CN114381507B - Graves disease marker microorganism and application thereof - Google Patents

Graves disease marker microorganism and application thereof Download PDF

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CN114381507B
CN114381507B CN202111414584.3A CN202111414584A CN114381507B CN 114381507 B CN114381507 B CN 114381507B CN 202111414584 A CN202111414584 A CN 202111414584A CN 114381507 B CN114381507 B CN 114381507B
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宋瑞雪
郑智俊
张笑笑
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Hangzhou Tuohong Biological Technology Co ltd
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Abstract

The present invention proposes a graves disease marker microorganism comprising a first set of microorganisms, and therefore further proposes a kit comprising reagents suitable for detecting at least one species of the first set of microorganisms, said first set of microorganisms consisting of: the genus chaetomium, the genus faecium, the genus praecox, the genus chaetomium, the genus praecox, eubacterium rectum, eubacterium johnsonii, enterococcus faecalis, and enterococcus faecalis clostridium tenella, clostridium, vibrio spike, burkholderia, laque bacillus, bifidobacterium adolescentis and vibrio butyrate. The microorganism provided by the invention has obvious difference in abundance in healthy people and Graves disease patients, and can be used as a marker for detecting and/or treating Graves disease.

Description

Graves disease marker microorganism and application thereof
Technical Field
The present invention relates to the field of biotechnology, in particular, to a graves disease marker microorganism and use thereof, and more particularly, to a kit, use of a reagent in preparation of a kit, a pharmaceutical composition or a food composition for preventing or treating graves disease, a method of determining whether an individual has graves disease, a device for determining whether an individual has graves disease, a device, a method of screening a drug.
Background
Graves' Disease refers to toxic diffuse goiter, an autoimmune Disease that causes hyperthyroidism, meaning that the human thyroid gland secretes too much thyroid hormone, resulting in a series of symptoms. In areas where iodine intake is sufficient, the prevalence of GD is about 0.5%, with 20-50 cases occurring per 10 thousands of people. GD prevalence is sex-related, 3% in women, 0.5% in men, and 20% in women over age as the main population.
Currently, the potential pathogenic factors of GD are not yet determined, and most researches consider that hyperthyroidism is related to genetic genes, the occurrence of hyperthyroidism is related to human leukocyte antigens (HLA class ii antigens), the detection rate of hyperthyroidism is different according to the species of people, and the hyperthyroidism is related to mental trauma and abnormal immune system, such as hyperexcitability or over depression, and can cause over secretion of thyroid hormone. The clinical manifestations of the affected population are not the same, most patients show thyromegaly, and nervous system symptoms such as mania and mental allergy can appear; or hypermetabolic syndrome, or herniation of the eyeball, increased heartbeat, dysphoria, dermatitis, skin thickening, oedema, weight loss, sensitivity to light, fragile hair, short menstrual cycle, and other various symptoms.
Diagnosis of hyperthyroidism is based on a characteristic clinical manifestation, serum thyrotropin, free thyroxine levels and thyrotropin receptor antibodies. While current diagnostic methods are adequate for the most severe patients, diagnosis based on clinical features and blood metrics is complex and time consuming, often delaying early diagnosis and treatment for mild GD patients. GD patients and parkinson patients share common clinical and biochemical diagnostic features that make differential diagnosis more difficult and may be confused with parkinson's disease, possibly masking the occurrence of one of the diseases during the other course. Therefore, a more convenient and accurate GD diagnostic method is urgently needed.
With the completion of human genome sequencing and the rapid development of high-throughput sequencing technology, gene screening is another direction of Graves disease diagnosis, and is found to be related to classification markers in intestinal microbiomes in small-scale test point researches. By studying the microorganisms of the fecal sample, the early diagnosis of Graves disease is facilitated, and the disclosure of the microorganism markers of Graves disease is facilitated.
Disclosure of Invention
The present application is made based on the discovery and recognition by the inventors of the following facts and problems:
Through extensive research in the early stage, the applicant of the application unexpectedly finds that some microorganisms can be used as marker microorganisms for detecting graves 'disease, reasonably and effectively apply the marker microorganisms, support the growth of beneficial intestinal bacteria, inhibit the potential pathogenic bacteria of the intestinal tract, and treat or relieve the clinical symptoms of graves' disease.
For this purpose, in a first aspect of the invention, the invention proposes a kit. According to an embodiment of the invention, a reagent is included which is adapted to detect at least one species of a first set of microorganisms, the first set of microorganisms consisting of: the genus helicobacter (Paraseterella exterminatum), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalibacterium_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Copribacterium_com), the genus Bacillus (Copribacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_F7, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_computer), the genus Bacillus (Bubali_1_E11), the genus Bacillus (Bubali_bacillus), the genus Bacillus (BlueTokida_bacteria), the genus Bacillus (Bluebacillus_7), the genus Bacillus (Bluefaciens_bacteria) and the genus Bacillus (Bluefacilis) may be the genus Bdinus (Bluebacteria) may be the genus Bdinus_bacteria). According to the kit provided by the embodiment of the invention, the reagent for detecting at least one strain in the first microorganism set can be accurately detected, so that Graves patients and healthy individuals can be accurately distinguished or diagnosed.
