CN114381534A - Fatty liver marker microorganism and application thereof - Google Patents

Fatty liver marker microorganism and application thereof Download PDF

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CN114381534A
CN114381534A CN202111411877.6A CN202111411877A CN114381534A CN 114381534 A CN114381534 A CN 114381534A CN 202111411877 A CN202111411877 A CN 202111411877A CN 114381534 A CN114381534 A CN 114381534A
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exiguobacterium
bacteroides
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fatty liver
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陈翔
朱剑梅
马玮
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Hangzhou Tuohong Biological Technology Co ltd
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Abstract

The present invention proposes a fatty liver marker microorganism and its use, the fatty liver marker microorganism comprising a first set of microorganisms, thus further providing a kit comprising reagents suitable for detecting at least one species in the first set of microorganisms, said first set of microorganisms consisting of: roseburia amine, atrophaeus minimalis, bacteroides ovatus, bacteroides, fetid odor bacillus, filobacter, xenobacter, rothidae, xenobacter saxatilis, bacteroides twinning coccus, globulobacter unusually, veillonella dispar and pasteurella gordonii. The abundance of the microorganism provided by the invention has obvious difference in healthy people and fatty liver patients, and can be used as a marker for effectively detecting and/or treating fatty liver.

Description

Fatty liver marker microorganism and application thereof
Technical Field
The present invention relates to the field of biotechnology, specifically, the present invention relates to a fatty liver marker microorganism and use thereof, and more specifically, the present application relates to a kit, use of a reagent in preparation of the kit, a pharmaceutical composition or a food composition for preventing or treating fatty liver, a method for determining whether an individual has fatty liver, a device, a method for screening a drug.
Background
Fatty liver (Fatty liver) is a pathological condition of excessive fat accumulation in liver cells due to various reasons, and is a common pathological change of liver rather than an independent disease. Fatty liver disease seriously threatens the health of people in China, is the second most serious liver disease of viral hepatitis, has continuously increased incidence rate and is younger in attack age. Normal human liver tissue contains a small amount of fat, such as triglycerides, phospholipids, glycolipids, and cholesterol, and its weight is about 3% to 5% of the weight of the liver, and if too much fat accumulates in the liver, it exceeds 5% of the weight of the liver or when there is steatosis in more than 50% of the liver cells histologically, it is called fatty liver. The mild case has no symptoms, and the severe case has fierce illness.
With the rapid improvement of living standard and the unreasonable trend development of dietary structure of people, the fatty liver is the second largest liver disease next to viral hepatitis, and the severe fatty liver patients may have liver cirrhosis and other chronic liver diseases, which are extremely harmful to the health development of human bodies.
In human body, choline participating in lecithin composition has important significance in methyl conversion and lipoprotein metabolism, and can be clinically treated by using appropriate dose of choline and choline chloride. The choline medicine with excessive dosage has strong hepatotoxicity and no effect on preventing and treating alcoholic fatty liver and hepatic fibrosis of primates. Methionine is used as an essential amino acid in human body, plays a role of a methyl donor in the choline synthesis process, and has important significance in phospholipid metabolism and maintenance of biological membrane functions.
The gut is populated with a large number of microorganisms ranging from bacteria, viruses, fungi and archaea to bacteriophages and protozoa. The gut microbiome can regulate nutrient metabolism upon dietary intake and produce a number of metabolites that interact with the host in a variety of ways, including regulating glucose and lipid metabolic pathways, affecting immune cell differentiation and function, affecting insulin sensitivity, and the like. The large body of human and animal data provides strong evidence that the gut microbiome and its metabolites play a crucial role in the development and progression of many metabolic diseases. The human intestinal microbiota is driven by dietary macronutrients to produce bioactive compounds consisting of bile acids, short chain fatty acids, ammonia, phenols, endotoxins, and the like. These microbiota-derived metabolites are vehicles for the microorganisms to communicate with the host, which is critical for maintaining host physiology. Indeed, the profile of metabolites associated with the gut microbiome provides further insight into the impact of lifestyle and dietary factors on disease. A number of tuple science methods have been developed to study the function of microbiota-derived metabolites, including metagenomics, macroproteomics, and metabolomics. These techniques enable us to define metabolic features, identify and quantify classes and compounds of interest, characterize small molecules produced by the gut microbiome and define biochemical pathways for metabolites, and play an important role in identifying microbial markers. Therefore, the research on the characteristics of the intestinal microbiota of the fatty liver patients has important significance for the detection and treatment of the fatty liver.
