CN114369671A - Marker microorganism for rheumatic arthritis and application thereof - Google Patents
Marker microorganism for rheumatic arthritis and application thereof Download PDFInfo
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- CN114369671A CN114369671A CN202111413310.2A CN202111413310A CN114369671A CN 114369671 A CN114369671 A CN 114369671A CN 202111413310 A CN202111413310 A CN 202111413310A CN 114369671 A CN114369671 A CN 114369671A
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Abstract
The invention provides a rheumatic arthritis marker microorganism and application thereof, wherein the rheumatic arthritis marker microorganism comprises a first microorganism set, and further provides a kit which comprises a reagent suitable for detecting at least one strain in the first microorganism set, and the first microorganism set comprises the following strains: acinetobacter johnsonii, Bifidobacterium adolescentis, Bifidobacterium animalis, Brevundimonas diminuta, Citrobacter freundii, Citrobacter, Enterobacter arrhizus, Klebsiella oxytoca, Klebsiella, Pasteurella, Weissella fusca and Weissella fusca. The abundance of the microorganism provided by the invention has obvious difference in healthy people and patients with rheumatoid arthritis, and can be used as a marker for effectively detecting and/or treating the rheumatoid arthritis.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a marker microorganism for rheumatoid arthritis and application thereof, and more particularly to a kit, application of a reagent in preparation of the kit, a pharmaceutical composition or a food composition for preventing or treating rheumatoid arthritis, a method for determining whether an individual has rheumatoid arthritis, a device and a method for screening drugs.
Background
Rheumatoid arthritis (rheumatoid arthritis) is a common acute or chronic inflammation of connective tissue. Generally, the rheumatic arthritis is one of the main manifestations of rheumatic fever, and is clinically characterized by joint and muscle wandering soreness, red swelling and pain. It is associated with group A type B hemolytic streptococcal infection, and cold, dampness and other factors can induce the disease. The large joints of the lower limbs such as the knee joints and the ankle joints are most frequently affected. Although the incidence of rheumatic fever has declined significantly in recent decades, atypical rheumatic fever and chronic rheumatic arthritis are not uncommon. According to symptoms, epidemiological and immunological analysis, the rheumatoid arthritis is considered to be closely related to human hemolytic streptococcus infection, the infection route is important, and pharyngeal streptococcus infection is a necessary condition for the pathogenesis. However, the pathogenesis of group A streptococcal bacteria causing rheumatic fever is not completely understood. It is also noted that viral infection is also associated with this disease. The pathological changes of the rheumatoid arthritis in the active period are edema of joint synovium and surrounding tissues, mydriasis in connective tissues under the synovium, cellulose-like degeneration and inflammatory cell infiltration, and atypical rheumatic bodies sometimes exist. After the activity period, the exudates in the joints can be absorbed, generally no adhesion is caused, and therefore sequelae such as joint deformation and the like are not generated. Therefore, the research on the characteristics of the intestinal microbiota of patients with rheumatoid arthritis has important significance for the detection and treatment of the rheumatoid arthritis.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following facts and problems:
through a large amount of previous researches, the applicant of the application unexpectedly finds that some microorganisms can be used as marker microorganisms for detecting the rheumatoid arthritis, and provides a non-invasive method for early detection of the rheumatoid arthritis; the marker microorganisms are reasonably and effectively applied, the growth of beneficial bacteria in the intestinal tract is supported, potential pathogenic bacteria in the intestinal tract are inhibited, and the clinical symptoms of the rheumatoid arthritis can be treated or relieved.
To this end, in a first aspect of the invention, the invention proposes a kit. According to an embodiment of the invention, reagents suitable for detecting at least one species in a first set of microorganisms are included, said first set of microorganisms consisting of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter albugineus (Enterobacter albureae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Sutterbacterium wadswhicensis), Lactobacillus fusiformis (Weissella fusibaria) and Weissella fusiformis (Weissella fusiformis). According to the kit provided by the embodiment of the invention, the rheumatoid arthritis patient and the healthy individual can be accurately distinguished.
In a second aspect of the invention, the invention proposes the use of a reagent suitable for detecting at least one species of a first collection of microorganisms in the preparation of a kit. According to an embodiment of the invention, the kit is for diagnosing rheumatoid arthritis or for detecting the therapeutic effect of rheumatoid arthritis, the first collection of microorganisms consisting of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter albugineus (Enterobacter albureae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Sutterbacterium wadswhicensis), Lactobacillus fusiformis (Weissella fusibaria) and Weissella fusiformis (Weissella fusiformis). According to the kit prepared by the reagent provided by the embodiment of the invention, at least one strain in the first microorganism set can be accurately detected, and a rheumatoid arthritis patient and a healthy individual can be very accurately distinguished, so that the diagnosis of the rheumatoid arthritis can be effectively carried out at an early stage, or the kit can be used for detecting the change of the rheumatoid arthritis in the treatment process, such as cure, aggravation or alleviation.