In a second aspect of the invention, the invention proposes the use of a reagent in the preparation of a kit, said reagent being suitable for detecting at least one species of a first set of microorganisms. According to an embodiment of the invention, the kit is for diagnosing graves 'disease or detecting the therapeutic effect of graves' disease, the first set of microorganisms consisting of the following species: the genus helicobacter (Paraseterella exterminatum), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalibacterium_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Copribacterium_com), the genus Bacillus (Copribacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_F7, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_computer), the genus Bacillus (Bubali_1_E11), the genus Bacillus (Bubali_bacillus), the genus Bacillus (BlueTokida_bacteria), the genus Bacillus (Bluebacillus_7), the genus Bacillus (Bluefaciens_bacteria) and the genus Bacillus (Bluefacilis) may be the genus Bdinus (Bluebacteria) may be the genus Bdinus_bacteria). According to the kit prepared by the reagent of the specific embodiment of the invention, at least one strain in the first microorganism set can be accurately detected, and Graves disease patients and healthy individuals can be extremely accurately distinguished, so that Graves disease diagnosis can be effectively carried out in early stage, or the kit can be used for detecting change of Graves disease in the treatment process.
In a third aspect of the invention, the invention provides a pharmaceutical or food composition for preventing or treating graves' disease. According to an embodiment of the invention, at least one species of a first set of microorganisms is contained, said first set of microorganisms consisting of: the genus helicobacter (Paraseterella exterminatum), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalibacterium_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Copribacterium_com), the genus Bacillus (Copribacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_F7, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_computer), the genus Bacillus (Bubali_1_E11), the genus Bacillus (Bubali_bacillus), the genus Bacillus (BlueTokida_bacteria), the genus Bacillus (Bluebacillus_7), the genus Bacillus (Bluefaciens_bacteria) and the genus Bacillus (Bluefacilis) may be the genus Bdinus (Bluebacteria) may be the genus Bdinus_bacteria). According to the Graves disease marker microorganism, the strain of the first microorganism set in the Graves disease marker microorganism can be noninvasively found in the early stage or assisted in detection of Graves disease, and the probability of the individuals suffering from Graves disease or the probability of the individuals in a healthy state can be determined; meanwhile, the probability of suffering from Graves disease can be reduced or Graves disease can be slowed down and cured by improving various strains in the first microorganism concentration in the intestinal tract of a high-risk group of Graves disease or a patient with Graves disease, so that the medicine or food composition containing at least one strain in the first microorganism concentration can be used for balancing intestinal flora and effectively preventing or treating Graves disease.
In a fourth aspect of the invention, the invention features a method of determining whether an individual has graves' disease. According to an embodiment of the invention, it comprises: (1) Determining an abundance of a marker microorganism in a fecal sample of the individual, the marker microorganism comprising at least one species of a first set of microorganisms and a second set of microorganisms; (2) Comparing the abundance obtained in step (1) to a predetermined threshold to determine if the individual has graves' disease; wherein the first set of microorganisms consists of the following species: the genus helicobacter (Parasepsis_excrementis), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalablock_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Propionibacterium_com), the genus Bacillus (Propionibacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_E11, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_cross), the genus Bacillus (Bluebacillus_1), the genus Bacillus (Bluebacillus) and the genus Bacillus (Bluefaciens_bacteria), the genus Bacillus (Bluefaciens_bacillus) and the genus Bacillus (Bluefaciens) are different; the second set of microorganisms consists of the following species: streptococcus parahaemolyticus (Streptococcus parasanguinis), megamonas (Megamonas rupellensis), eglinium lentum (Eggerthella lenta), bacillus faecalis (coprobacter sp.) 3_3_56faa. The method according to the embodiment of the invention can determine whether an individual has graves disease according to the abundance of the marker microorganism in the stool sample of the individual, wherein the marker microorganism is determined by verifying a large number of stool samples in a known state and analyzing the abundance of various intestinal microorganisms in the stool samples of the graves disease group and the healthy group through difference comparison.
In a fifth aspect of the invention, the invention features an apparatus for determining whether an individual has graves' disease. According to an embodiment of the invention, it comprises: 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 in order to determine whether the individual has graves' disease; wherein the first set of microorganisms consists of the following species: the genus helicobacter (Parasepsis_excrementis), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalablock_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Propionibacterium_com), the genus Bacillus (Propionibacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_E11, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_cross), the genus Bacillus (Bluebacillus_1), the genus Bacillus (Bluebacillus) and the genus Bacillus (Bluefaciens_bacteria), the genus Bacillus (Bluefaciens_bacillus) and the genus Bacillus (Bluefaciens) are different; the second set of microorganisms consists of the following species: streptococcus parahaemolyticus (Streptococcus parasanguinis), megamonas (Megamonas rupellensis), eglinium lentum (Eggerthella lenta), bacillus faecalis (coprobacter sp.) 3_3_56faa. The marker microorganism is determined by analyzing the abundance of various intestinal microorganisms in stool samples of Graves patients and healthy people through difference comparison and through analysis and verification of a large number of stool samples in known states, and the device according to the embodiment of the invention can accurately determine whether an individual is a high-risk group of Graves disease or a Graves patient.
In a sixth aspect of the invention, the invention provides an apparatus. According to an embodiment of the invention, it comprises: a computer-readable storage medium having stored thereon a computer program for executing the method of the fourth aspect; and one or more processors configured to execute the program in the computer-readable storage medium. The device according to the embodiment of the invention can accurately determine whether an individual is a high-risk group of graves 'disease or graves' disease patient.