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 discovers a fatty liver marker microorganism and provides a non-invasive method for early detection of fatty liver; 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 fatty liver 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, the kit comprises reagents suitable for detecting at least one species in a first set of microorganisms consisting of: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophaeus (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceus (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sarmentosum (Alisterii _ shahii), Exiguobacterium sp. AP11, Geellaria twins (Gemelalla _ unclarified), Aneudiobolus vulgaris (Subdariella vularia _ variella), Exigus williamella (Veillonella _ vestris) and Suaedella terrestris (Subdariella terrestris). According to the kit provided by the embodiment of the invention, at least one strain in the first microorganism set can be accurately detected, so that the fatty liver patient and the healthy individual can be accurately distinguished or diagnosed.
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 present invention, the kit is for diagnosing fatty liver or detecting the therapeutic effect of fatty liver, and the first microbial pool is composed of the following species: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophaeus (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceus (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sarmentosum (Alisterii _ shahii), Exiguobacterium sp. AP11, Geellaria twins (Gemelalla _ unclarified), Aneudiobolus vulgaris (Subdariella vularia _ variella), Exigus williamella (Veillonella _ vestris) and Suaedella terrestris (Subdariella terrestris). 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 fatty liver patients and healthy individuals can be very accurately distinguished, so that fatty liver diagnosis can be effectively carried out at an early stage, or the kit can be used for detecting the change of fatty liver in a treatment process.
In a third aspect of the present invention, the present invention provides a pharmaceutical composition or a food composition for preventing or treating fatty liver. 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: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophaeus (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceus (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sarmentosum (Alisterii _ shahii), Exiguobacterium sp. AP11, Geellaria twins (Gemelalla _ unclarified), Aneudiobolus vulgaris (Subdariella vularia _ variella), Exigus williamella (Veillonella _ vestris) and Suaedella terrestris (Subdariella terrestris). According to the embodiment of the invention, the strains of the first microorganism group in the fatty liver marker microorganisms can be non-invasively found or assisted to detect the fatty liver in an early stage, and the probability that an individual has the fatty liver or the probability that the individual is in a healthy state is determined; meanwhile, the various strains in the first microorganism concentration in the intestinal tract of the high-risk people with fatty liver or the patients with fatty liver are improved, so that the probability of fatty liver diseases can be reduced, or the fatty liver can be slowed down and cured, therefore, the medicine or food composition containing at least one strain in the first microorganism concentration can be used for balancing the intestinal flora, and the fatty liver can be effectively prevented or treated.
In a fourth aspect of the invention, a method of determining whether an individual has fatty liver 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 fatty liver; wherein the first microbial collection consists of the following species: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophicum (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceum (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sakesii (Alisteri _ shahii), Exiguobacterium sp. AP11, Micrococcus bacterium twins (Gemelassella _ unclarified), Exiguella vulgaris (Subdellulirus _ variella), Exigus williamsii Veillella (Veillonella _ and Thermus terrestra (Suydans); the second microbial set consists of the following species: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis). The method according to the embodiment of the invention can determine whether the individual has the fatty liver according to the abundance of the marker microorganisms in the fecal sample of the individual, wherein the marker microorganisms are 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 the fatty liver group and the healthy group through difference comparison.
In a fifth aspect of the invention, an apparatus for determining whether an individual has fatty liver 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 resulting abundance with a predetermined threshold value in order to determine whether the individual has fatty liver; wherein the first microbial collection consists of the following species: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophicum (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceum (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sakesii (Alisteri _ shahii), Exiguobacterium sp. AP11, Micrococcus bacterium twins (Gemelassella _ unclarified), Exiguella vulgaris (Subdellulirus _ variella), Exigus williamsii Veillella (Veillonella _ and Thermus terrestra (Suydans); the second microbial set consists of the following species: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis). The marker microorganisms are determined by analyzing the abundance of various intestinal microorganisms in the fecal samples of fatty liver patients and healthy people through difference comparison and verifying a large number of fecal samples with known states, and the device provided by the embodiment of the invention can accurately determine whether the individual is a high-risk group of fatty liver or a fatty liver patient.