In a third aspect of the present invention, the present invention provides a pharmaceutical composition or a food composition for preventing or treating rheumatoid arthritis. According to an embodiment of the invention, at least one species of a first microbial collection is contained, said first microbial collection consisting of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter albugineus (Enterobacter albureae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Sutterbacterium wadswhicensis), Lactobacillus fusiformis (Weissella fusibaria) and Weissella fusiformis (Weissella fusiformis). According to the embodiment of the invention, the strains of the first microorganism group in the rheumatoid arthritis marker microorganisms can be non-invasively discovered or assisted to detect the rheumatoid arthritis in an early stage, and the probability that an individual suffers from the rheumatoid arthritis is determined or the probability that the individual is in a healthy state is determined; meanwhile, the abundance of various strains in the first microorganism group in the intestinal tract of a high-risk group with rheumatoid arthritis or a patient with rheumatoid arthritis is improved, so that the probability of suffering from rheumatoid arthritis can be reduced, or the rheumatoid arthritis can be slowed down and cured, and therefore, the medicine or food composition containing at least one strain in the first microorganism group can be used for balancing the intestinal flora and effectively preventing or treating the rheumatoid arthritis.
In a fourth aspect of the invention, a method of determining whether an individual has rheumatoid arthritis is presented. According to an embodiment of the invention, comprising: (1) determining an abundance of a marker microorganism in a fecal sample of the individual, the marker microorganism comprising at least one species of the first set of microorganisms and the second set of microorganisms; (2) comparing the abundance obtained in step (1) to a predetermined threshold value to determine whether the individual has rheumatoid arthritis; wherein the first microbial collection consists of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter alberii (Enterobacter alberiae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Suttera wadswhensis), Lactobacillus fusiformis (Weissella fusiformis) and Weissella fusiformis (Weissella fusiformis) (Weissella fusiformis fus); the second microbial set consists of the following species: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA. The method according to the embodiment of the invention can determine whether the individual is a high-risk group with rheumatoid arthritis or has rheumatoid arthritis according to the abundance of the marker microorganism in the fecal sample of the individual, wherein the marker microorganism is determined by verifying a large number of fecal samples with known states and analyzing the abundance of various intestinal microorganisms in the fecal samples of a rheumatoid arthritis group and a healthy group through difference comparison.
In a fifth aspect of the invention, an apparatus for determining whether an individual has rheumatoid arthritis is presented. According to an embodiment of the invention, comprising: an abundance determination unit for determining an abundance of a marker microorganism in a fecal sample of the individual, the marker microorganism comprising at least one species of the first set of microorganisms and the second set of microorganisms; a comparison unit for comparing the obtained abundance with a predetermined threshold value so as to determine whether the individual has rheumatoid arthritis; wherein the first microbial collection consists of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), and Bifidobacterium animalis
(Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter Citrobacter sp A1, Enterobacter avermitis (Enterobacter asburiae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella oxytoca (Klebsiella sp.) RC7, Salmonella lavandustriolata (Suttera wadswordensis), Lactobacillus weissensis (Weissella cibaria) and Weissella fusiformis (Weissella convulsa); the second microbial set consists of the following species: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA. The marker microorganisms are determined by analyzing the abundance of various intestinal microorganisms in the stool samples of rheumatoid arthritis patients and healthy people through difference comparison and verifying a large number of stool samples with known states, and the device provided by the embodiment of the invention can accurately determine whether individuals are high-risk groups of rheumatoid arthritis or patients with rheumatoid arthritis.
In a sixth aspect of the invention, an apparatus is presented. According to an embodiment of the invention, comprising: a computer-readable storage medium having stored thereon a computer program for executing the method of the fourth aspect; and one or more processors for executing the program in the computer-readable storage medium. The device provided by the embodiment of the invention can accurately determine whether the individual is a high-risk group of rheumatoid arthritis or a rheumatoid arthritis patient.
In a seventh aspect of the invention, a method of screening for a drug is presented. According to an embodiment of the invention, the medicament is for the treatment or prevention of rheumatoid arthritis, the method comprising: administering a candidate drug to a subject, said subject's stool being tested for abundance of a marker microorganism comprising at least one species of a first set of microorganisms and a second set of microorganisms before and after administration, wherein a candidate drug satisfying at least one of the following conditions is suitable for use in the treatment or prevention of rheumatoid arthritis: (1) said abundance of at least one species in said first collection of microorganisms is increased following said administering; and (2) said abundance of at least one species in said second set of microorganisms is reduced after said administering; wherein the first microbial collection consists of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter alberii (Enterobacter alberiae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Suttera wadswhensis), Lactobacillus fusiformis (Weissella fusiformis) and Weissella fusiformis (Weissella fusiformis) (Weissella fusiformis fus); the second microbial set consists of the following species: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA. According to the method provided by the embodiment of the invention, the medicines which promote the growth of various strains in the first microorganism set in the marker microorganisms and/or inhibit the growth of various strains in the second microorganism set in the intestinal marker microorganisms can be produced or screened, and the method has important significance for assisting in relieving the clinical symptoms of rheumatoid arthritis.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a schematic diagram of an experimental analysis procedure for screening rheumatoid arthritis marker microorganisms according to an embodiment of the present invention; and
FIG. 2 is a schematic diagram showing the evaluation results of the marker microorganism combination indicator AUC according to the embodiment of the present invention, wherein Specificity represents Specificity, i.e., prediction is positive and actually positive, true positive, Sensitivity represents Sensitivity, i.e., true negative, and Confidence interval represents Confidence interval:
2-A is a result graph of AUC values and confidence intervals under ROC curves of 113 sample data in the first stage;
2-B is a graph of AUC values and confidence interval results under the ROC curve for the 64 sample data in the second stage.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
The term "optionally" is used for descriptive purposes only and is not to be construed as indicating or implying relative importance. Thus, a feature defined as "optionally" may or may not explicitly include the feature.