In a seventh aspect of the invention, the invention provides a method of screening for a drug. According to an embodiment of the invention, the medicament is for the treatment or prevention of graves' disease, the method comprising: administering a candidate drug to a subject, detecting the abundance of a marker microorganism in the subject's stool before and after administration, the marker microorganism comprising at least one species of the first set of microorganisms and the second set of microorganisms, wherein the candidate drug that satisfies at least one of the following conditions is suitable for treating or preventing graves' disease: (1) After said administering, said abundance of at least one species of said first set of microorganisms increases; and (2) said abundance of at least one species of said second microorganism set decreases following said administering; wherein the first set of microorganisms consists of the following species: the genus helicobacter (Parasepsis_excrementis), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalablock_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Propionibacterium_com), the genus Bacillus (Propionibacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_E11, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_cross), the genus Bacillus (Bluebacillus_1), the genus Bacillus (Bluebacillus) and the genus Bacillus (Bluefaciens_bacteria), the genus Bacillus (Bluefaciens_bacillus) and the genus Bacillus (Bluefaciens) are different; the second set of microorganisms consists of the following species: streptococcus parahaemolyticus (Streptococcus parasanguinis), megamonas (Megamonas rupellensis), eglinium lentum (Eggerthella lenta), bacillus faecalis (coprobacter sp.) 3_3_56faa. The method according to the embodiment of the invention can produce or screen out drugs which promote the growth of various strains in the first microorganism set in the marker microorganism and/or inhibit the growth of various strains in the second microorganism set in the intestinal marker microorganism, and has important significance for assisting in alleviating the clinical symptoms of Graves disease.
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 foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic diagram of an experimental analysis procedure for screening for Graves disease marker microorganisms according to an embodiment of the present invention; and
FIG. 2 is a graph showing the results of evaluation of the AUC of a microorganism-labeled composite index according to an embodiment of the present invention, wherein the abscissa indicates Specificity and the ordinate indicates Sensitivity:
2-A is a graph of AUC values and confidence interval results under the ROC curve of 103 sample data in the first period;
2-B is a graph of AUC values and confidence interval results under the ROC curve for the 49 sample data of the second phase.
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 by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined 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 "optional" may explicitly or implicitly include or exclude that feature.
Biological markers are cell/biochemical or molecular changes that can be detected from biological media. Biological agents 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, e.g., the extent of that microorganism in a population of intestinal microorganisms, can be expressed as the content of that microorganism in that population.
According to one embodiment of the present invention, there is provided a kit comprising reagents suitable for detecting at least one species of a first set of microorganisms consisting of: the genus helicobacter (Paraseterella exterminatum), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalibacterium_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Copribacterium_com), the genus Bacillus (Copribacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_F7, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_computer), the genus Bacillus (Bubali_1_E11), the genus Bacillus (Bubali_bacillus), the genus Bacillus (BlueTokida_bacteria), the genus Bacillus (Bluebacillus_7), the genus Bacillus (Bluefaciens_bacteria) and the genus Bacillus (Bluefacilis) may be the genus Bdinus (Bluebacteria) may be the genus Bdinus_bacteria).
According to a specific embodiment of the invention, the kit further comprises reagents suitable for detecting at least one species of a second set of microorganisms consisting of the following species: streptococcus parahaemolyticus (Streptococcus parasanguinis), megamonas (Megamonas rupellensis), eglinium lentum (Eggerthella lenta), bacillus faecalis (coprobacter sp.) 3_3_56faa.
According to a specific embodiment of the invention, the kit comprises reagents suitable for detecting all of the species in the first set of microorganisms.
According to a specific embodiment of the invention, the kit comprises reagents suitable for detecting all of the species in the second set of microorganisms.
According to a specific embodiment of the present invention, the marker microorganism is determined by comparing and analyzing and verifying the difference of the abundance of microorganisms in stool samples of a large number of individuals suffering from graves 'disease and a large number of healthy control individuals, and the marker microorganism related to graves' disease in intestinal microorganisms is clarified. The kit comprising the reagent for detecting the marker microorganism can be used for determining the probability of the individuals suffering from Graves disease or the probability of the individuals suffering from Graves disease, and can be used for noninvasive early detection or auxiliary detection of Graves disease.
According to a specific embodiment of the present invention, the reagent suitable for detecting the first microorganism-set or the second microorganism-set is not particularly limited, and any reagent that can detect the microorganism-species is included in the scope of the present invention, such as a reagent that detects the microorganism-species by morphological characteristics, physiological and biochemical reaction characteristics, ecological characteristics, and serological reactions, sensitivity to phage, molecular biology, and the like, in particular, such as antibodies, enzymes, nucleic acid molecules, and the like.
Herein, the morphological characteristics of the microorganism refer to: the shape, size, arrangement, etc. of the microorganism, the cell structure, the gram stain reaction, the movement, the site and number of the flagellum, the presence or absence of spores and capsules, the size and position of spores, the shape, structure, the number, shape, size, color, surface characteristics, etc. of the actinomycetes and the reproductive organs of fungi are observed under a microscope.
Herein, the microbial physiological biochemical reaction characteristics refer to: the ability of the microorganism to utilize the substance, the specificity of the metabolite, such as whether H is produced 2 S, indole and CO 2 Alcohol, organic acid, whether nitrate can be reduced, whether milk can be coagulated, frozen, etc., growth environment (temperature, humidity, concentration of gases such as oxygen and carbon dioxide, pH, whether or not) Hypertonic resistance, halophilicity, etc.), and other biological relationships (e.g.: symbiotic, parasitic, host range, and pathogenic conditions), and the like.
Herein, the microbiological serological reaction refers to: the highly sensitive specific reaction of antigen and antibody is used to identify similar species or to identify microorganisms of the same species, such as antisera made with known species, types or strains, with the presence or absence of specific serological reactions with the microorganism to be identified.