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 fatty liver or a fatty liver 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 treating or preventing fatty liver, the method comprising: administering a drug candidate to a subject, wherein the subject's stool comprises an abundance of a marker microorganism comprising at least one species of the first set of microorganisms and the second set of microorganisms, and wherein a drug candidate satisfying at least one of the following conditions is suitable for use in treating or preventing fatty liver: (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: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophicum (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceum (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sakesii (Alisteri _ shahii), Exiguobacterium sp. AP11, Micrococcus bacterium twins (Gemelassella _ unclarified), Exiguella vulgaris (Subdellulirus _ variella), Exigus williamsii Veillella (Veillonella _ and Thermus terrestra (Suydans); the second microbial set consists of the following species: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis). 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 fatty liver.
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 fatty liver 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 78 sample data in the first period;
2-B is a graph of AUC values and confidence interval results under the ROC curve for 78 samples 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 the present invention there is provided a kit comprising reagents suitable for detecting at least one species in a first collection of microorganisms consisting of: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophaeus (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceus (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sarmentosum (Alisterii _ shahii), Exiguobacterium sp. AP11, Geellaria twins (Gemelalla _ unclarified), Aneudiobolus vulgaris (Subdariella vularia _ variella), Exigus williamella (Veillonella _ vestris) and Suaedella terrestris (Subdariella terrestris).
According to a particular embodiment of the invention, the kit further comprises reagents suitable for detecting at least one species of a second microbiome consisting of: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis).
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 set of microorganisms.
According to a specific embodiment of the present invention, the marker microorganism is determined by comparative analysis and verification of the differences of the abundance of the microorganism in the fecal samples of a large number of individuals suffering from fatty liver and a large number of healthy control individuals, and the fatty liver-related marker microorganism in the intestinal microorganisms is determined. The kit containing the reagent for detecting the marker microorganism can determine the probability that an individual is in a fatty liver state or in a healthy state, and can be used for non-invasive early detection or auxiliary detection of fatty liver.
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 an agent provided according to the invention for the preparation of a kit suitable for detecting at least one species in a first microbial set for diagnosing fatty liver or for detecting the therapeutic effect of fatty liver, said first microbial set consisting of the following species: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophaeus (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceus (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sarmentosum (Alisterii _ shahii), Exiguobacterium sp. AP11, Geellaria twins (Gemelalla _ unclarified), Aneudiobolus vulgaris (Subdariella vularia _ variella), Exigus williamella (Veillonella _ vestris) and Suaedella terrestris (Subdariella terrestris).
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 fecal samples of a large number of individuals suffering from fatty liver and a large number of healthy control individuals, and the fatty liver-related microorganism marker in the intestinal microorganism is determined. The reagent for detecting the marker microorganism can determine the probability that an individual has fatty liver or the probability that the individual is in a healthy state, and can be used for non-invasive early detection or auxiliary detection of fatty liver.
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, the second set of microorganisms consisting of: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis).
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 and organic acid, whether nitrate can be reduced, whether milk can be coagulated or frozen, the growth environment (temperature and humidity suitable for growth, concentration of gases such as oxygen and carbon dioxide, PH, whether hypertonic resistance exists, whether halophilic property exists, and the like), the relationship with other organisms (such as symbiosis, parasitism, host range and pathogenic condition), 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.
According to the present invention, there is provided a pharmaceutical composition or food composition for preventing or treating fatty liver, comprising at least one species of a first microbial population consisting of the following species: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophaeus (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceus (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sarmentosum (Alisterii _ shahii), Exiguobacterium sp. AP11, Geellaria twins (Gemelalla _ unclarified), Aneudiobolus vulgaris (Subdariella vularia _ variella), Exigus williamella (Veillonella _ vestris) and Suaedella terrestris (Subdariella terrestris).
