FR2990954A1 - New Archaea Methanomassiliicoccus luminyensis isolated and established in culture, comprising 16S ribosomal DNA gene-specific sequence or a complementary sequence, useful for diagnostic purposes - Google Patents

Abstract

Archaea Methanomassiliicoccus luminyensisisolated and established in culture, comprising 16S ribosomal DNA (rDNA) gene-specific sequence of SEQ ID NO. 1 of the Sequence Listing or a complementary sequence, is new, where the sequence not defined here may be found at ftp://ftp.wipo.int/pub/published-pct-sequences/publication. Independent claims are included for: (1) the preparation of Archaea; (2) a method (I) of determining the presence of Archaea in a biological sample, preferably in a sample of human or animal feces, comprising extracting the DNA from the biological sample and detecting, preferably specifically quantifying Archaea DNA by enzymatic amplification of PCR type, preferably of real-time PCR with a pair of primers, preferably probe, sequences from the 16S rDNA sequence of SEQ ID NO: 1; and (3) a kit useful for the above method (I), comprising reagents for amplifying DNA comprising amplification primers and probe for detecting Archaea Methanomassiliicoccus luminyensis, including constituents of the oligonucleotides primers and probes derived from SEQ ID NO: 1, preferably SEQ ID NOs: 2 (5'-gttcgtagccggtctggtaa-3'), 3 (5'-ccaagtctggcagtctcctc-3') or 4 (5'-tcgggaaggttaactttccgacttcc-3'), preferably any specific primers of Archaea of sequence comprising SEQ ID NOs: 5 (5'-agccgccgcggtaayac-3') and 6 (5'-cyggcgttgavtccaatt-3'), and reagents for extracting DNA comprising at least one pulverulent abrasive product, preferably glass powder and a reagent of chemical and/or enzymatic lysis.

Description

The present invention relates to a novel culture medium and method for culturing methanogenic archaea. The Archaea are prokaryotes constituting one of the four areas of life alongside eukarya, bacteria and giant DNA viruses [16]. In particular, methanogenic Archaea (hereafter referred to as methanogens) are strict anaerobic archaea. Initially isolated from the environment and in particular the extreme environments that earned them their name extremophiles, Archaea were then detected in various invertebrate and vertebrate animals including birds and mammals. In mammals, it is the methanogens capable of detoxifying hydrogen with methane synthesis that have been essentially detected, and methanogens are therefore of considerable importance now since it is considered that the methane emission by the report cattle constitutes the source. destruction of the ozone layer in the environment. In humans as well, metagenomic analyzes have shown the presence of specific DNA signatures of several Archaea species that belong essentially to both phyla Euryarcheaota and halophilic Archaea; more particularly, the sequences detected in humans indicate the presence of Methanosarcina, Thermoplasma, Crenarchaeaota, and halophilic Archaea as well as a last order of methanogens [25-30].

Of all these Archaea, only four species of Archaea have been isolated from man: Methanobrevibacter srnithll [31], Methanosphaera stadtmaniae [32], Methanobrevibacter oralis [5] and Methanornassilicoccus / uminyensis and only M. srnithii, M. stadtmanae and M. / uminyensis were isolated from the gastrointestinal tract.

In humans, these Archaea methanogens participate in the homeostasis of the digestive flora by detoxifying hydrogen with methane production and thus participate in the regulation of pH.

Also, some Archaea are involved in oral pathology in polymicrobial infection during periodontal disease. It has been shown that the digestive carriage of Archaea methanogens was constant in all individuals [11].

