METHOD AND KIT FOR THE DETECTION OF CHRISTENSENELLA MINUTA

Description

Technical field

[0001] The invention relates to a method in vitro to detect or diagnose the presence of at least one bacterium of the species Christensenella minuta in a subject, by detecting at least a portion of a specific sequence of the reference genome Christensenella minuta. The present invention also relates to methods and kits for identifying and monitoring the development of C . minute in a subject. State of the art

[0002] Our intestinal microbiota or "intestinal flora" is made up of a set of non-pathogenic bacteria, viruses, parasites and fungi, or 10 12< to 10 14< microorganisms present in the digestive tract of each individual, which represents 2 to 10 times more than the number of cells present in our body.

[0003] In 2012, a microbiologist identified and cultivated a new species from human feces : Christensenella minuta,belonging to the gram-negative Clostridiales. (Morotomi et al., Description of Christensenella minuta gen. nov., sp. nov., isolated from human faeces, which forms a distinct branch in the order Clostridiales, and proposal of Christensenellaceae fam. nov., International Journal of Systematic and Evolutionary Microbiology (2012), 62, 144-149).

[0004] In 2014, Goodrich et al. identified Christensenella minuta as the most heritable bacterial taxon in humans and also suggested its therapeutic potential.

[0005] To date, it is accepted by the scientific community that the human intestinal microbiota is linked to the appearance of certain so-called "non-transmissible" pathologies, which defines a dysbiosis of the microbiota.

[0006] Microbial ecology has been increasingly studied in recent years as a regulator of metabolic functions and to identify its central role in establishing a healthy ecosystem, in symbiosis with its host.

[0007] Some bacteria of the genus Christensenellaceae have thus been associated with low body mass index in many human cohorts and other studies have suggested a protective role in regulating inflammation.

[0008] An absence or deficiency of bacteria of the genus Christensenellaceaewould be involved in particular in obesity, metabolic diseases, heart and vascular diseases, liver and bile duct diseases, kidney diseases, joint diseases linked to overweight, cancers in particular cancers linked to metabolism and / or dysbiosis of the microbiota, autoimmune diseases, atopic dermatological diseases, chronic inflammatory bowel diseases, pneumonia, infectious diarrhea, food allergies, inflammatory nephrology and neurological diseases including degenerative diseases or neuropsychiatric diseases such as anxiety-related disorders or eating disorders (e.g. bulimia).

[0009] The detection and identification of a germ of bacterial origin is traditionally carried out by so-called conventional methods, such as the culture of biological samples, microscopic examination after staining, blood culture, detection of bacterial antigens or detection by mass spectrometry such as MALDI-TOF. Although these methods are mainly used routinely in clinics and allow the identification of the bacterial germ, they require a culture of at least 24 to 48 hours of pure bacterial culture, that is to say a culture comprising a single germ. However, this is only possible if the bacteria is cultivable and if the sample does not contain a large number of distinct germs. Thus, in certain cases, a faster diagnosis, independent of culture conditions and from a more complex sample (presence of several germs) is required.

[0010] Molecular diagnosis also allows the identification of a bacterial germ and is based on the extraction and amplification of a specific gene fragment and then its detection, in particular a 16S DNA fragment or a ribosomal DNA fragment. Molecular techniques are known to be very sensitive and specific but require quality extraction in order to reduce the risks of contamination reducing the quality of the amplification. In addition, its cost is still high and they require that the fragment of interest be accessible and specific to the bacterial germ of interest. However, many species of bacteria have a very similar 16S DNA which overestimates the presence of said bacteria in a sample, such as bacteria of the species C . minute.Vasana Jinatham et al. (2018) and Zou Yuanqiang et al. (2020) describe a molecular method for identifying C. minuta based on the extraction and amplification of a 16S DNA gene fragment.

[0011] Arnold Jason et al. (2018) and Chandran Archana et al. (2013) describe methods for amplification of the BSH gene by RT-qPCR to detect different strains of probiotic bacteria.

[0012] There is therefore a clinical need for a new method for identifying bacteria of the species C . minute which is simple, effective, specific, fast, sensitive and economical. This is the objective of the present invention. Summary of the invention

[0013] To meet this objective, the invention proposes a method in vitro to detect the presence of at least one bacterium Christensenella minutain a sample through the detection of a specific sequence located between the 1,921,147 pdb and 2,014,152 pdb limits of the reference genome C . minute deposited under GenBank number CP029256.1.

[0014] For the purposes of the invention, “base pair” or “base pair” means the pairing of two nucleic bases located on two complementary strands of DNA or RNA.