The marker microorganisms are determined by analyzing the abundance of various intestinal microorganisms in the fecal samples of the fatty liver disease group and the healthy group through difference comparison and verifying a large number of fecal samples with known states. The first microbiome species of the marker microorganisms is significantly enriched in the healthy population group compared to the fatty liver patient group, wherein significantly enriched is statistically significantly higher or significantly substantially higher than the abundance of the species in the healthy group compared to the abundance in the fatty liver patient group; the substance capable of improving the abundance of the partial strains can be used for treating fatty liver or is beneficial to patients with fatty liver to take, and the substance capable of improving the abundance is not limited to medicines for treating fatty liver and functional foods beneficial to the balance of intestinal flora. The marker microorganism provided by the embodiment can be used for preparing a medicament for treating the fatty liver and/or preparing a functional food, a health-care medicament and the like which are beneficial to balancing intestinal flora, wherein the medicament or the food can effectively treat or relieve the fatty liver.
According to the invention, a method for determining whether an individual has fatty liver comprises 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: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophicum (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceum (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sakesii (Alisteri _ shahii), Exiguobacterium sp. AP11, Micrococcus bacterium twins (Gemelassella _ unclarified), Exiguella vulgaris (Subdellulirus _ variella), Exigus williamsii Veillella (Veillonella _ and Thermus terrestra (Suydans); the second microbial set consists of the following species: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis).
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 (U)S)+Ab(MS) Wherein S represents the number of the marker microorganism, Ab (S) represents the abundance of the marker microorganism S, Ab (U)S)＝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,
Figure RE-GDA0003534473060000081
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,
Figure RE-GDA0003534473060000082
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) And comparing the abundance to determine whether the individual has fatty liver.
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 fatty liver.
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 value may be a value or a range of values, the setting manner of the threshold value is not particularly limited, and any threshold value capable of determining whether the individual has the inflammatory bowel disease may be used, for example, based on the average abundance of the marker microorganism in the individual with the known diseased or healthy state, the threshold value corresponding to the microorganism may be set as a 95% confidence interval of the average abundance.
The confidence interval refers to an estimation interval of the overall parameter constructed by the sample statistic. In statistics, the Confidence interval (Confidence interval) of a probability sample is an interval estimate for some overall parameter of this 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 present invention, when the abundance of the marker microorganism determined in step (1) reaches the threshold of the abundance of fatty liver, the individual is determined to have fatty liver, and when the abundance of the marker microorganism determined in step (1) reaches the threshold of the abundance of fatty liver, the individual is determined to have no fatty liver.
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 fatty liver individual or a healthy individual according to the obtained comparison result. Provides a non-invasive auxiliary detection or auxiliary intervention treatment method for early fatty liver discovery.
All or a part of the steps of the method for determining whether an individual has fatty liver 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 fatty liver, 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 resulting abundance with a predetermined threshold value in order to determine whether the individual has fatty liver; wherein the first microbial collection consists of the following species: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophicum (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceum (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sakesii (Alisteri _ shahii), Exiguobacterium sp. AP11, Micrococcus bacterium twins (Gemelassella _ unclarified), Exiguella vulgaris (Subdellulirus _ variella), Exigus williamsii Veillella (Veillonella _ and Thermus terrestra (Suydans); the second microbial set consists of the following species: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis). The above description of the technical features and advantages of the method for determining whether an individual has fatty liver using marker microorganisms according to any embodiment of the present invention is equally applicable to the apparatus according to this aspect of the present invention, and will not be repeated 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 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 (U)S)+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,
Figure RE-GDA0003534473060000111
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,
Figure RE-GDA0003534473060000112
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 above description of the technical features and advantages of the method for determining whether an individual has fatty liver using marker microorganisms according to any embodiment of the present invention is equally applicable to the apparatus according to this aspect of the present invention, and will not be repeated herein.