A biological marker is a cellular/biochemical or molecular change that can be detected from a biological medium. Biological media include various body fluids, tissues, cells, feces, hair, breath, and the like.
The abundance of a microorganism refers to the abundance of that microorganism in a population of microorganisms, for example, the population of gut microorganisms, and can be expressed as the amount of that microorganism in that population.
According to a particular embodiment of the invention, a kit is provided comprising reagents suitable for detecting at least one species of a first microbial collection consisting of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter albugineus (Enterobacter albureae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Sutterbacterium wadswhicensis), Lactobacillus fusiformis (Weissella fusibaria) and Weissella fusiformis (Weissella fusiformis).
According to a particular embodiment of the invention, the composition further comprises reagents suitable for detecting at least one species of a second microbial set consisting of: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA.
According to a particular embodiment of the invention, said kit comprises reagents suitable for detecting all of said species in said first microbial collection.
According to a particular embodiment of the invention, said kit comprises reagents suitable for detecting all of said species in said second microbial collection.
According to the specific embodiment of the invention, the marker microorganism is determined by comparing, analyzing and verifying the difference of the abundance of the microorganism in the excrement samples of a large number of individuals suffering from rheumatoid arthritis and a large number of healthy control individuals, and the marker microorganism related to rheumatoid arthritis in the intestinal microorganism is determined. The kit containing the reagent for detecting the marker microorganism can determine the probability that an individual is in a state with rheumatoid arthritis or in a healthy state, and can be used for non-invasive early detection or auxiliary detection of rheumatoid arthritis.
According to a specific embodiment of the present invention, the reagent suitable for detecting the first microorganism collection or the second microorganism collection is not particularly limited, and any reagent capable of detecting the microorganism species is included in the scope of the present invention, such as reagents for detecting the microorganism species through morphological characteristics, physiological biochemical reaction characteristics, ecological characteristics, serological reactions, sensitivity to bacteriophage, molecular biology, and the like, specifically, antibodies, enzymes, nucleic acid molecules, and the like.
Herein, the microbial morphological characteristics refer to: the shape, arrangement, etc. of the microorganism, the cell structure, gram staining reaction, the ability to move, the number and position of flagella, the presence or absence of spores, 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. were observed under a microscope.
Herein, the physiological and biochemical reaction characteristics of the microorganisms refer to: the ability of the microorganism to utilize substances, specificity of metabolites, e.g. production of H2S, indole, CO2Alcohol, organic acid, whether to reduce nitrate, whether to coagulate or freeze milk, growth environment (temperature, humidity, concentration of oxygen, carbon dioxide, and other gases, pH, whether to be hypertonic, whether to have halophilicity, etc.), relationship with other organisms (such as symbiosis, parasitism, host range, and pathogenic conditions), and the like.
Herein, the microbial serological response refers to: identification of similar species is achieved by highly sensitive specific reaction of antigen and antibody, or by typing of the same species of microorganism, such as antisera made with known species, type or strain, and serological reaction of specificity with the microorganism to be identified.
Herein, the detection of microorganisms by molecular biological methods mainly comprises: PCR technology, high-throughput sequencing and other methods are utilized.
Use of a reagent provided according to a particular embodiment of the invention for the preparation of a kit suitable for detecting at least one species of a first microbial population for the diagnosis of rheumatoid arthritis or for detecting the therapeutic effect of rheumatoid arthritis, said first microbial population consisting of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter albugineus (Enterobacter albureae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Sutterbacterium wadswhicensis), Lactobacillus fusiformis (Weissella fusibaria) and Weissella fusiformis (Weissella fusiformis).
According to the specific embodiment of the invention, the marker microorganism is determined by comparing, analyzing and verifying the difference of abundance of microorganisms in the fecal samples of a large number of individuals suffering from rheumatoid arthritis and a large number of healthy control individuals, and the microorganism marker related to rheumatoid arthritis in the intestinal microorganism is determined. The reagent for detecting the marker microorganism can determine the probability that an individual has rheumatoid arthritis or is in a healthy state, and can be used for non-invasive early detection or auxiliary detection of rheumatoid arthritis.
According to some specific embodiments of the invention, the reagent is further adapted to detect at least one species of a second set of microorganisms, said second set of microorganisms consisting of: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA.
According to a specific embodiment of the present invention, the reagent suitable for detecting the first microorganism collection or the second microorganism collection is not particularly limited, and reagents capable of detecting the microorganism species are all included in the scope of the present invention, such as reagents for detecting the microorganism species through morphological characteristics, physiological biochemical reaction characteristics, ecological characteristics, serological reactions, sensitivity to bacteriophage, molecular biology, and the like, specifically, antibodies, enzymes, nucleic acid molecules.
Herein, the microbial morphological characteristics refer to: the shape, arrangement, etc. of the microorganism, the cell structure, gram staining reaction, the ability to move, the number and position of flagella, the presence or absence of spores, 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. were observed under a microscope.