[0015] Of the entire reference genome comprising nearly 3 million pdb, the inventors were able to identify a specific portion located between the limits 1,921,147 pdb and 2,014,152 pdb. This portion is particularly suitable for specifically identifying, detecting and diagnosing the presence of at least one bacterium of the species C . minute. This is the object of the present invention.

[0016] Thus, the invention relates to a method in vitro to detect the presence of at least one bacterium Christensenella minutain a sample, said method comprising the detection of the sequence SEQ ID NO: 1 located between the limits 1,921,147 pdb and 2,014,152 pdb of the reference genome C . minute deposited under GenBank number CP029256.1. Such a portion also avoids overestimating the abundance of the species C . minute in the target sample. Preferably, said method comprises the detection of at least 70 bpd of the sequence SEQ ID NO: 1.

[0017] Advantageously, the sequence of interest that one seeks to detect is located in the Bile Salt Hydrolase (BSH) gene located between the terminals 1,933,575 pdb and 1,934,552 pdb of SEQ ID NO: 2 of said reference genome.

[0018] In another aspect, the invention relates to a method for detecting / diagnosing a disease or a subject susceptible to developing a disorder such as a disease and / or dysfunction and / or imbalance, said disorder perhaps, for example, intestinal dysbiosis, obesity or an inflammatory disease.

[0019] The invention also relates to a method for monitoring the evolution of the quantity of C . minute in a subject, that is to say the evolution of the quantity of C . minute in the intestinal microbiota of a subject, said method comprising: (a) the implementation of a method to determine and quantify the presence of C . minute according to the invention, in a biological sample of said subject, at a time (t0) b) implementing a method for determining and quantifying the presence of C . minuteaccording to the invention, in a biological sample of said subject, at a time (t1), and c) comparing the quantity measured in a) with the quantity measured in b).

[0020] The invention also relates to a method for determining or adapting a therapeutic or dietary regime intended to prevent or combat intestinal dysbiosis in C. minute and / or a deficiency in C . minute in a subject, said method comprising: (a) the implementation of a method for determining a level or quantity of C . minute according to the invention, in a biological sample of said subject, and b) comparing it with a level or quantity of C . minute, in a biological sample of said subject after administration of a treatment to the subject, c) adapting / modifying the therapeutic or dietary regime of said subject on the basis of the comparison of steps a) and b).

[0021] Finally, the present application relates to specific primer pairs corresponding to the primers of SEQ ID NO: 6 and SEQ ID NO: 7 and / or the primers of SEQ ID NO: 8 and SEQ ID NO: 9 and / or the primers of SEQ ID NO: 10 and SEQ ID NO: 11 making it possible to target SEQ ID NO: 2. Preferably, said primer pairs make it possible to target respectively the sequences SEQ ID NO: 3 and SEQ ID NO: 4 and SEQ ID NO: 5 of the BSH gene.

[0022] The invention also relates to a kit comprising at least one pair of primers chosen from the pairs of primers cited above and optionally at least one probe corresponding to SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 15. Such a kit is capable of implementing one of the methods according to the invention.

[0023] Other characteristics and advantages will emerge from the detailed description of the invention, the examples and the figures which follow. Brief description of the Figures

[0024] There Figure 1 is a circular representation of the genome of Christensenella minuta DSM 22607 including the location of a conserved portion among the species Christensenella minuta. There Figure 2A represents the results of qPCR (Syber) amplification of the BSH gene, in particular the standard range for the primer pairs of SEQ ID NO: 6 and SEQ ID NO: 7. The Figure 2B represents the results of qPCR (Syber) amplification of the BSH gene, in particular the melting curve for the primer pairs of SEQ ID NO: 6 and SEQ ID NO: 7. The Figure 2C represents the results of qPCR (Syber) amplification of the BSH gene, in particular the standard range for the primer pairs of SEQ ID NO: 8 and SEQ ID NO: 9. The 2D figures represents the results of qPCR (Syber) amplification of the BSH gene, in particular the melting curve for the primer pairs of SEQ ID NO: 8 and SEQ ID NO: 9. The Figure 3 represents the results of qPCR amplification (Syber) of the bacteriaC . minute 1 in CFU / g of fecal matter in samples enriched or not with a quantity of C. minute 1 reference for the primer pairs of SEQ ID NO: 6 and SEQ ID NO: 7 ( Figure 3A ) and for the primer pairs of SEQ ID NO: 8 and SEQ ID NO: 9 ( Figure 3B ). Detailed description of the invention Definition

[0025] For the purposes of the invention, the term "subject" means a mammal, preferably a human or an animal. The human subject may be healthy, i.e. having no symptoms of intestinal dysbiosis or deficiency in C . minute, or may be likely to develop or suspect that they have intestinal dysbiosis or deficiency C . minute.