Herein, the molecular biological method for detecting microorganisms mainly includes: PCR technology, high throughput sequencing and other methods are utilized.
The use of a reagent according to the invention for the preparation of a kit suitable for detecting at least one species of a first set of microorganisms for diagnosing graves 'disease or for detecting the therapeutic effect of graves' disease, said first set of microorganisms consisting of: the genus helicobacter (Paraseterella exterminatum), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalibacterium_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Copribacterium_com), the genus Bacillus (Copribacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_F7, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_computer), the genus Bacillus (Bubali_1_E11), the genus Bacillus (Bubali_bacillus), the genus Bacillus (BlueTokida_bacteria), the genus Bacillus (Bluebacillus_7), the genus Bacillus (Bluefaciens_bacteria) and the genus Bacillus (Bluefacilis) may be the genus Bdinus (Bluebacteria) may be the genus Bdinus_bacteria).
According to a specific embodiment of the present invention, the marker microorganism is determined by comparing and analyzing and verifying the difference of the abundance of the microorganism in the stool samples of a large number of individuals suffering from graves 'disease and a large number of healthy control individuals, and the marker of graves' disease related microorganism in the intestinal microorganism is clarified. The use of the reagent for detecting the marker microorganism can determine the probability of an individual suffering from Graves 'disease or the probability of an individual being in a healthy state, and can be used for noninvasive early detection or auxiliary detection of Graves' disease.
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 consisting of: streptococcus parahaemolyticus (Streptococcus parasanguinis), megamonas (Megamonas rupellensis), eglinium lentum (Eggerthella lenta), bacillus faecalis (coprobacter sp.) 3_3_56faa.
According to a specific embodiment of the present invention, the reagent suitable for detecting the first microorganism-set or the second microorganism-set is not particularly limited, and reagents that can detect the microorganism-species are included in the scope of the present invention, such as reagents that detect the microorganism-species by morphological characteristics, physiological biochemical reaction characteristics, ecological characteristics, and serological reactions, sensitivity to phage, molecular biology, and the like, specifically, such as antibodies, enzymes, nucleic acid molecules.
Herein, the morphological characteristics of the microorganism refer to: the shape, size, arrangement, etc. of the microorganism, the cell structure, the gram stain reaction, the movement, the site and number of the flagellum, the presence or absence of spores and capsules, the size and position of spores, the shape, structure, the number, shape, size, color, surface characteristics, etc. of the actinomycetes and the reproductive organs of fungi are observed under a microscope.
Herein, the microbial physiological biochemical reaction characteristics refer to: the ability of the microorganism to utilize the substance, the specificity of the metabolite, such as whether H is produced 2 S, indole and CO 2 Alcohol, organic acid, whether nitrate can be reduced, whether milk can be coagulated, frozen, etc., the growth environment (temperature, humidity, concentration of gases such as oxygen and carbon dioxide, pH, high permeability resistance, halophilicity, etc. suitable for growth), the relationship with other organisms (such as symbiosis, parasitism, host range and pathogenic condition), etc.
Herein, the microbiological serological reaction refers to: the highly sensitive specific reaction of antigen and antibody is used to identify similar species or to identify microorganisms of the same species, such as antisera made with known species, types or strains, with the presence or absence of specific serological reactions with the microorganism to be identified.
Herein, the molecular biological method for detecting microorganisms mainly includes: PCR technology, high throughput sequencing and other methods are utilized.
According to the present invention, there is provided a pharmaceutical or food composition for preventing or treating graves' disease, comprising at least one species of a first microorganism group consisting of: the genus helicobacter (Paraseterella exterminatum), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalibacterium_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Copribacterium_com), the genus Bacillus (Copribacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_F7, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_computer), the genus Bacillus (Bubali_1_E11), the genus Bacillus (Bubali_bacillus), the genus Bacillus (BlueTokida_bacteria), the genus Bacillus (Bluebacillus_7), the genus Bacillus (Bluefaciens_bacteria) and the genus Bacillus (Bluefacilis) may be the genus Bdinus (Bluebacteria) may be the genus Bdinus_bacteria).
The marker microorganisms were determined by the inventors by comparing and analyzing the abundance of various intestinal microorganisms in stool samples of Graves disease group and healthy group by difference, and by verifying a large number of stool samples in known state. The species in the marker microorganism is significantly enriched in the healthy population group compared to the graves 'disease patient population, wherein the significant enrichment means that the abundance of the species in the healthy group is statistically significantly higher or significantly, substantially higher than the abundance in the graves' disease patient group; the substance capable of increasing the abundance of the partial strain can be used for treating graves ' disease or is beneficial for graves ' disease patients to take, and the substance capable of increasing the abundance is not limited to a drug for treating graves ' disease and a functional food beneficial for intestinal flora balance. The marker microorganism provided by the embodiment can be used for preparing a medicine for treating graves 'disease and/or a functional food, health care medicine and the like beneficial to balance intestinal flora, and the medicine or the food can effectively treat or relieve graves' disease.
According to the present invention there is provided a method of determining whether an individual has graves' disease comprising steps (1) and (2).
(1) Determining the abundance of a marker microorganism in a fecal sample of the individual.