Archaea are microorganisms of medical and veterinary interest given their role in physiology and possibly in pathology. The development of new laboratory techniques for strict anaerobic culture, including the Hungate technique [1, 2], was an important technological advance that allowed the isolation and characterization of several new methanogen species [38]. However, methanogens are fastidious microorganisms whose isolation and then culture require very specific conditions, in particular strict anaerobiosis [1, 2, 9]. Moreover, each methanogenic species requires the preparation of a specific culture medium which is extremely time consuming and is not compatible with the use of such media in routine laboratory. Finally, the growth of methogens is slow (more than 5 days). Knowledge of nutrient conditions is an important factor in the culture of methanogens [10]. The presence of an atmosphere consisting of 80% H2 and 20% CO2 is one of the many growth factors required to allow the optimal proliferation of these micro-organisms [11]. The growth of Methanobrevibacter smithii [7] and Methanobrevibacter oralis [5] require rumen fluid [10], possibly with acetate, branched-chain fatty acids [12] and coenzyme M (2-mercaptoethanesulfonic acid). ) [13]. Methanosphaera stadtrnanae [8] has the lowest energy metabolism among all human methanogens, they produce methane only by reducing methanol with H2 and depend on acetate as a carbon source [14]. The inventors have recently reported that a solution of selenite and / or tungstate was necessary for the growth of Methanornassilicoccus / uminyensis [4]. Other species require aromatic amino acids such as tryptophan, vitamin thiamine, pyridoxine, and p-aminobenzoic acid (PABA), branched chain fatty acids, acetate [15.16]. Methanogens also require a series of metal ions, especially nickel [17], which are cofactors present in the F430 carbon monoxide dehydrogenase [18.19]. The culture of these microorganisms is very tedious. Specifically, to isolate M. smithii, the authors [31] used a faecal sample inoculated in Balch medium 1 (composition shown in Table 2 below) [33] under an atmosphere of H2-0O2. (80:20) pressurized to 2 atmospheres (202.6 kPa) with stirring. After 2 to 3 days, 0.5 mL samples of the atmosphere were analyzed for the presence of methane by gas chromatography. Methane-producing and fluorescent isolates for factor-420 (as determined by epifluorescence microscopy) were subcultured on Balch medium 1 supplemented with 2% agar until pure culture was obtained. ; purity criteria were uniform cell morphology, methane production, factor-420 fluorescence of all cells, and absence of growth in a complex medium (SMS) containing substrateless carbohydrates for methanogenesis. To isolate M. stadtmanae, the authors (5) used an El culture medium (Table 3) modified by the addition of 30% (v / v) rumen fluid, clindamycin and cephalothin, respectively, of 0.4 % methanol and an atmosphere composed of N2, 80% and CO2, 20%. The culture was maintained by monthly transfers (10% vol.) In the same medium. A fluorescent spherical morphology by factor-420 fluorescence was the dominant morphotype. At day 60, the culture was subcultured in Balch's Modified Medium 1 by addition of rumen fluid, NH4Cl, cepthalothin and clindamycin (7) with methanol under an atmosphere of 80% H2: and CO2, 20%. A pure culture of a methanogenic Archaea has been obtained. To isolate M. oralis, the authors used substantially the same culture medium as that used to isolate M. smithii (9).

To isolate M. luminyensis, the inventors have recently reported that methanol supplementation of the culture medium was necessary for the growth of Archaea Methanomassilicoccus luminyensis [4]. But, these conditions were not sufficient to allow the deposit of the strain in a collection of deposit.