Herein, the physiological and biochemical reaction characteristics of the microorganisms refer to: the ability of the microorganism to utilize substances, specificity of metabolites, e.g. production of H2S, indole, CO2Alcohol, organic acid, whether nitrate can be reduced, whether milk can be coagulated or frozen, and the like, growth environment (temperature, humidity, concentration of gas such as oxygen and carbon dioxide, pH, whether hyperosmotic resistance exists, whether halophilic property exists, and the like suitable for growth)And relationships with other organisms (e.g., symbiosis, parasitism, host range, and pathogenic conditions).
Herein, the microbial serological response refers to: identification of similar species is achieved by highly sensitive specific reaction of antigen and antibody, or by typing of the same species of microorganism, such as antisera made with known species, type or strain, and serological reaction of specificity with the microorganism to be identified.
Herein, the detection of microorganisms by molecular biological methods mainly comprises: PCR technology, high-throughput sequencing and other methods are utilized.
According to the present invention, there is provided a pharmaceutical composition or a food composition for preventing or treating rheumatoid arthritis comprising at least one species of a first microorganism group, said first microorganism group consisting of: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter albugineus (Enterobacter albureae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Sutterbacterium wadswhicensis), Lactobacillus fusiformis (Weissella fusibaria) and Weissella fusiformis (Weissella fusiformis).
The marker microorganisms are determined by analyzing the abundance of various intestinal microorganisms in the fecal samples of the rheumatoid arthritis disease group and the healthy group through difference comparison and verifying a large number of fecal samples with known states. The strain in the marker microorganism is remarkably enriched in the healthy group compared with the rheumatoid arthritis patient group, wherein the remarkable enrichment refers to that the abundance of the strain in the healthy group is statistically significantly higher or obviously and substantially higher than that in the rheumatoid arthritis patient group compared with that in the rheumatoid arthritis patient group; the substance capable of improving the abundance of the partial strains can be used for treating rheumatoid arthritis or is beneficial to patients with rheumatoid arthritis to take, and the substance capable of improving the abundance is not limited to a medicament for treating the rheumatoid arthritis and a functional food beneficial to the balance of intestinal flora. Therefore, the marker microorganism provided by the embodiment can be used for preparing a medicament for treating rheumatoid arthritis and/or preparing a functional food, a health-care medicament and the like which are beneficial to balancing intestinal flora, and the medicament or the food can effectively treat or relieve the rheumatoid arthritis.
According to a specific embodiment of the present invention, there is provided a method for determining whether an individual has rheumatoid arthritis, comprising the steps (1) and (2).
(1) Determining the abundance of marker microorganisms in a fecal sample of the individual.
The marker microorganism includes at least one species of the first set of microorganisms and the second set of microorganisms. Wherein the first microbial collection consists of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter alberii (Enterobacter alberiae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Suttera wadswhensis), Lactobacillus fusiformis (Weissella fusiformis) and Weissella fusiformis (Weissella fusiformis) (Weissella fusiformis fus); the second microbial set consists of the following species: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA.
According to some specific embodiments of the present invention, the step (1) further comprises: obtaining nucleic acid sequencing data in a stool sample of the individual; aligning the sequencing data to a reference genome; determining the abundance of the marker microorganism based on the results of the alignment.
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:
ab (S) ═ Ab (us) + Ab (ms), where S denotes the number of the marker microorganism, Ab (S) denotes the abundance of the marker microorganism S, and Ab (U)S)＝US/lS，USNumber of reads, l, uniquely aligned to the reference genome of the marker microorganism S in the sequencing dataSIs the total length of the reference genome of the marker microorganism S,
MSfor the number of reads in the sequencing data that are non-uniquely aligned to the reference genome of the marker microorganism S, i represents the number of the non-uniquely aligned reads, CoiThe abundance coefficient corresponding to the ith read,
Coi,srepresenting the abundance coefficient of the non-uniquely aligned reads i for the marker microorganism S, N being the total number of microorganisms that the non-uniquely aligned reads i can align to, j representing the number of microorganisms that the non-uniquely aligned reads i can align to.
The alignment can be performed by using known alignment software, such as SOAP, BWA, TeraMap, etc., in the alignment process, the alignment parameters are generally set, one or a pair of reads (reads) is set to allow at most s base mismatches (mismatches), for example, s is set to be less than or equal to 2, and if more than s bases in the reads are mismatched, it is considered that the reads cannot be aligned (aligned) to the assembled fragment. The obtained comparison result comprises comparison conditions of each read and the reference genome of each species, and comprises information of whether the read can be compared with the reference genome of a certain or some species, only one species or multiple species, the position of the reference genome of the species, the unique position or multiple positions of the reference genome of the species, and the like.