The marker microorganism includes at least one species of the first microorganism set and the second microorganism set. Wherein the first set of microorganisms consists of the following species: the genus helicobacter (Paraseterella exterminatum), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalibacterium_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Copribacterium_com), the genus Bacillus (Copribacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_F7, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_computer), the genus Bacillus (Bubali_1_E11), the genus Bacillus (Bubali_bacillus), the genus Bacillus (BlueTokida_bacteria), the genus Bacillus (Bluebacillus_7), the genus Bacillus (Bluefaciens_bacteria) and the genus Bacillus (Bluefacilis) may be the genus Bdinus (Bluebacteria) may be the genus Bdinus_bacteria).
According to some specific embodiments of the invention, step (1) further comprises: obtaining nucleic acid sequencing data in a fecal sample of the individual; comparing the sequencing data to a reference genome; based on the results of the alignment, the abundance of the marker microorganism is determined.
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 (U) S )+Ab(M S ) 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 For the number of reads in the sequencing data that are uniquely compared to the reference genome of the marker microorganism S, l S For the total length of the reference genome of the marker microorganism S,M S for 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 For the corresponding abundance coefficient of the ith read,/->Co i,s Representing the abundance ratio of the non-uniquely aligned reads i for the marker microorganism S, N being the total number of microorganisms to which the non-uniquely aligned reads i can be aligned, j representing the number of microorganisms to which the non-uniquely aligned reads i can be aligned.
The alignment can be performed by using known alignment software, such as SOAP, BWA, teraMap, etc., and 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 (mismatch), for example, s.ltoreq.2, and if more than s bases in the reads are mismatched, the reads are considered to be unable to align to (align with) the assembled fragment. The obtained comparison result comprises the comparison condition of each read and the reference genome of each species, and comprises information such as whether the reads can be compared with the reference genome of a certain species or a certain species, whether the reads are compared with the reference genome of a single species or a plurality of species only, the positions of the reference genomes of the species, the unique positions of the reference genomes of the species or a plurality of positions 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 class to which a pre-obtained sample to be tested belongs or is included, for example, the target is a microorganism in the sample to be tested, the reference sequence may be selected from a reference genome of various microorganisms in an NCBI database and/or a DACC intestinal reference genome disclosed in HMP and MetaHIT projects, further, a resource library including more reference sequences may be pre-configured, for example, a sequence which is more similar to the reference sequence may be selected or determined according to factors such as a state, a region, and the like of an individual from which the sample to be tested is derived. According to one embodiment of the invention, the reference genomes of various microorganisms are obtained from a public database, typically, one microorganism has multiple versions of the reference genome, i.e., one microorganism has multiple public reference genomes.
reads can be aligned with a reference genome of a species, which can be divided into two parts: a) Unique reads (U): uniquely comparing the reference genome of the previous species; these reads are called unique reads. That is, if the reference genomes on the reads are all from the same species, define these reads as unique reads; b) Multiplex reads (M): the reference genome of more than one species is aligned and defined as multiple reads. That is, reads are defined as multiple reads if the reference genome on which they are aligned is from at least two species.
(2) Abundance comparisons to determine if an individual has graves' disease.
According to one embodiment of the invention, the abundance obtained in step (1) is compared with a predetermined threshold in order to determine whether the individual has graves' disease.
According to some embodiments of the invention, the threshold is preset. Comparing the abundance of the marker microorganism in the sample of the individual to be detected with the threshold value, and determining the state of the individual to be detected. The threshold may be a value or a range of values, and the threshold setting manner is not particularly limited, and any threshold that can determine whether the individual suffers from inflammatory bowel disease may be used, for example, based on the average abundance value of a marker microorganism in an individual with known disease or health status, the threshold corresponding to the microorganism may be set to a confidence interval (Confidence interval) of 95% of the average abundance value.
The confidence interval refers to an estimated interval of the overall parameters constructed by the sample statistics. In statistics, the confidence interval of a probability sample is an interval estimate of some overall parameter of the sample. The confidence interval reveals the extent to which the true value of this parameter falls around the measurement with a certain probability. The confidence interval gives the degree of confidence in the measured value of the measured parameter, i.e. the "certain probability" as required before, this probability being referred to as the confidence level.
According to some embodiments of the invention, the individual is determined to have graves 'disease when the abundance of the marker microorganism determined in step (1) reaches the graves' disease abundance threshold and does not reach the graves 'disease abundance threshold, and the individual is determined to not have graves' disease when the abundance of the marker microorganism determined in step (1) reaches the graves 'disease abundance threshold and does not reach the graves' disease abundance threshold.
It should be noted that, depending on the purpose or requirement, there may be different requirements for determining the confidence level of the individual status result, and those skilled in the art may select different significance levels or thresholds.
The method is based on detecting the abundance of various strains in a marker microorganism in a fecal sample of an individual, comparing the abundance of various strains determined by detection with a threshold value thereof, and determining the probability that the individual is a Graves disease individual or a healthy individual according to the obtained comparison result. A non-invasive method for assisting detection or assisting intervention therapy is provided for early detection of Graves' disease.
All or part of the steps of the method of determining whether an individual has graves' disease using a marker microorganism in any of the above embodiments may be performed using an apparatus/system comprising detachable corresponding unit functional modules, or the method may be programmed, stored on a machine-readable medium, which is run using a machine.