In the unpublished French patent application No. 11,56069, the inventors succeeded in cultivating the bacterium M. uminyensis by adding greater concentrations of methanol and a solution of selenite / tungstate, which made it possible to deposit the M. luminyensis strain in the DSMZ microorganism deposit collection (Germany) under the number DSM 24529. The object of the present invention was to provide a novel culture medium composition with the objective of developing the same and unique culture medium allowing (1) the cultivation of all methanogenic archaea, and (2) significantly reducing the cultivation time of archaea methanogens. To do this, the inventors have determined specific media and conditions for isolation and culture of said methanogenic Archaea for isolating and establishing in culture all methanogenic Archaea, including Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, Methanomassilficoccus luminyensis, Methanobacterium. beijingense and Methanosaeta concluded, Methanosarcina acetivorans. More specifically, the subject of the present invention is a sterilized culture medium useful for the cultivation of archaea comprising a methanogenic Archaea culture base medium, characterized in that it further comprises the following components: a-selenite salt at a concentration of 5.10-3 mM to 0.5 mM, preferably 5.10-2 to 0.5 mM, and b- tungstate salt at a concentration of 5.10-3 mM to 0.5 mM, preferably 5.10-2 to 0.5 mM, and c- molybdate salt at a concentration of 10-3 to 10-2 mM, nickel salt at a concentration of 10-3 to 10-2 mM, and e-methanol at a concentration of at least 10 mM, preferably from 10 to 100 mM. Preferably, said culture medium comprises a concentration of 10 to 50 mM of methanol. More particularly, the medium according to the present invention comprises the following components: a-Selenite salt Na 2 SiO 3, at a concentration of 0.001 mg / l to 0.1 mg / l, preferably 0.01 to 0.1 mg / l, more preferably 0.015 mg / l, more preferably in the form of hydrate Na2Se03.5H20, and b- tungstate salt Na2W04, at a concentration of 0.001 mg / l to 0.1 mg / l, more preferably 0.02 mg / l, more preferably in the form of hydrate Na2W04.2H20, c- molybdate salt Na2MoO4, at a concentration of 0.01 to 0.1 mg / l, preferably 0.02 mg / l, preferably still in the form of Na2MoO4.2H2O hydrate, and NiCl2 salt at a concentration of 0.01 to 0.1 mg / l, preferably 0.7 mg / l, preferably in the form of hydrate NiCl2. 6:20.

According to another particular characteristic, the culture medium according to the invention further comprises the following component: formate salt, at a molar concentration of 1 mM to 10 mM, preferably 6 mM, more particularly NaHCO 2 at a concentration of 0.1 to 1 g / l, preferably 0.4 g / l. Preferably, the culture medium further comprises volatile fatty acids (hereinafter abbreviated "AGV") at a molar concentration of 1 to 10 mM, preferably 5 to 7 mM for each of said volatile fatty acids.

More particularly, the volatile fatty acids are fatty acids with short carbon chains, less than 6 carbon atoms, more particularly from 4 to 6 carbon atoms, more particularly the fatty acids chosen from valeric acid (C5H1002), acid isovaleric (C5H1002), 2-methylbutyric acid (C5H1002), isobutyric acid (C4H802), 2-methylvaleric acid (C6H1202). In the concentration values above: 1 M = 1 mol / I More particularly, said Archaea culture base medium comprises at least: several sources of carbon and nitrogen, preferably chosen from a yeast extract , an acetate salt, a formate salt and a tryptic peptone, - a source of phosphorus, preferably a phosphate salt, - a source of nitrogen, preferably an ammonium salt, - at least one salt of each of the metals K, Mg, Na, Ca, preferably NaCl, - a reducing substance, more particularly chosen from cysteine-HCl and Na 2 S, - a pH-regulating buffer substance of 7 to 7.5, preferably KOH 3 to adjust the pH at 7.5; trace elements, preferably salts selected from Fe, Ni, W, Se, Mo, Mn, Cu, Zn, B, Co salts, more preferably Widdel trace elements, vitamins, preferably vitamins from the Balch medium, preferably an oxidation-reduction indicator for controlling the year aerobic, preferably resazurin. The trace elements of Balch are listed in Table 4 below and include NaCl, FeSO4, ZnSO4, MgSO4, MnSO4, CoSO4, H3B03, NiCl2, Na2MoO4, CaCl2. The Widdel trace elements are listed in Table 5 below and include FeCl 2, CoCl 2, MnCl 2, ZnCl 2, H 3 BO 3, Na 2 MoO 4, NiCl 2 and CuCl 2. The vitamins of Balch's medium are listed in Table 6 below and include biotin, folic acid, pyridoxine hydrochloride, thiamine hydrochloride, riboflavin, nicotinic acid, DL-calcium pantothenate, vitamin B12, p-aminobenzoic acid, lipoic acid. More particularly, said culture medium according to the invention contains the following species: KH2PO4, K2PO4, MgSO47H2O, NaCl, KCl, KOH, NH4Cl, CaCl2.2H2O, KOH, NaCOOCH3 cysteine, yeast extract, trypticase peptone, NaOH, NaHCO2 , CH3OH, Na2S, NaHCO3, NaHCO2, Na2SeO3.5H2O, Na2W04.2H2O, Na2MoO4-21-120, NiC12-6H2O, FeSO4.7H2O, widdel oligoelements, Balch vitamins and preferably resazurin; the pH being adjusted to 7.5.