The reference genome of the strain/microorganism refers to a predetermined sequence of the microorganism species, and may be any reference template of a biological category to which a sample to be tested belongs or which is obtained in advance, for example, the target is a microorganism in the sample to be tested, the reference sequence may be a reference genome of various microorganisms in an NCBI database and/or a DACC intestinal reference genome disclosed in HMP and MetaHIT projects, and further, a resource library including more reference sequences may be configured in advance, for example, a more similar sequence is selected or determined and assembled as the reference sequence according to factors such as a state of an individual from which the sample to be tested is derived, a region, and the like. According to one embodiment of the invention, the reference genomes of the various microorganisms are obtained from public databases, typically, the reference genome of a microorganism has multiple versions, i.e., a microorganism has multiple public reference genomes.
reads are aligned to the reference genome of the species, which can be divided into two parts: a) unique reads (U): uniquely aligning the reference genome of the previous species; these reads are referred to as unique reads. That is, if the reference genomes on the reads alignment are from the same species, the reads are defined as unique reads; b) multiple reads (M): aligning reference genomes of more than one species, defined as multiple reads. That is, if the reference genome on which reads align is from at least two species, these reads are defined as multiple reads.
(2) Abundance comparisons to determine whether an individual has rheumatoid arthritis.
According to one embodiment of the invention, the abundance obtained in step (1) is compared to a predetermined threshold value in order to determine whether the individual has rheumatoid arthritis.
According to some embodiments of the invention, the threshold is predetermined. And comparing the abundance of the marker microorganisms in the sample of the individual to be tested with the threshold value to determine the state of the individual to be tested. The threshold may be a value or a range of values, the setting of the threshold is not particularly limited, and any threshold capable of determining whether the individual has inflammatory bowel disease may be used, for example, the threshold corresponding to the microorganism may be set to a 95% Confidence interval (Confidence interval) of the mean abundance based on the mean abundance of the marker microorganism in the individual with known disease or health state.
The confidence interval refers to an estimation interval of the overall parameter constructed by the sample statistic. In statistics, the confidence interval for a probability sample is an interval estimate for some overall parameter of the sample. The confidence interval exhibits the extent to which the true value of this parameter has a certain probability of falling around the measurement. The confidence interval indicates the degree of plausibility of the measured value of the measured parameter, i.e. the "certain probability" required above, which is referred to as the confidence level.
According to some embodiments of the invention, the individual is determined to have rheumatoid arthritis when the abundance of the marker microorganism determined in step (1) reaches the abundance threshold for rheumatoid arthritis and does not reach the abundance threshold for non-rheumatoid arthritis, and the individual is determined not to have rheumatoid arthritis when the abundance of the marker microorganism determined in step (1) reaches the abundance threshold for non-rheumatoid arthritis and does not reach the abundance threshold for rheumatoid arthritis.
It is to be noted that, depending on the purpose or requirement, there may be different requirements on the confidence level of the result of determining the state of an individual, and that a person skilled in the art may select different significance levels or thresholds.
The method is based on detecting the abundance of various strains in the marked microorganisms in the fecal sample of the individual, respectively comparing the detected abundance of various strains with the threshold value thereof, and determining the probability that the individual is a rheumatoid arthritis individual or a healthy individual according to the obtained comparison result. Provides a non-invasive auxiliary detection or auxiliary intervention treatment method for early detection of the rheumatoid arthritis.
All or a part of the steps of the method for determining whether an individual has rheumatoid arthritis using marker microorganisms in any of the above embodiments may be performed using an apparatus/system including detachable functional modules of the respective units, or may be implemented by programming the method, storing the method in a machine-readable medium, and operating the readable medium by a machine.
According to the present invention there is provided an apparatus for determining whether an individual has rheumatoid arthritis, the apparatus comprising: an abundance determination unit for determining the abundance of marker microorganisms in a stool sample of the individual; a comparison unit for comparing the obtained abundance with a predetermined threshold value so as to determine whether the individual has rheumatoid arthritis; wherein the first microbial collection consists of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter alberii (Enterobacter alberiae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Suttera wadswhensis), Lactobacillus fusiformis (Weissella fusiformis) and Weissella fusiformis (Weissella fusiformis) (Weissella fusiformis fus); the second microbial set consists of the following species: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA. The above description of the technical features and advantages of the method for determining whether an individual has rheumatoid arthritis using marker microorganisms according to any of the embodiments of the present invention is equally applicable to the apparatus according to this aspect of the present invention, and will not be described herein again.
According to an embodiment of the invention, the abundance determination unit is adapted to determine the abundance by: obtaining nucleic acid sequencing data in a stool sample of the individual; aligning the sequencing data to a reference genome; determining the abundance of the marker microorganism based on the results of the alignment.
The alignment can be performed by using known alignment software, such as SOAP, BWA, TeraMap, etc., in the alignment process, the alignment parameters are generally set, one or a pair of reads (reads) is set to allow at most s base mismatches (mismatches), for example, s is set to be less than or equal to 2, and if more than s bases in the reads are mismatched, it is considered that the reads cannot be aligned (aligned) to the assembled fragment. The obtained comparison result comprises comparison conditions of each read and the reference genome of each species, and comprises information of whether the read can be compared with the reference genome of a certain or some species, only one species or multiple species, the position of the reference genome of the species, the unique position or multiple positions of the reference genome of the species, and the like. According to one embodiment of the invention, alignment is performed using SOAPalign 2.21 with the setting parameter-r 2-m 100-x 1000.