According to the present invention there is provided an apparatus for determining whether an individual has graves' disease, the apparatus comprising: an abundance determination unit for determining the abundance of the marker microorganism in the individual's stool sample; a comparison unit for comparing the obtained abundance with a predetermined threshold in order to determine whether the individual has graves' disease; wherein the first set of microorganisms consists of the following species: the genus helicobacter (Parasepsis_excrementis), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalablock_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Propionibacterium_com), the genus Bacillus (Propionibacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_E11, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_cross), the genus Bacillus (Bluebacillus_1), the genus Bacillus (Bluebacillus) and the genus Bacillus (Bluefaciens_bacteria), the genus Bacillus (Bluefaciens_bacillus) and the genus Bacillus (Bluefaciens) are different; the second set of microorganisms consists of the following species: streptococcus parahaemolyticus (Streptococcus parasanguinis), megamonas (Megamonas rupellensis), eglinium lentum (Eggerthella lenta), bacillus faecalis (coprobacter sp.) 3_3_56faa. The above description of the technical features and advantages of the method for determining whether an individual has graves' disease using a marker microorganism according to any of the embodiments of the present invention applies equally to the apparatus according to this aspect of the present invention and will not be described in detail herein.
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 fecal sample of the individual; comparing the sequencing data to a reference genome; based on the results of the alignment, the abundance of the marker microorganism is determined.
The alignment can be performed by using known alignment software, such as SOAP, BWA, teraMap, etc., and 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 (mismatch), for example, s.ltoreq.2, and if more than s bases in the reads are mismatched, the reads are considered to be unable to align to (align with) the assembled fragment. The obtained comparison result comprises the comparison condition of each read and the reference genome of each species, and comprises information such as whether the reads can be compared with the reference genome of a certain species or a certain species, whether the reads are compared with the reference genome of a single species or a plurality of species only, the positions of the reference genomes of the species, the unique positions of the reference genomes of the species or a plurality of positions and the like. According to one embodiment of the invention, the alignment is performed using a SOAPAlign 2.21, setting the parameters to-r 2-m 100-x 1000.
The reference genome of the microorganism refers to a predetermined sequence of the microorganism species, and may be any reference template of a biological class to which a pre-obtained sample to be tested belongs or is included, for example, the target is the microorganism in the sample to be tested, the reference sequence may be selected from the reference genome of various microorganisms in an NCBI database and/or the DACC intestinal genome disclosed in HMP and MetaHIT projects, further, a resource library including more reference sequences may be pre-configured, for example, a sequence which is closer to the reference sequence may be selected or determined and assembled according to factors such as the state, region, and the like of an individual from which the sample to be tested is derived. According to one embodiment of the invention, the reference genomes of various microorganisms are obtained from a public database, typically, one microorganism has multiple versions of the reference genome, i.e., one microorganism has multiple public reference genomes.
reads can be aligned with a reference genome of a species, which can be divided into two parts: a) Unique reads (U): uniquely comparing the genome of the previous species; these reads are called unique reads. That is, reads are defined as unique reads if the reference genomes on the reads are all from the same species. b) Multiplex reads (M): the reference genome of more than one species is aligned and defined as multiple reads. That is, reads are defined as multiple reads if the reference genome on which they are aligned is from at least two species.
According to a specific embodiment of the present invention, the abundance of the marker microorganism is determined according to the following formula: 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, ab (U) S )=U S /l S ,U S For the number of reads in the sequencing data that are uniquely compared to the reference genome of the marker microorganism S, l S For the total length of the reference genome of the marker microorganism S,M S for 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 For the corresponding abundance coefficient of the ith read,/->Co i,s Representing the abundance ratio of the non-uniquely aligned reads i for the marker microorganism S, N being the total number of microorganisms to which the non-uniquely aligned reads i can be aligned, j representing the number of microorganisms to which the non-uniquely aligned reads i can be aligned. The above description of the technical features and advantages of the method for determining whether an individual has graves' disease using a marker microorganism according to any of the embodiments of the present invention applies equally to the apparatus according to this aspect of the present invention and will not be described in detail herein.
According to the present invention there is provided an apparatus comprising: a computer readable storage medium having stored thereon a computer program for performing one of the methods of determining whether an individual has graves' disease as described above; and one or more processors configured to execute the program in the computer-readable storage medium.
According to the present invention there is provided a method of screening for a drug for the treatment or prevention of graves' disease, comprising: administering a candidate drug to a subject, detecting the abundance of a marker microorganism in the subject's stool before and after administration, the marker microorganism comprising at least one species of the first set of microorganisms and the second set of microorganisms, wherein the candidate drug that satisfies at least one of the following conditions is suitable for treating or preventing graves' disease: (1) After said administering, said abundance of at least one species of said first set of microorganisms increases; and (2) said abundance of at least one species of said second microorganism set decreases following said administering; wherein the first set of microorganisms consists of the following species: the genus helicobacter (Parasepsis_excrementis), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalablock_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Propionibacterium_com), the genus Bacillus (Propionibacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_E11, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_cross), the genus Bacillus (Bluebacillus_1), the genus Bacillus (Bluebacillus) and the genus Bacillus (Bluefaciens_bacteria), the genus Bacillus (Bluefaciens_bacillus) and the genus Bacillus (Bluefaciens) are different; the second set of microorganisms consists of the following species: streptococcus parahaemolyticus (Streptococcus parasanguinis), megamonas (Megamonas rupellensis), eglinium lentum (Eggerthella lenta), bacillus faecalis (coprobacter sp.) 3_3_56faa.
By using the method for producing or screening the medicines for treating the Graves disease, which is disclosed by the invention, the medicines capable of supporting the growth of beneficial intestinal bacteria and/or inhibiting potential pathogenic bacteria of the intestinal tract can be obtained by reasonably and effectively applying the determined Graves disease biomarkers for screening, and the method has important significance for assisting in alleviating clinical symptoms of the Graves disease.