According to yet another embodiment of 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 foregoing methods of determining whether an individual has fatty liver; 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 fatty liver, the method including: administering a drug candidate to a subject, wherein the subject's stool comprises an abundance of a marker microorganism comprising at least one species of the first set of microorganisms and the second set of microorganisms, and wherein a drug candidate satisfying at least one of the following conditions is suitable for use in treating or preventing fatty liver: (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: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophicum (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceum (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sakesii (Alisteri _ shahii), Exiguobacterium sp. AP11, Micrococcus bacterium twins (Gemelassella _ unclarified), Exiguella vulgaris (Subdellulirus _ variella), Exigus williamsii Veillella (Veillonella _ and Thermus terrestra (Suydans); the second microbial set consists of the following species: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis).
By utilizing the method for producing or screening the medicament for treating the fatty liver, disclosed by the invention, 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 by reasonably and effectively applying the determined fatty liver biomarker for screening, and the method has important significance for assisting in relieving the clinical symptoms of the fatty liver.
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 marker microorganisms
In this example, the inventors studied fecal samples of 43 fatty liver patients and 35 healthy controls to obtain the characteristics of the microbial community and functional components of the intestinal flora. In summary, the inventors downloaded about 428.09Gb high quality sequencing data (control healthy people) and 294Gb high quality sequencing data obtained by experimental sequencing to construct a fatty liver reference gene set, and constructed a more complete gene set with the downloaded LC gene set and IGC gene set. Metagenomic analysis shows that 36 microbial species are closely related to fatty liver diseases, 24 of them are enriched in intestinal microbes of healthy people, and 12 are enriched in intestinal microbes of fatty liver patients.
1. Obtaining sequencing data:
fatty liver disease and normal control samples were taken from published articles PMID: 22699609, PMID: 25758642, PMID: 28467925.
with reference to the experimental procedure shown in fig. 1, relevant biomarkers of fatty liver were identified, wherein omitted steps or details are well known to those skilled in the art, and several important steps are described below.
2. Microbial species abundance analysis
2.1 sequence optimization statistics
1) Firstly, performing first-stage sequencing, acquiring data of 78 samples in the current-stage sequencing, filtering the data after acquiring the sequencing data of the 78 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) And (3) processing the downloaded data of the healthy people by adopting the method 1).
3) The LC gene set was obtained from Qin, N.et al.Alterations of the human gut microbiome in liver cirrhosis.Nature 513, 59-64 (2014.) and the IGC gene set was obtained from the IGC gene setftp:// climb.genomics.cn/pub/10.5524/100001_101000/100064/1.GeneCatalogs/ IGC.fa.gz。
2.2 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,
Figure RE-GDA0003534473060000131
MSis the number of sequencingAccording to the number of reads that are non-uniquely aligned with the reference genome of the marker microorganism S,
Coithe abundance coefficient corresponding to the ith read,
Figure RE-GDA0003534473060000132
i represents the number of the non-unique alignment reads,
Coi,smeans that with respect to the marker microorganism S,
abundance coefficients of the non-uniquely aligned reads i,
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 fatty liver diseases, the inventor utilizes the abundance data of intestinal microbial species of two groups, namely a fatty liver patient (RA) group (63 cases) and a normal person (Health) group (50 cases) to carry out a research on the correlation between the species and the diseases at the species level. Based on the abundance of species table obtained in step 2.2, the inventors set criteria as follows: (1) the median abundance of species in the fatty liver patient group or healthy human group must be greater than 0.00001; (2) the p-value and q-value of the correlation of each species and fatty liver disease were obtained by Wilcoxon rank sum test combined with multiple tests of Benjamini Hochberg; (3) screening was performed using the above parameters using a strict threshold p _ values < 0.05. The inventor obtains 36 intestinal microorganism species closely related to fatty liver diseases, wherein 12 species of microorganisms are enriched in intestinal tracts of fatty liver patients, 24 species of microorganisms are enriched in healthy people, and the 36 microorganism species markers are shown in table 1.
Table 1:
Figure RE-GDA0003534473060000141
example 2 validation of microbial species markers
To verify the findings in example 1, the inventors further analyzed the abundance of the 36 genera in fecal samples from 35 healthy persons and 43 patients with fatty liver in the validation population, and selected the 36 microbial species markers based on the enrichment of each species in the healthy and disease groups, and the DNA extraction, sequencing, and species abundance analyses of the validation population were performed with reference to example 1.