Even more particularly, the medium according to the present invention comprises the following components at the following concentrations: NiCl 2 x 6H 2 O 0.07 mg / l Na 2 MoO 4 x 2H 2 O 0.02 mg / I FeSO 4 x 7H 2 O mg / I MgSO 4 x 7H 2 O 0.01 g / I K2HPO4 0.5 g / I KH2PO4 0.5 g / I KCl 0.05 g / I CaCl2 0.05 g / I NaCl 1.5 g / I NH4Cl 1 g / I Na-Acetate 1 g / I Yeast extract 1 g / I Biotrypticase 1 g / I L-Cysteine, HCI 0.5 g / I Oligoelements solution of 2 m1 / 1 Widdel Oligoelements solution of 10 m1 / 1 Balch Resazurin 1 mg / I Na2Se2O3. 5H20 0.015 mg / I Na2WO4.2H20 0.02 mg / I NiC12.6H20 0.07 mg / I NaHCO3 4 g / I Na2S 0.5 g / I Na-Formate 0.4 g / I Methanol 40 mM Vitamin solution of 10 ml / 1 Balch medium Valeric acid 0.6 g / I Isovaleric acid 0.6 g / I 2-methylbutyric acid 0.6 g / I Isobutyric acid 0.6 g / I 2-methylvaleric acid 0.6 g The culture medium according to the invention can be in the liquid state in which case it comprises distilled water or in a freeze-dried state capable of being regenerated. addition of distilled water or in the solid state, especially in agar form, more particularly by adding agar, more particularly agar to 2% final. The subject of the present invention is also a process for the preparation of a sterilized culture medium according to the invention, characterized in that the following successive steps are carried out in which: one places a said culture medium that does not contain the components capable of being degraded by heating comprising said methanol, said vitamins, labile sodium salts comprising Na HCO2, Na HCO3 and Na2S and, where appropriate, said volatile fatty acids (VFA), hereinafter referred to as "incomplete culture medium under anaerobic atmosphere, preferably under a flow of nitrogen, in at least one sealed tube, and said tube is sealed, and preferably the gaseous atmosphere of said sealed tube is replaced by a N 2 / mixture. 80/20% CO2, and 3-is sterilized by heating said incomplete culture medium, preferably in an autoclave, a temperature above 100 ° C for at least 15 minutes, and 4-cooling said culture medium incomplete at room temperature and said components which may be degraded by sterilization in an autoclave comprising methanol, said vitamins, said labile sodium salts comprising Na HCO3, Na HCO2 and Na2S are added to said incomplete culture medium. More particularly, the temperature in step 3 above is 120 ° C for at least 20 minutes or 110 ° C for at least 45 minutes. The subject of the present invention is also a process for cultivating an archaea with a culture medium according to the invention in which the following successive steps are carried out: a biological sample capable of containing a said archaea is inoculated in said medium; in anaerobic culture, preferably in a nitrogen atmosphere, and 2-placing said culture medium under an atmosphere of anaerobiosis and containing at least 50% hydrogen at a pressure of less than 2 atm, and 3- is carried out incubation at a temperature of 20 to 40 ° C, preferably at 37 ° C to reach an exponential growth phase with evolution of methane in at least 1 day, preferably in no more than 15 days, preferably no more than 7 days. Preferably, in step 2, said culture medium is placed under a H 2 / CO 2 compound atmosphere in a volume proportion of 80/20% at a pressure of 1 to 2 atm. The media and method of cultivation above made it possible to establish in culture said archaea, that is to say that it can thus be under cultivated indefinitely. More particularly, it is possible to obtain an exponential growth phase culture in 1 to 10 days, preferably not more than 7 days, this growth phase being characterized by: (1) a methane production of at least 10 pM, as determined by gas chromatography, and (2) fluorescence microscopic visualization of spherical microorganisms with a size of 0.5 to 1 μm, and self fluorescent at 420 nm.