[0026] For the purposes of the invention, the term "biological sample" means samples likely to contain bacteria, in particular bacteria of the species C . minute,for example, samples of feces, stool, colon biopsy or colon effluent.

[0027] By "evolution of the quantity of C . minute " within the meaning of the invention, we mean an increase or a decrease in the quantity of C . minute in a sample, preferably a biological sample.

[0028] For the purposes of the invention, the term "primers" means isolated nucleic acid molecules that can hybridize or specifically hybridize to 5' or 3' regions of a complementary target sequence. In general, they are about 10 to 30 nucleotides in length and hybridize to both ends of a region containing about 50 to 200 nucleotides, preferably 70 nucleotides. Under appropriate conditions and with appropriate reagents, these primers allow the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. Since they must be used in pairs, they are often referred to as a "primer pair" or "primer set" (e.g., SEQ ID NO: 6-7; SEQ ID NO: 8-9, SEQ ID NO: 10-11).

[0029] For the purposes of the invention, the term "probes" means molecules capable of specifically hybridizing to a sequence of interest (for example, of SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5). They are useful for detecting the presence of said sequence of interest in biological samples. These probes may comprise at least one non-natural nucleotide, for example a peptide nucleic acid (PNA), a peptide nucleic acid having a phosphate group (PHONA), a bridged nucleic acid or a locked nucleic acid (BNA or LNA), and a morpholino nucleic acid. Unnaturally occurring nucleotides also include chemically modified nucleic acids or nucleic acid analogs such as methylphosphonate DNA or RNA, phosphorothioate DNA or RNA, phosphoramidate DNA or RNA, and 2'-O-methyl-DNA or RNA. Method

[0030] The present invention therefore relates to a method in vitroto detect the presence of at least one bacterium in a sample Christensenella minuta, said method comprising the detection of at least 70 pdb of the sequence SEQ ID NO: 1 located between the limits 1,921,147 pdb and 2,014,152 pdb of the reference genome C . minute deposited under GenBank number CP029256.1.

[0031] The reference strain of Christensenella minuta has been deposited at the Leibniz Institute DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, in French German Collection of Microorganisms and Cell Cultures GmbH) under number DSM 22607 and its genome is accessible under number CP029256.1 in GenBank.

[0032] The genome was visualized using SnapGene Viewer (version 5.2). The start of the genome was thus indicated as 0, the position indicated as 0 being the position of the first base of the sequence of the FASTA file of the genome. The gene coding for 16S ribosomal RNA 1 is located between positions 468,243 and 468,318 (locus tag B1H56_02250) and the gene coding for 16S ribosomal RNA 2 is located between positions 1,735,562 and 1,737,097 (locus tag B1H56_08210) were also indicated. The position of the conserved DNA portion is between nucleotides 1,921,147 and 2,014,152 (cf. Figure 1 ).

[0033] This portion of DNA from the genome Christensenella minutaDSM22607 has been determined, through bioinformatics analyses, to be particularly conserved in a number of bacterial strains of this species. The results that will be presented below demonstrate its interest for the purposes of identification, detection, diagnosis and quantification of this bacterium in complex samples, using techniques such as PCR amplification of a fragment of this portion.

[0034] A particularly suitable sequence in the context of the present invention is the sequence SEQ ID NO: 2. It is located between the limits 1,933,575 pdb and 1,934,552 pdb corresponding to the BSH gene and is present in the conserved sequence of SEQ ID NO: 1. According to one embodiment, the method according to the invention therefore comprises the detection of at least 70 pdb of the sequence SEQ ID NO: 2. According to a variant, it may be the detection of the entire sequence SEQ ID NO: 2.

[0035] The BSH gene encodes a protein involved in the deconjugation of bile acids. This gene is only present in certain prokaryotic bacteria. The BSH gene of Christensenella minuta is unique both in its nucleotide sequence and in the amino acid sequence for which it codes. It is located between the 1,933,575 bpd and 1,934,552 bpd boundaries of the reference genome C . minute deposited under GenBank number CP029256.1. This gene has the GenBank identifier AYH40638.1. The sequence of SEQ ID NO: 2 gives very good amplification performance for the BSH gene present in all bacteria belonging to the species Christensenella minuta. [Table 1] SEQ ID NO: 2

[0036] Particularly preferably, a specific sequence of interest of C . minuteis SEQ ID NO: 3 and / or SEQ ID NO: 4 and / or SEQ ID NO: 5. These sequences are in fact, according to the invention, particularly suitable for identifying a bacterium belonging to the species C . minute.