The reference genome of the microorganism refers to a predetermined sequence of the species of the microorganism, and may be any reference template of a biological category to which a sample to be tested belongs or contains, which is obtained in advance, for example, the target is the microorganism in the sample to be tested, the reference sequence may be a reference genome of various microorganisms in an NCBI database and/or a DACC intestinal genome disclosed in HMP and MetaHIT projects, and further, a resource library containing more reference sequences may be configured in advance, for example, a more similar sequence is selected or determined to be assembled as the reference sequence according to factors such as the state of an individual from which the sample to be tested is derived, a region, and the like. According to one embodiment of the invention, the reference genomes of the various microorganisms are obtained from public databases, typically, the reference genome of a microorganism has multiple versions, i.e., a microorganism has multiple public reference genomes.
reads are aligned to the reference genome of the species, which can be divided into two parts: a) unique reads (U): uniquely aligning the genome of the previous species; these reads are referred to as unique reads. That is, if the reference genomes on the reads alignments are from the same species, the reads are defined as unique reads. b) Multiple reads (M): aligning reference genomes of more than one species, defined as multiple reads. That is, if the reference genome on which reads align is from at least two species, these reads are defined as multiple reads.
According to one embodiment of the invention, the abundance of the marker microorganism is determined according to the following formula:
Ab(S)＝Ab(US)+Ab(MS) Wherein S represents the number of the marker microorganism, Ab (S) represents the abundance of S of the marker microorganism, Ab (U)S)＝US/lS，USNumber of reads, l, uniquely aligned to the reference genome of the marker microorganism S in the sequencing dataSIs the total length of the reference genome of the marker microorganism S,
MSfor the number of reads in the sequencing data that are non-uniquely aligned to the reference genome of the marker microorganism S, i represents the number of the non-uniquely aligned reads, CoiThe abundance coefficient corresponding to the ith read,
Coi,srepresenting the abundance coefficient of the non-uniquely aligned reads i for the marker microorganism S, N being the total number of microorganisms that the non-uniquely aligned reads i can align to, j representing the number of microorganisms that the non-uniquely aligned reads i can align to.
According to yet another embodiment of the present invention, there is provided an apparatus including: a computer readable storage medium having stored thereon a computer program for executing a method of determining whether an individual has rheumatoid arthritis as described above; and one or more processors for executing the program in the computer-readable storage medium.
According to still another embodiment of the present invention, there is provided a method of screening a drug for treating or preventing rheumatoid arthritis, the method including: administering a candidate drug to a subject, said subject's stool being tested for abundance of a marker microorganism comprising at least one species of a first set of microorganisms and a second set of microorganisms before and after administration, wherein a candidate drug satisfying at least one of the following conditions is suitable for use in the treatment or prevention of rheumatoid arthritis: (1) said abundance of at least one species in said first collection of microorganisms is increased following said administering; and (2) said abundance of at least one species in said second set of microorganisms is reduced after said administering; wherein the first microbial collection consists of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter alberii (Enterobacter alberiae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Suttera wadswhensis), Lactobacillus fusiformis (Weissella fusiformis) and Weissella fusiformis (Weissella fusiformis) (Weissella fusiformis fus); the second microbial set consists of the following species: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA.
By utilizing the method for producing or screening the medicament for treating the rheumatoid arthritis, the determined rheumatoid arthritis biomarker is reasonably and effectively applied for screening, the medicament capable of supporting the growth of beneficial bacteria in the intestinal tract and/or inhibiting potential pathogenic bacteria in the intestinal tract can be obtained, and the method has important significance for assisting in relieving clinical symptoms of the rheumatoid arthritis.
The embodiments will be described in detail below. The reagents, sequences, software and equipment not specifically submitted to the following examples are all conventional commercial products.
Example 1 identification of biomarkers
In this example, the inventors studied stool samples of 63 rheumatoid arthritis patients and 50 healthy controls to obtain characteristics of the microbial community and functional components of the intestinal flora. In summary, the inventors obtained 589.521Gb high quality sequencing data by experimental sequencing. Metagenomic analysis shows that 15 microbial species are closely related to rheumatoid arthritis diseases, 13 bacteria are enriched in intestinal microorganisms of healthy people, and 2 bacteria are enriched in intestinal microorganisms of rheumatoid arthritis patients.
1. Obtaining sequencing data:
the rheumatoid arthritis patients and the healthy controls are from Zhejiang Chinese medicine university, 63 feces samples of Chinese rheumatoid arthritis patients and 50 feces samples of healthy controls are collected in the experiment, wherein fresh feces samples of each individual are divided into 200 mg/part and 5 parts in total, and are immediately stored in a refrigerator at the temperature of-80 ℃.
Total DNA was extracted from the fecal samples of 63 Chinese patients with rheumatoid arthritis and 50 healthy controls. DNA is extracted by a method of extracting DNA by phenol trichloromethane treatment.
2. Construction of DNA libraries and sequencing
DNA banking was performed according to the instructions of the Instrument manufacturer (Illumina). The library was sequenced at PE2 × 150bp, and the Illumina HiSeq2500(Illumina, San Diego, CA) platform sequenced the library of 113 samples. Each sample yielded on average 5.217Gb high quality sequencing results, totaling 589.521 sequencing data volume.
The identification of biomarkers of relevance to rheumatoid arthritis is carried out by reference to the experimental procedure shown in FIG. 1, where the steps omitted or the details are well known to those skilled in the art, and several important steps are described below.