The embodiments will be described in detail below. The reagents, sequences, software and instrumentation referred to in the examples below, which are not specifically addressed, are all conventional commercial products.
Example 1 identification of biomarkers
In this example, the inventors have studied stool samples from 61 graves patients and 42 healthy controls to obtain the microbiota and functional component characteristics of the intestinal microbiota. The inventor obtains about 354.6Gb high-quality healthy human data and 502.7Gb high-quality Graves disease patient sequencing data through sequencing and quality control to construct a Graves disease reference gene set, and constructs a more complete gene set with the IGC gene set. Metagenomic analysis showed that 42 microbial species are closely related to graves 'disease, of which 35 bacteria are enriched in intestinal microorganisms in healthy humans and 7 bacteria are enriched in intestinal microorganisms in graves' disease patients.
1. Acquisition of sequencing data:
all sample sequencing data were downloaded from literature (PMID: 34079079), graves patients and healthy controls were all from Hainan province people hospital, haikou, china, aged between 24-69, subjects of each healthy group collected stool samples prior to the first meal in the morning, and GD patients collected stool samples during clinical visits. After determining the fecal weight, sample protectants (CW 0592M, CWBIO, china) were added in a 5:1 ratio to stabilize the nucleotides. The samples were stored at 20 ℃ until further processing.
DNA was extracted from fecal samples using QIAamp DNA Stool Mini Kit (Qiagen, hilden, germany) and the extracted DNA was identified by 0.8% agarose gel electrophoresis and the OD 260/280 was determined spectrophotometrically, following the protocol of the kit.
2. Construction of DNA library and sequencing
DNA banking was performed according to the instructions of the instrument manufacturer (Illumina). The library was PE150 bp sequenced and a library of 103 samples was sequenced on an Illumina Hiseq2500 (Illumina, san Diego, calif.) platform. Each sample produced on average 8.1Gb (sd. ±1.5 Gb) sequencing results, totaling 860.5Gb sequencing data volume.
Referring to the experimental procedure shown in fig. 1, relevant biomarkers of graves' disease are identified, wherein omitted steps or 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, carrying out first-stage sequencing, acquiring data of 103 samples in the first-stage sequencing, filtering the data after obtaining the sequencing data of the 103 samples in the first stage, and carrying out quality control according to the following standard: a) Removing reads greater than 5N bases; b) Removing more than 50% of reads of low quality bases (Q20); c) Tail low mass (Q20) and N bases were removed. Missing paired reads sequences are considered as a single reads for assembly.
Wherein the IGC gene set was downloaded from ftp.cngb.org/pub/SciRAID/Microbiome/humanGut_9.9M/GeneCatalog/IGC.fa.gz.
3.2 species abundance analysis
SOAPalign 2.21 is used to match the paired-end clean reads against redundant genomes, here called redundant genomes from reference genomes of bacteria disclosed in the respective databases, with alignment parameters of-r 2-m 200-x 1000. The comparison result of Reads and redundant genome can be divided into two parts: a) Unique reads (U): reads aligned only to the genome of the last 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) Multiplex reads (M): if reads align the genomes of two or more species, it is defined as multiple reads. That is, if the genomes on the alignment are from different species, the inventors define these reads as multiple reads.
For species S, the abundance is Ab (S), and the abundance is calculated as follows, in relation to the unique U reads and the shared M reads:
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,
Ab(U S )=U S /l S
U S for the number of reads in the sequencing data that are uniquely compared to the reference genome of the marker microorganism S,
l S for the total length of the reference genome of the marker microorganism S,
M S micro-generating the sequencing data with the markersThe number of reads of the reference genome of organism S that are not uniquely aligned,
Co i for the corresponding abundance ratio of the ith read,
i represents the number of the non-uniquely aligned reads,
Co i,s represents the presence of a microorganism S directed to said marker,
the abundance ratio of the non-uniquely aligned reads i,
n is the total number of microorganisms to which the non-uniquely aligned reads i can be aligned,
j represents the number of microorganisms to which the non-uniquely aligned reads i can be aligned.
And (3) obtaining a normalized species abundance table after dividing all the calculated species abundance values in each sample by the total abundance of each sample.
3.3 screening of microbial species markers
In order to obtain intestinal microbial species markers closely related to graves disease, the inventors made a study on the species level using the abundance data of intestinal microbial species from both graves disease patient (GD) group (61 cases) and normal human (Health) group (42 cases). Based on the species abundance table obtained in step 3.2, the inventors set the criteria as follows: (1) The median of abundance of species from the graves' patient group or healthy human group must be greater than 0.0001; (2) The correlation p value of each species and graves disease was obtained by combining the Wilcoxon rank sum test of Benjamini Hochberg multiplex test; (3) Screening was performed using the above parameters using a threshold p_values < 0.005. The inventors have obtained 42 species of intestinal microorganisms closely related to graves disease, wherein 7 species of microorganisms are enriched in the intestinal tract of graves disease patients and 35 species are enriched in healthy persons, and the 42 species markers are shown in table 1.
Table 1:
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example 2 validation of microbial species markers
To verify the findings in example 1, the inventors further analyzed the abundance of the 42 genera in fecal samples of 20 healthy persons and 29 graves' disease patients in the verified population, and made deletions of the 42 microorganism species markers according to the enrichment of each species in healthy and disease groups, DNA extraction, sequencing, and analysis of species abundance in the verified population were performed with reference to example 1.
The verification result is as follows: the P-value results of the above-described validation of 35 species enriched in healthy humans, 17 resulting in high quality validation (P __ values < 0.01) in the validation set, and the enriched microbial species markers for healthy humans are shown in table 2.