[0037] Thus, according to a particularly preferred embodiment, the method according to the invention aims to detect the presence in a sample of at least one bacterium Christensenella minuta, said method preferably comprising the detection of the sequence SEQ ID NO: 3 and / or SEQ ID NO: 4 and / or SEQ ID NO: 5.

[0038] Table 2 below describes the said sequences of interest. [Table 2] SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5

[0039] In the context of the invention, the sample in which the presence of C. minuteis preferably a biological sample from a subject. Alternatively, the sample may also be a sample of soil, sediment, food, water, a drug, or probiotics. A sample of soil, sediment, food, or water can thus be used to test for possible contamination. Testing for the presence of bacteria in a drug or probiotics is also of interest for quality control purposes.

[0040] When the sample is a biological sample, the sample is preferentially chosen from feces, stools, colon biopsies and colon effluents of a subject.

[0041] In the context of the invention, the subject may be a human or a mammal, preferably an animal. Thus, the method according to the invention is of interest for human and / or veterinary medicine.

[0042] The method therefore aims to detect a specific sequence belonging to a bacterium C. minute and thus identify the presence of such a bacterium in a given sample, whether for diagnostic purposes, to check the contamination of a sample such as an agri-food product or for quality control purposes, for example of a drug or a probiotic.

[0043] Various techniques are well known to those skilled in the art and they will be able to determine the most suitable technique for detecting the sequence of interest according to the invention in a given sample.

[0044] Preferably, the detection of the sequence of interest is determined by a molecular amplification method such as PCR and its derivatives and / or a sequencing method such as NGS.

[0045] When it comes to a molecular amplification method, it can be a PCR ( Polymerase Chain Reactionin English or Polymerase Chain Reaction in French) or its derivatives.

[0046] For the purposes of the invention, derivative PCR means a variant of conventional PCR. It may therefore be a so-called multiplex PCR, a meta-PCR, a nested PCR, an asymmetric PCR, a quantitative PCR (qPCR) or a digital PCR in micro-compartments (dPCR).

[0047] Alternatively, the detection of the sequence of interest can also be determined by sequencing such as Next Generation Sequencing or NGS.

[0048] Preferably, the detection of the sequence of interest is determined by PCR or qPCR or dPCR.

[0049] According to a particularly preferred subject, the detection of the sequence is determined by PCR using the primers of SEQ ID NO: 6 and SEQ ID NO: 7 and / or the primers of SEQ ID NO: 8 and SEQ ID NO: 9 and / or the primers of SEQ ID NO: 10 and SEQ ID NO: 11.

[0050] When the primer pair is of SEQ ID NO: 6 and SEQ ID NO: 7, it is capable of amplifying the sequence of interest SEQ ID NO: 1 or SEQ ID NO: 2, preferably the sequence of interest SEQ ID NO: 3.

[0051] When the primer pair is of SEQ ID NO: 8 and SEQ ID NO: 9, it is capable of amplifying the sequence of interest SEQ ID NO: 1 or SEQ ID NO: 2, preferably the sequence of interest SEQ ID NO: 4.

[0052] When the primer pair is of SEQ ID NO: 10 and SEQ ID NO: 11, it is capable of amplifying the sequence of interest SEQ ID NO: 1 or SEQ ID NO: 2, preferably the sequence of interest SEQ ID NO: 5.

[0053] When the detection of the sequence of interest is determined by qPCR, the method also includes probes of SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14.

[0054] Thus, the detection of the sequence of interest is determined by qPCR using the primer pairs of SEQ ID NO: 6 and SEQ ID NO: 7 and the probe of SEQ ID NO: 12 and / or the primer pairs of SEQ ID NO: 8 and SEQ ID NO: 9 and the probe of SEQ ID NO: 13 and / or the primer pairs of SEQ ID NO: 10 and SEQ ID NO: 11 and the probe of SEQ ID NO: 14.

[0055] Table 3 below describes the sequences of the primers and probes according to the invention. [Table 3] Name Amorce 5'>3' sequence SEQ ID NO: 6 CTCGGTCAATGTGCAAGTGT SEQ ID NO: 7 GGAATCTCGCTGGTCAGAA SEQ ID NO: 12 AGTGAAGAGCTGCCGCTTTCTCCATT SEQ ID NO: 8 AAGAACCCCCTGCAAATACA SEQ ID NO: 9 AACGCTGCTTTCACGAAATCT SEQ ID NO: 13 ACTGTTCAAGTTCCAATTCGGCAGCAA SEQ ID NO: 10 TGCTCCCTTTGAATTTATTCC SEQ ID NO: 11 CCAAGTTAATCTGTTCTAAGAGA SEQ ID NO: 14 CACACTTGCACATTGACCGAGTATCCA

[0056] Alternatively, the detection and quantification of the bacteria C. minutecan also be carried out by an antibody or an aptamer specifically recognizing a portion present in the sequence SEQ ID NO: 1 and / or SEQ ID NO: 2 and / or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5.