The verification results are as follows: the above 24 species enriched in healthy people, 14 in the validation set, are validated with high quality (P _ values <0.05), and the mean and P value results of the validation of the microorganism species markers enriched in healthy people are shown in table 2, wherein bacteroides exist in human intestinal tract, and mainly help to decompose food to provide nutrition and energy required by human body.
Table 2:
Figure RE-GDA0003534473060000151
for the 12 species enriched in fatty liver patients described above, 6 of them were high quality validated in the validation set (p-value <0.05), and the cases of p-value and q-value validated for 2 microbial markers enriched in fatty liver patients in the validation set data are shown in table 3.
Dysfunction of the intestinal epithelial barrier and gut microbiota may be associated with the pathogenesis of liver cancer, with a significantly higher number of amplicon sequence variations in tumor-associated microbiota than in non-tumor regions of the liver, including bacteria of the genus Lachnospiraceae (Lachnospiraceae _ bacterium), and studies have shown that Lachnospiraceae is most abundant at the family level in the metabolism-associated fatty liver group (MAFLD).
Table 3:
Figure RE-GDA0003534473060000152
the inventor believes that the 14 microbial species markers enriched from healthy people can be used as a reverse index for fatty liver disease, or used as a microbial preparation medicinal flora component for treating fatty liver, or used as a recovery index for detecting fatty liver and monitoring the treatment process of fatty liver; the 6 microbial species markers enriched in fatty liver patients are used as positive indexes of fatty liver disease, and are particularly used for non-invasive detection and diagnosis of fatty liver disease.
The inventor utilizes the 20 microbial species markers to construct a comprehensive index, estimates the area AUC under the ROC (Receiver-operating characteristic) curve, and the larger the AUC is, the higher the diagnostic capability is, and evaluates the diagnostic capability of the comprehensive score corresponding to the fatty liver. By evaluating 78 samples in the first stage (the first stage) and 78 samples in the second stage (the second stage), as shown in fig. 2, the diagnosis ability is very good, the AUC obtained in the first stage is 88%, as shown in fig. 2-a, and the confidence interval is 79.9% -96.18%; the second phase gave an AUC of 97.5% with a confidence interval of 94.83% to 100% as shown in figure 2-B.
Example 3 detection of Individual State
In this example, the inventors performed the detection of the individual status of the sample source using 37 stool samples.
The abundances of Streptococcus paracasei (Streptococcus _ paraanguinis), Streptococcus salivarius (Streptococcus _ salivariaus), Eubacterium _ recipient, Coprococcus chaperone (Coprococcus _ comes), lachnospiriceae (Lachnospiraceae _ bacterium) _5163FAA), and Roseburia (Roseburia _ hominis) shown in table 3 in each stool sample were determined with reference to the method of example 2, whether the 6 strains in each sample were abundantly determined to fall within a confidence interval of 95% of the abundance of each of the disease control group or the healthy control group, the status of the individual corresponding to the sample in which the 6 strains were abundantly determined to fall within the corresponding interval of the disease group was determined to be a fatty liver patient, and the status of the individual corresponding to the sample in which the 6 strains were abundantly determined to fall within the corresponding interval of the healthy group was determined to be a non-hepatic fatty liver fatty patient.
The result shows that the method of the embodiment can be used for judging the individual state of 26 samples, and the judgment of the individual state corresponding to 22 samples in the 26 samples is consistent with the recorded state of the individual from which the sample is derived.
In addition, the inventors found that the combined detection of the species in table 2 and table 3, for example, the detection of the enriched species markers in table 3, and the non-enriched species markers in table 2, can more accurately determine and find the fatty liver patients or susceptible people.
In the scheme of treating fatty liver by using the marker, the inventor finds that the growth of the species marker in the step 3 is inhibited or eliminated, and the species marker in the step 2 is enriched, so that the treatment effect is excellent.
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: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophaeus (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceus (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sarmentosum (Alisterii _ shahii), Exiguobacterium sp. AP11, Geellaria twins (Gemelalla _ unclarified), Aneudiobolus vulgaris (Subdariella vularia _ variella), Exigus williamella (Veillonella _ vestris) and Suaedella terrestris (Subdariella terrestris).