Table 2:
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of the 7 species enriched in Graves patients, 4 gave high quality validation (P value < 0.01) in validation sets, and the P values of the 4 microbial markers enriched in Graves patients in validation populations are shown in Table 3 as Streptococcus parahaemolyticus (Streptococcus parasanguinis), pseudomonas (Megamonas rupellensis), eggeratel lentus (Eggerthella lenta), bacillus faecalis (Coprobacillus sp.3_3_56FAA), respectively.
Table 3:
the inventor believes that 17 microorganism species markers enriched from healthy people can be used as reverse indexes of diseases of Graves disease, or as microbial preparation drug flora components for developing treatment of Graves disease, or as recovery indexes for detecting Graves disease and monitoring the treatment progress of Graves disease; the 4 microorganism species markers enriched by Graves disease patients are used as forward indicators of the disease of Graves disease, and are particularly used for non-invasive detection and diagnosis of the disease of Graves disease.
The inventor utilizes the 21 microorganism species markers to construct a comprehensive index, estimates the area under ROC (Receiver-operating characteristic) curve AUC, and the larger the AUC is, the higher the diagnostic capability is, and the evaluation comprehensive score corresponds to the diagnostic capability of the marker on Graves disease. By evaluating 103 samples of the first stage (first stage) and 49 samples of the second stage (second stage), as shown in fig. 2, both showed good diagnostic ability, with auc=95.6% obtained at the first stage, with a confidence interval of 92.2% -98.9% as shown in fig. 2-a; auc=90.7% was obtained in stage two, with confidence intervals of 82.6% -98.8% as shown in fig. 2-B.
Example 3 detection of individual State
In this example, the inventors used 10 stool samples for the detection of the individual status of the sample source.
The abundance of streptococcus parahaemolyticus (Streptococcus parasanguinis), megamonas (Megamonas rupellensis), eglinium tarda (Eggerthella lenta) and bacillus sp 3_3_56faa shown in table 3 in each fecal sample was determined by the method of example 2, whether the abundance of these 4 strains in each sample fell within a confidence interval of 95% of the abundance of the disease control group or the healthy control group, the state of the individual corresponding to the sample in which the abundance of these 4 strains fell within the corresponding interval of the disease group was determined to be graves 'disease, and the state of the individual corresponding to the sample in which the abundance of these 4 strains fell within the corresponding interval of the healthy group was determined to be non-graves' disease.
The results show that the method described in this example can be used to determine the individual status of 10 samples, including 7 samples with graves' disease and 3 healthy samples, and that the determination of the status of 8 samples in the 10 samples corresponding to the individual is consistent with the recorded status of the individual from which the sample originated.
In addition, the inventors found that the combined detection of the species in table 2 and table 3, e.g., detecting that the species markers in table 3 are enriched, while the species markers in table 2 are not enriched, can more accurately determine whether graves' disease patients or susceptible populations are found.
In the case of using the markers for the treatment of graves' disease, the inventors found that the species markers in table 3 are inhibited or cleared, and at the same time the species markers in table 2 are enriched, resulting in a better therapeutic effect.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means 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 present invention. In this specification, schematic representations of the above terms are not necessarily directed 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, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (2)

1. A kit comprising reagents suitable for detecting all species in a first set of microorganisms, said first set of microorganisms consisting of all of said species: the genus helicobacter (Parasepsis_excrementis), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalablock_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Propionibacterium_com), the genus Bacillus (Propionibacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_E11, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_cross), the genus Bacillus (Bluebacillus_1), the genus Bacillus (Bluebacillus) and the genus Bacillus (Bluefaciens_bacteria), the genus Bacillus (Bluefaciens_bacillus) and the genus Bacillus (Bluefaciens) are different;
The kit further comprises reagents suitable for detecting all of said species in a second set of microorganisms consisting of: streptococcus parahaemolyticus (Streptococcus parasanguinis), megamonas (Megamonas rupellensis), eglinium lentum (Eggerthella lenta), bacillus faecalis (coprobacter sp.) 3_3_56faa.
2. Use of a reagent adapted to detect all species in a first set of microorganisms for diagnosing graves' disease in the preparation of a kit consisting of: the genus helicobacter (Parasepsis_excrementis), the genus helicobacter (Lachnospiraceae_bacteria) _5_1_63FAA, the genus fecal (Faechalaea_cylinddes), the genus Bacillus (Faechalablock_prausiiti), the genus Bacillus (Eubacterium_rectale), the genus Bacillus (Eubacterium_haliii), the genus Bacillus (Eubacterium_companion), the genus enterococcus (Propionibacterium_com), the genus Bacillus (Propionibacterium_cata), the genus Clostridium (Clostridium_bacillus), the genus Clostridium (Clostridium_bacteria) _5_1_E11, the genus Bacillus (Bubali_bacillus), the genus Bacillus (Bubali_cross), the genus Bacillus (Bluebacillus_1), the genus Bacillus (Bluebacillus) and the genus Bacillus (Bluefaciens_bacteria), the genus Bacillus (Bluefaciens_bacillus) and the genus Bacillus (Bluefaciens) are different;
The reagent is further adapted to detect all species in a second set of microorganisms, the second set of microorganisms consisting of: streptococcus parahaemolyticus (Streptococcus parasanguinis), megamonas (Megamonas rupellensis), eglinium lentum (Eggerthella lenta), bacillus faecalis (coprobacter sp.) 3_3_56faa.
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