[0057] According to another object, the method of the invention comprises a step of quantifying the C bacteria. minute detected in the sample. Quantification of C. bacteria. minute present in a sample can allow the method according to the invention to be used for diagnostic purposes, patient monitoring, treatment monitoring, adapting a therapeutic and / or dietary regime.

[0058] When the method is used for diagnostic purposes, it makes it possible to identify a subject suffering from a disease or imbalance or a subject likely to develop a disease or imbalance.

[0059] Thus, the invention also relates to a method according to the invention for detecting / determining, preferably intestinal dysbiosis in C . minute and / or a deficiency of C . minute in a subject. Said subject may be healthy or sick.

[0060] Preferably, intestinal dysbiosis in C . minute and / or the lack of C . minute is detected / determined in a subject, when the quantity of bacteria C . minute detected is less than 1%, preferably 0.4%, of the microbiota of said subject. The percentage is understood as the number of bacteria in relation to the total number of bacteria detected. 1% is equivalent to 10 9< CFU of C. minute per gram of feces, the total number of bacteria admitted being in fact 10 11< CFU / g of feces. Also, one embodiment of the invention is a method in which the quantity of bacteria C . minutedetected is less than 10 9< CFU / g of feces of said subject.

[0061] The method according to the invention is preferably capable of detecting / determining a disease or a subject likely to develop a disease, said disease being preferably chosen from: Metabolic diseases, selected from non-insulin-dependent diabetes, gestational diabetes, NASH, hepatic steatosis, pancreatic steatosis, hyperlipidemia, hypercholesterolemia, infertility related to overweight, urinary incontinence related to overweight, Other chronic metabolic diseases, including thyroiditis, cardiac and vascular diseases, selected from atherosclerosis, thrombopathies, acute pericarditis and chronic constrictive pericarditis, arterial hypertension, vasculitis, liver and bile duct diseases, selected from hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, cirrhosis, hepatic encephalopathy, gallstones, joint diseases related to overweight, selected from osteopenia, osteoporosis, osteoarthritis, disc inflammation spinal neurodegenerative diseases, chosen from Alzheimer's disease, Parkinson's disease,motor neuron diseases such as amyotrophic lateral sclerosis, primary lateral sclerosis and Kennedy's disease cancers linked to metabolism and / or dysbiosis of the microbiota, chosen from hepatocarcinomas, cancers of the digestive tract such as esophagus, stomach and colorectal cancer, pancreatic carcinoma, neuroendocrine tumors (NETs) of the gastroenteropancreatic system, liver tumors, tumors of the gallbladder and biliary tract, kidney tumors, glioblastomas, lymphomas, multiple myelomas, chronic myeloid leukemia, chronic myeloproliferative diseases, lung carcinomas autoimmune diseases, chosen from insulin-dependent diabetes, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, lupus disseminated erythematosus, polyendocrine autoimmune syndrome, atopic dermatological diseases, chosen from eczema,psoriasis chronic inflammatory bowel diseases, selected from Crohn's disease, ulcerative colitis, diverticulitis, esophagitis, gastritis, pancreatitis, peptic ulcer, irritable bowel syndrome respiratory disorders, selected from asthma, cystic fibrosis, chronic obstructive pulmonary disease and chronic bronchitis, interstitial lung disease and pulmonary fibrosis, sleep apnea syndrome (SAOS) pneumonia, selected from infectious pneumonia, influenza pneumonia and avian influenza, severe acute respiratory syndrome (SARS), pneumocystis pneumonia infectious diarrhea, selected from Clostridium difficile infection, EHEC infection, salmonella gastroenteritis, campylobacter enteritis, foodborne infections by enterotoxin-producing bacteria, cholera, yersiniosis, shigellosis,cryptosporidiosis, listeriosis Food allergies, chosen from celiac disease, lactose intolerance, bile salt malabsorption syndrome Inflammatory nephrologies or those linked to microbiota dysbiosis, chosen from urethritis, chronic renal failure, urolithiasis Other inflammatory disorders, chosen from multiple sclerosis, lymphangitis Neurological diseases linked to microbiota dysbiosis, chosen from anorexia, bulimia, depression, bipolar syndrome, autism, schizophrenia, Tourette's syndrome.