3. Microbial species abundance analysis
3.1 sequence optimization statistics
1) Firstly, performing first-stage sequencing, collecting data of 113 samples in the current-stage sequencing, filtering the data after obtaining the sequencing data of the 113 samples in the first stage, and performing quality control according to the following standards: a) removing reads greater than 5N bases; b) (ii) removing reads greater than 50% of the low quality base (Q20); c) the tail low mass (Q20) and N bases were removed. Missing pairs of reads sequences are considered a single read for assembly.
2) Data from healthy persons were also processed using the method described in 1).
3) The IGC gene set was downloaded from ftp. cngb. org/pub/SciRAID/Microbiome/humanGut-9.9M/GeneCatalog/IGC. fa. gz link.
3.2 species abundance analysis
SOAPalign 2.21 was used to match paired-end clean reads against redundant genomes, here called redundant genomes from reference genomes of bacteria disclosed in each database, with alignment parameters-r 2-m 200-x 1000. The comparison between Reads and redundant genomes can be divided into two parts: a) unique reads (U): reads align the genomes of only the previous species; these reads are defined as unique reads. That is, if the genomes are from the same species, the inventors define these reads as unique reads. b) Multiple reads (M): multiplex reads are defined if they align the genomes of two or more species. That is, if the aligned genomes are from different species, the inventors define these reads as multiple reads.
For species S, the abundance is ab (S), related to the characteristic U reads and shared M reads, and is calculated as follows:
Ab(S)＝Ab(US)+Ab(MS)，
wherein S represents the number of the marker microorganism,
ab (S) represents the abundance of the marker microorganism S,
Ab(US)＝US/lS，
USthe number of reads in the sequencing data that are uniquely aligned with the reference genome of the marker microorganism S,
lSis the total length of the reference genome of the marker microorganism S,
MSis the number of reads in the sequencing data that are non-uniquely aligned to the reference genome of the marker microorganism S,
Coithe abundance coefficient corresponding to the ith read,
i represents the number of the non-unique alignment reads,
Coi,srepresenting the abundance coefficient of the non-uniquely aligned reads i for the marker microorganism S,
n is the total number of microorganisms that the non-uniquely aligned reads i are capable of aligning,
j represents the number of microorganisms that the non-uniquely aligned reads i are capable of aligning.
And (4) obtaining a normalized species abundance table after all dividing the species abundance value obtained by calculation in each sample by the total abundance of each sample.
3.3 screening microbial species markers
In order to obtain intestinal microbial species markers closely related to rheumatoid arthritis diseases, the inventor utilizes the abundance data of intestinal microbial species of two groups, namely a rheumatoid arthritis patient (RA) group (63 cases) and a normal person (Health) group (50 cases) to carry out a research on the correlation between species and diseases. Based on the abundance of species table obtained in step 3.2, the inventors set criteria as follows: (1) the median abundance of species in the rheumatoid arthritis patient group or healthy human group must be greater than 0.00001; (2) the p-value of the correlation between rheumatoid arthritis and healthy individual samples for each species was obtained by Wilcoxon rank-sum test combined with multiple tests of Benjamini Hochberg; (3) screening was performed using the parameters described above using a strict threshold q _ values < 0.001. The inventor obtains 15 intestinal microorganism species closely related to rheumatoid arthritis diseases, wherein 2 species of microorganisms are enriched in intestinal tracts of patients with rheumatoid arthritis, 13 species are enriched in healthy people, and the 15 microorganism species markers are shown in table 1.
Table 1:
example 2 validation of microbial species markers
To verify the findings in example 1, the inventors further analyzed and verified the abundance of the 15 genera in stool samples of 32 healthy persons and 32 rheumatoid arthritis patients in the population, and selected the 15 microbial species markers based on the enrichment of each species in the healthy and disease groups, and verified the DNA extraction, sequencing and analysis of the abundance of the species with reference to example 1.
The verification results are as follows: the 13 species enriched in healthy people all received high quality verification in the verification set (P _ values <0.05), and the mean and P value results of the microorganism species marker verification enriched in healthy people are shown in table 2.
Table 2:
the high quality of the validation set (p-value <0.05) for all 2 species enriched in rheumatoid arthritis patients as described above verifies that the p-value and q-value profiles of the validation of 2 microbial markers enriched in rheumatoid arthritis patients are shown in table 3 in the validation set data.
Table 3:
the inventor believes that 13 microbial species markers enriched from healthy people can be used as a reverse index of the disease of the rheumatoid arthritis, or used as a microbial preparation medicinal flora component for developing and treating the rheumatoid arthritis, or used as a recovery index for detecting the rheumatoid arthritis and monitoring the treatment progress of the rheumatoid arthritis; 2 microorganism species markers enriched in patients with rheumatoid arthritis are used as positive indexes for the diseases of the rheumatoid arthritis, and are particularly used for non-invasive detection and diagnosis of the diseases of the rheumatoid arthritis.