2. The kit of claim 1, further comprising reagents suitable for detecting at least one species in a second collection of microorganisms consisting of: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis).
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 an agent suitable for detecting at least one species in a first collection of microorganisms for diagnosing fatty liver or for detecting the therapeutic effect of fatty liver, for the preparation of a kit, said first collection consisting of the following species: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophaeus (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceus (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sarmentosum (Alisterii _ shahii), Exiguobacterium sp. AP11, Geellaria twins (Gemelalla _ unclarified), Aneudiobolus vulgaris (Subdariella vularia _ variella), Exigus williamella (Veillonella _ vestris) and Suaedella terrestris (Subdariella terrestris).
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: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis).
7. A pharmaceutical or food composition for the prevention or treatment of fatty liver, comprising at least one species of a first microbial population consisting of: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophaeus (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceus (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sarmentosum (Alisterii _ shahii), Exiguobacterium sp. AP11, Geellaria twins (Gemelalla _ unclarified), Aneudiobolus vulgaris (Subdariella vularia _ variella), Exigus williamella (Veillonella _ vestris) and Suaedella terrestris (Subdariella terrestris).
8. A method of determining whether an individual has fatty liver, 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 fatty liver;
wherein the first microbial collection consists of the following species: rosemonobacterium aminopeptone (Rothia _ mucor), Atopobium atrophicum (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacillus _ ph8), Microbacterium viscidum (Odobacter _ splanchnicus), Arthrobacter pterus (Alisteries _ finegoldii), Arthrobacter Odonnaeus (Alisteries _ ondendokii), Arthrobacter putida (Alisteries _ pureinis), Arthrobacter sakesii (Alisteries _ shahii), Arthrobacter allobacter sp _ AP11, Micrococcus twins (Gemelassella _ unclarified), Annormally cocci; (Subdoligranum _ variabilie), Veillonella dispar (Veillonella _ dispar) and Salmonella lavandustria (Sutterella _ wadsworthensis);
the second microbial set consists of the following species: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis).
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 fatty liver, 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 resulting abundance with a predetermined threshold value in order to determine whether the individual has fatty liver;
wherein the first microbial collection consists of the following species: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophicum (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceum (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sakesii (Alisteri _ shahii), Exiguobacterium sp. AP11, Micrococcus bacterium twins (Gemelassella _ unclarified), Exiguella vulgaris (Subdellulirus _ variella), Exigus williamsii Veillella (Veillonella _ and Thermus terrestra (Suydans);
the second microbial set consists of the following species: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis).
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 treating or preventing fatty liver, 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 fatty liver:
(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: rosemophilus aminolytica (Rothia _ mucor), Atopobium atrophicum (Atopobium _ parvulum), Bacteroides ovatus (bacteriodes _ ovatus), Bacteroides Bacteroides (Bacteroides _ bacterium _ ph8), Deuterobacter visceral putida (Odobacter _ splanchnicus), Exiguobacterium fingoldii (Alisterides _ finegoldii), Exiguobacterium anserinaceum (Alisterides _ ondokoukii), Exiguobacterium destructor (Alisterides _ pureinnis), Exiguobacterium sakesii (Alisteri _ shahii), Exiguobacterium sp. AP11, Micrococcus bacterium twins (Gemelassella _ unclarified), Exiguella vulgaris (Subdellulirus _ variella), Exigus williamsii Veillella (Veillonella _ and Thermus terrestra (Suydans);
the second microbial set consists of the following species: streptococcus paracasei (Streptococcus _ paraanguitis), Streptococcus salivarius (Streptococcus _ salivarius), Eubacterium _ rectangle, Coprococcus (Coprococcus _ comes), Lachnospiraceae bacteria (Lachnospiraceae _ bacteria) _5163FAA, and Roseburia hominis (Roseburia _ hominis).
CN202111411877.6A 2021-11-25 2021-11-25 Fatty liver marker microorganism and application thereof Pending CN114381534A (en)

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