[0062] According to another object, the invention relates to a method for monitoring or following the evolution of the quantity of C . minute in a subject, that is to say the evolution of the quantity of C. minuta in the microbiota of a subject, said method comprising: (a) implementing a method according to any of the preceding embodiments for determining and quantifying the presence of C . minutes, in a biological sample from said subject, at a time (t0), b) implementing a method according to any one of the preceding embodiments for determining and quantifying the presence of C. minuta, in a biological sample of said subject, at a time (t1), and c) comparing the quantity measured in a) with the quantity measured in b).

[0063] The subject may be suffering from a disease or imbalance such as intestinal dysbiosis and may be likely to develop such a disease or imbalance.

[0064] According to another object, the invention relates to a method for determining or adapting a therapeutic or dietary regime intended to prevent or combat an intestinal disease or dysbiosis in C. minutain a subject, said disease being chosen from one of the diseases previously cited and said method comprising: (a) implementing a method according to any of the preceding embodiments for determining a level or quantity of C. minuta, in a biological sample of said subject, and (b) comparing it with a level or quantity of C. minuta, in a biological sample of said subject after administration of a treatment to the subject, c) adapting / modifying the therapeutic or dietary regime of said subject on the basis of the comparison of steps a) and b).

[0065] Preferably, the treatment administered to the subject may be drug treatment, surgical intervention, or diet.

[0066] In particular, said treatment is effective if the level or quantity of C. minutes,in a biological sample of said subject is greater or increased compared to the level or quantity of C . minute determined before treatment. Primers, probes and kit

[0067] According to another aspect, the present application also relates to primers suitable for use in the present invention.

[0068] Thus, the primers, in particular the primer pairs, are capable of specifically targeting and amplifying one of the sequences chosen from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, preferably the sequence SEQ ID NO: 3 of the BSH gene and / or the sequence SEQ ID NO: 4 of the BSH gene and / or the sequence SEQ ID NO: 5 of the BSH gene. Preferably, the primer pairs consist of the primers of SEQ ID NO: 6 and SEQ ID NO: 7 and / or the primers of SEQ ID NO: 8 and SEQ ID NO: 9 and / or the primers of SEQ ID NO: 10 and SEQ ID NO: 11.

[0069] When the primer pair of SEQ ID NO: 6 and SEQ ID NO: 7 is used, it targets and amplifies the sequence SEQ ID NO: 3 of the BSH gene. When the primer pair of SEQ ID NO: 8 and SEQ ID NO: 9 is used, it targets and amplifies the sequence SEQ ID NO: 4 of the BSH gene. When the primer pair of SEQ ID NO: 10 and SEQ ID NO: 11 is used, it targets and amplifies the sequence SEQ ID NO: 5 of the BSH gene.

[0070] Thus, the kit comprises at least two primers specifically targeting the sequence SEQ ID NO: 3 of the BSH gene and / or the sequence SEQ ID NO: 4 and / or the sequence SEQ ID NO: 5 of the BSH gene, said kit comprising the primers of SEQ ID NO: 6 and SEQ ID NO: 7 and / or primers of SEQ ID NO: 8 and SEQ ID NO: 9 and / or primers of SEQ ID NO: 10 and SEQ ID NO: 11.

[0071] According to another subject, the invention relates to a kit comprising at least one pair of primers (or two primers), said pair(s) of primers is chosen from the pair of primers of SEQ ID NO: 6 and SEQ ID NO: 7, the pair of primers of SEQ ID NO: 8 and SEQ ID NO: 9, the pair of primers of SEQ ID NO: 10 and SEQ ID NO: 11 and their mixture.

[0072] Preferably, the kit may also comprise at least one probe specifically targeting the sequence SEQ ID NO: 3 of the BSH gene and / or the sequence SEQ ID NO: 4 of the BSH gene and / or the sequence SEQ ID NO: 5 of the BSH gene, said kit comprising at least one probe chosen from the probe of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0073] When the probe is of SEQ ID NO: 12, it is associated with the primers of sequence SEQ ID NO: 6 and SEQ ID NO: 7. When the probe is of SEQ ID NO: 13, it is associated with the primers of sequence SEQ ID NO: 8 and SEQ ID NO: 9. When the probe is of SEQ ID NO: 13, it is associated with the primers of sequence SEQ ID NO: 10 and SEQ ID NO: 11.

[0074] Finally, said kit according to the invention is suitable for implementing one of the methods according to the invention.

[0075] The invention is now illustrated by non-limiting examples of compositions according to the invention and by results. Examples Example 1

[0076] qPCR amplification test (Syber) of the BSH gene with the primers according to the invention (SEQ ID NO: 6 and SEQ ID NO: 7; and SEQ ID NO: 8 and SEQ ID NO: 9). Such a test makes it possible to demonstrate the efficiency of quantification on a sample comprising only strains C. minutaand on a sample comprising a complex mixture of different bacteria.