The inventor utilizes the 15 microbial species markers to construct a comprehensive index, estimates the area AUC under the ROC (Receiver-operating characteristic) curve, and evaluates the diagnosis capability of the comprehensive index corresponding to the diagnosis capability of the rheumatoid arthritis, wherein the higher the AUC is, the higher the diagnosis capability is. By evaluating 113 samples in the first stage (first stage) and 64 samples in the second stage (second stage), as shown in fig. 2, the diagnosis ability is good, and the AUC obtained in the first stage is 85% (the AUC obtained in the first stage is good at more than 85%), as shown in fig. 2a, the confidence interval is 78.07% -92.22%; the second phase resulted in an AUC of 79.59%, with a confidence interval of 68.72% to 90.46%, as shown in fig. 2 b.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (13)
1. A kit comprising reagents suitable for detecting at least one species in a first collection of microorganisms consisting of: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter albugineus (Enterobacter albureae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Sutterbacterium wadswhicensis), Lactobacillus fusiformis (Weissella fusibaria) and Weissella fusiformis (Weissella fusiformis).
2. The kit of claim 1, further comprising reagents suitable for detecting at least one species in a second collection of microorganisms consisting of: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA.
3. The kit of claim 1, comprising reagents suitable for detecting all of said species in said first collection of microorganisms.
4. The kit of claim 2, comprising reagents suitable for detecting all of said species in said second collection of microorganisms.
5. Use of a reagent suitable for detecting at least one species in a first collection of microorganisms for diagnosing rheumatoid arthritis or for detecting the therapeutic effect of rheumatoid arthritis in the manufacture of a kit, said first collection of microorganisms consisting of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter albugineus (Enterobacter albureae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Sutterbacterium wadswhicensis), Lactobacillus fusiformis (Weissella fusibaria) and Weissella fusiformis (Weissella fusiformis).
6. Use according to claim 5, characterized in that said reagent is further suitable for detecting at least one species of a second group of microorganisms consisting of: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA.
7. A pharmaceutical or food composition for the prevention or treatment of rheumatoid arthritis, comprising at least one species of a first microbial population consisting of: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter albugineus (Enterobacter albureae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Sutterbacterium wadswhicensis), Lactobacillus fusiformis (Weissella fusibaria) and Weissella fusiformis (Weissella fusiformis).
8. A method of determining whether an individual has rheumatoid arthritis, comprising:
(1) determining an abundance of a marker microorganism in a fecal sample of the individual, the marker microorganism comprising at least one species of the first set of microorganisms and the second set of microorganisms;
(2) comparing the abundance obtained in step (1) to a predetermined threshold value to determine whether the individual has rheumatoid arthritis;
wherein the first microbial collection consists of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter alberii (Enterobacter alberiae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Suttera wadswhensis), Lactobacillus fusiformis (Weissella fusiformis) and Weissella fusiformis (Weissella fusiformis) (Weissella fusiformis fus);
the second microbial set consists of the following species: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA.
9. The method of claim 8, wherein step (1) further comprises:
obtaining nucleic acid sequencing data in a stool sample of the individual;
aligning the sequencing data to a reference genome;
determining the abundance of the marker microorganism based on the results of the alignment.
10. An apparatus for determining whether an individual has rheumatoid arthritis, comprising:
an abundance determination unit for determining an abundance of a marker microorganism in a fecal sample of the individual, the marker microorganism comprising at least one species of the first set of microorganisms and the second set of microorganisms;
a comparison unit for comparing the obtained abundance with a predetermined threshold value so as to determine whether the individual has rheumatoid arthritis;
wherein the first microbial collection consists of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter alberii (Enterobacter alberiae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Suttera wadswhensis), Lactobacillus fusiformis (Weissella fusiformis) and Weissella fusiformis (Weissella fusiformis) (Weissella fusiformis fus);
the second microbial set consists of the following species: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA.
11. The apparatus of claim 10, wherein the abundance determination unit is adapted to determine the abundance by:
obtaining nucleic acid sequencing data in a stool sample of the individual;
aligning the sequencing data to a reference genome;
determining the abundance of the marker microorganism based on the results of the alignment.
12. An apparatus, comprising:
a computer-readable storage medium having stored thereon a computer program for executing the method of claim 8 or 9;
and one or more processors for executing the program in the computer-readable storage medium.
13. A method of screening for a drug for use in treating or preventing rheumatoid arthritis, the method comprising:
administering a candidate drug to the subject,
detecting an abundance of a marker microorganism in the subject's stool, the marker microorganism comprising at least one species of the first set of microorganisms and the second set of microorganisms, before and after administration,
wherein a drug candidate satisfying at least one of the following conditions is suitable for use in the treatment or prevention of rheumatoid arthritis:
(1) said abundance of at least one species in said first collection of microorganisms is increased following said administering; and
(2) said abundance of at least one species in said second collection of microorganisms is reduced after said administering;
wherein the first microbial collection consists of the following species: acinetobacter johnsonii (Acinetobacter johnsonii), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Brevundimonas diminuta (Brevundimonas diminuta), Citrobacter freundii (Citrobacter freundii), Citrobacter Citrobacter sp 302, Citrobacter sp A1, Enterobacter alberii (Enterobacter alberiae), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella sp.) OBRC7, Klebsiella warfara (Suttera wadswhensis), Lactobacillus fusiformis (Weissella fusiformis) and Weissella fusiformis (Weissella fusiformis) (Weissella fusiformis fus);
the second microbial set consists of the following species: bacillus faecalis (Copro bacillus sp.)3356FAA and Bacillus faecalis (Copro bacillus sp.)8254 BFAA.
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