker microorganisms in the stool sample of the individual, wherein the marker microorganisms include species in the first microorganism set and the second microorganism set; a comparing unit, configured to compare the obtained abundance with a predetermined threshold value, so as to determine whether the individual suffers from schizophrenia; Wherein, the first microorganism set consists of the following species: Eubacterium sp. CAG: 115, Eubacterium sp. CAG: 252, Firmicutes bacterium CAG: 227, Pseudomonas agarici; The second microorganism set consists of the following species: Actinomyces sp. oral taxon 180, Akkermansia muciniphila CAG:154, Akkermansia sp. UNK.MGS-1, Alloscardovia omnicolens, Bacillus vallismortis, Cryptobacterium curtum, Kytococcus sedentarius, and Trueperella pyogenes.

3. The device according to claim 2, characterized in that The abundance determination unit is adapted to determine the abundance by: obtaining nucleic acid sequencing data from a stool sample of the individual; Comparing the sequencing data with a reference genome; Based on the results of the comparison, the abundance of the marker microorganism is determined.

4. A device, characterized in that: include: A computer-readable storage medium having stored thereon a computer program for executing a method for determining whether an individual suffers from schizophrenia; and one or more processors for executing the program in the computer-readable storage medium; Wherein, the method for determining whether an individual suffers from schizophrenia comprises: (1) determining the abundance of marker microorganisms in a stool sample of the individual, wherein the marker microorganisms include species in a first microorganism set and a second microorganism set; (2) comparing the abundance obtained in step (1) with a predetermined threshold value to determine whether the individual suffers from schizophrenia; Wherein, the first microorganism set consists of the following species: Eubacterium sp. CAG: 115, Eubacterium sp. CAG: 252, Firmicutes bacterium CAG: 227, Pseudomonas agarici; Subdoligranulum_variabile, Veillonella_dispar and Sutterella_wadsworthensis; The second microorganism set is composed of the following species: Actinomyces sp. oral taxon 180, Akkermansia muciniphila CAG:154, Akkermansia sp. UNK.MGS-1, Alloscardovia omnicolens, Bacillus vallismortis, Cryptobacterium curtum, Kytococcus sedentarius, Trueperella pyogenes, Step (1) further comprises: obtaining nucleic acid sequencing data from a stool sample of the individual; Comparing the sequencing data with a reference genome; Based on the results of the comparison, the abundance of the marker microorganism is determined.

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