[0077] The standard ranges were performed on genomic DNA samples extracted from mouse feces and enriched with 10 9< Colony Forming Units (CFU) of Christensenella minuta 1.

[0078] The primers were used on genomic DNA extracted from mouse fecal samples (fed with a placebo). These samples were previously artificially enriched with 10 9< Colony Forming Units (CFU) of Christensenella minuta 1. This template was then serially diluted by factors of 10 to 10 million to obtain a standard range for each DNA primer pair and quantitative PCR reactions targeting our gene of interest were carried out ( Figure 2A And 2C). These standard ranges allow us to determine the linearity, efficiency, sensitivity and reproducibility of our test. Thus, remarkable efficiency (E) and confidence (R 2 < ) scores were obtained for the BSHqPCR2 and 3 primers on the standard ranges (respectively E = 97.3%; R 2 < = 0.999 and E = 98.7%; R 2 < = 0.994). The qPCR reactions were carried out with Applied BiosystemsTM Power SYBRTM Green PCR Master Mix on the CFX96 Touch Real-Time PCR Detection System instrument (Bio-Rad). The thermal cycler conditions were as follows: an initial denaturation of 3 minutes at 98°C followed by 40 cycles of 15 seconds at 98°C, and 15 seconds at 60°C. A melting curve was also performed to verify the presence of a single amplified PCR product ( Figure 2B And 2D ). Three technical replicates were performed for each condition. Example 2

[0079] qPCR amplification test (Syber) of the BSH gene with the primers according to the invention (SEQ ID NO: 6 and SEQ ID NO: 7; and SEQ ID NO: 8 and SEQ ID NO: 9). Such a test makes it possible to demonstrate the efficiency of quantification on a sample comprising only strains C. minuta and on a sample comprising a complex mixture of different bacteria.

[0080] Two genomic DNA samples extracted from mouse feces were then used to validate our method for strain detection and quantification. Christensenella minuta 1: a non-enriched negative control in C. minuta, and a reference extract previously enriched with a determined quantity of C. minuta ( Figure 3A and 3B ). Three technical replicates were performed for each condition. The quantity of C. minuta expressed in CFU / g of fecal matter could be determined using the standard range. Thus, the quantification of C . minute1 in the “enriched” sample C . minute 1" robustly reflects the expected quantity, represented by the sample " C. minuta 1 reference. The primer pairs of SEQ ID NO: 8 and SEQ ID NO: 9 demonstrate better results in the quantification of Christensenella minuta 1 within this complex matrix ( Figure 3B ). Indeed, the quantification delta between the expected number of CFU / g and the number found in our “enriched” sample C. minuta 1 » is more robust ( Figure 3B ).

Claims

1. An in vitro method for detecting the presence of at least one Christensenella minuta bacterium in a sample, said method comprising detecting at least 70 bp of the sequence SEQ ID NO: 2 located between the boundaries 1,933,575 bp and 1,934,552 bp corresponding to the BSH gene of the reference genome C. minuta filed under GenBank number CP029256.1.

2. The method according to the preceding claim, characterized in that the sample is a biological sample of a subject or a sample of soil or a medication or a probiotic.

3. The method according to the preceding claim, characterized in that the subject is a human or an animal.

4. The method according to the preceding claim, wherein said method comprises the detection of the sequence SEQ ID NO: 3 and / or SEQ ID NO: 4 and / or SEQ ID NO: 5.

5. The method according to one of claims 2 to 4, wherein the biological sample is a sample selected from feces, stools, colon biopsies and colon effluents of said subject.

6. The method according to one of the preceding claims, wherein the detection of the sequence is carried out by a molecular amplification method known as PCR and / or by a sequencing method.

7. The method according to one of the preceding claims, wherein the detection of the sequence is carried out by PCR using the primers of SEQ ID NO: 5 and SEQ ID NO: 6 and / or the primers of SEQ ID NO: 7 and SEQ ID NO: 8 and / or the primers of SEQ ID NO: 9 and SEQ ID NO: 10.

8. The method according to one of the preceding claims, wherein the method comprises a step of quantifying the C. minuta bacteria detected in the sample.

9. The method according to one of claims 2 to 8, for detecting C. minuta intestinal dysbiosis and / or a C. minuta deficiency in a subject.

10. The method according to the preceding claim, wherein the amount of C. minuta bacterium detected is less than 109 CFU / g of feces of said subject.

11. The method according to one of claims 9 or 10, for detecting a disease or a subject likely to develop a disease, said disease is selected from: - metabolic diseases, selected from non-insulin-dependent diabetes, gestational diabetes, NASH, hepatic steatosis, pancreatic steatosis, hyperlipidemia, hypercholesterolemia, infertility linked to excess weight, urinary incontinence linked to excess weight, - other chronic metabolic diseases, including thyroiditis, - cardiac and vascular diseases, selected from atherosclerosis, thrombopathies, acute pericarditis and chronic constrictive pericarditis, arterial hypertension, vasculitides - liver and bile duct diseases, selected from hepatitises, primary biliary cirrhosis, primary sclerosing cholangitis, cirrhosis, hepatic encephalopathy, vesicular lithiases - joint diseases linked to excess weight, selected from osteopenia, osteoporosis, osteoarthritis, vertebral disc inflammation - neurodegenerative diseases, selected from Alzheimer's disease, Parkinson's disease, motor neuron diseases such as amyotrophic lateral sclerosis, primitive lateral sclerosis and Kennedy disease - cancers linked to metabolism and / or to dysbiosis of the microbiota, selected from hepatocellular carcinomas, gastrointestinal tract cancers such as esophageal, stomach and colorectal cancer, pancreatic carcinoma, neuroendocrine tumors (NETs) of the gastrointestinal-pancreatic system, hepatic tumors, gallbladder and bile duct tumors, renal tumors, glioblastomas, lymphomas, multiple myelomas, chronic myeloid leukemia, chronic myeloproliferative diseases, lung carcinomas - autoimmune diseases, selected from insulin-dependent diabetes, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, systemic lupus erythematosus, autoimmune polyendocrine syndrome - atopic skin diseases, selected from eczema, psoriasis - chronic inflammatory bowel diseases, selected from Crohn's disease, hemorrhagic ulcerative colitis, diverticulitis, esophagitis, gastritis, pancreatitides, gastrointestinal ulcers, irritable bowel syndrome - respiratory function disorders, selected from asthma, cystic fibrosis, chronic obstructive pulmonary diseases and chronic bronchitis, interstitial lung diseases and pulmonary fibroses, sleep apnea syndrome (OSAS) - pneumonias, selected from infectious pneumonias, influenza pneumonia and avian flu, severe acute respiratory syndrome (SARS), pneumocystis pneumonia - infectious diarrhea, selected from Clostridium difficile infection, EHEC infection, salmonella gastroenteritis, campylobacter enteritis, food poisoning by enterotoxin-producing bacteria, cholera, yersiniosis, shigellosis, cryptosporidiosis, listeriosis - food allergies, selected from celiac disease, lactose intolerance, bile salt malabsorption syndrome - inflammatory kidney disorders or others related to dysbiosis of the microbiota, selected from urethritis, chronic renal failure, urolithiasis - other inflammatory disorders, selected from multiple sclerosis, lymphangitis - neurological diseases related to dysbiosis of the microbiota, selected from anorexia, bulimia, depression, bipolar syndrome, autism, schizophrenia, Tourette syndrome.

12. A method for monitoring the evolution of the amount of C. minuta in a subject, said method comprising: a) implementing a method according to one of the preceding claims to determine and quantify the presence of C. minuta, in a biological sample of said subject, at a time (t0) b) implementing a method according to one of the preceding claims to determine and quantify the presence of C. minuta, in a biological sample of said subject, at a time (t1), and c) comparing the amount measured in a) to the amount measured in b).

13. A method for determining or adapting a therapeutic or dietary regimen intended to prevent or combat C. minuta intestinal dysbiosis in a subject, said method comprising: a) implementing a method according to one of claims 1 to 11 to determine a level or an amount of C. minuta in a biological sample of said subject, and b) comparing it with a level or an amount of C. minuta in a biological sample of said subject after administering treatment to the subject, c) adapting / modifying the therapeutic or dietary regimen of said subject based on the comparison of steps a) and b).

14. A kit comprising at least two primers specifically targeting the sequence SEQ ID NO: 3 of the BSH gene and / or the sequence SEQ ID NO: 4 and / or the sequence SEQ ID NO: 5 of the BSH gene, said kit comprising the primers of SEQ ID NO: 6 and SEQ ID NO: 7 and / or primers of SEQ ID NO: 8 and SEQ ID NO: 9 and / or primers of SEQ ID NO: 10 and SEQ ID NO: 11.

15. The kit according to the preceding claim, also comprising at least one probe specifically targeting the sequence SEQ ID NO: 3 of the BSH gene or the sequence SEQ ID NO: 4 of the BSH gene or the sequence SEQ ID NO: 5 of the BSH gene, said kit comprising the probes of SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14.