More particularly, the subcultures will be carried out by subculture at 1/10 of the culture in said growth phase. Finally, the present invention also has a method for determining the presence of an Archaea in a biological sample, preferably in a sample of animal or human stools according to the invention or an Archaea grown according to the process of the invention. characterized in that the DNA is extracted from said biological sample and the DNA of said Archaea is detected and preferably specifically quantified by PCR type PCR-type enzymatic amplification in real time with a pair of primers and preferably a probe of sequences derived from the 16S rDNA gene. The inventors have discovered that the extraction methods used in the state of the art to extract the DNA from the stool were not sufficiently effective to lead to reliable results in terms of quantification, particularly when seeks to quantify thick-walled Archaea prokaryotes. The present invention therefore provides a high performance automated protocol for extracting Archaea DNAs that is efficient, reproducible and easy to implement in a laboratory routine in order to facilitate the analysis of a large number of samples for a purpose both diagnostic than epidemiological, allowing the extraction of all the DNA. To do this, the inventors have developed a technique for semi-automated extraction of DNA by alternating mechanical lysis and enzymatic lysis with at least two mechanical lyses. The inventors optimized all steps of extraction of bacterial lysis to the choice of the extraction method whether manual or automatic, taking into account the efficiency and reproducibility of the method. Finally, the inventors compared the extraction technique retained with that used by the main authors who have published on this subject, as described in Example 2. More specifically, according to the present invention, to carry out the extraction of the Prokaryotic DNA of said stool sample, the steps are performed in which: 1) a homogeneous suspension of said stool sample is made in a buffer solution, at a dilution of 0.01 to 1 g / l, preferably at a dilution of 0 , 2 g / ml, and 2) automated mechanical lysis is carried out by mixing and stirring said suspension of step 1) with an abrasive spraying product, preferably preferably acid washed glass powder, in an apparatus performing a strong agitation, in particular by multidirectional rotation. 3) more preferably, the resulting suspension is heated to at least 100 ° C for at least 10 minutes, and 4) chemical and / or enzymatic lysis is carried out by mixing and stirring the supernatant of the suspension obtained in step 2 or 3) with one or more chemical and / or enzymatic lysis buffers, then 5) the mechanical lysis step 2) is repeated but with the mixture obtained in step 4), and 6) preferably, it is repeated step 4) of chemical and / or enzymatic lysis with the mixture obtained in step 5). In a known manner, the DNA is finally separated either by known methods such as by attachment to a column containing silicate and then washing to elute said DNA by separating it from the column or by washing and centrifugation to recover said DNA fragments. . The realization of the first step of mechanical lysis makes it possible to optimize and promote the work of chemical or enzymatic lysing agents in step 2) by promoting their penetration into the prokaryotic microorganisms allowing the thick walls of the cells to be partially degraded. thick wall prokaryotic microorganisms, and the complete degradation of the thick walls is obtained only by at least a second mechanical lysis after said first chemical or enzymatic lysis. In step 3), the heating makes it possible to finalize the degradation of the components of the walls and membranes of the microorganisms. More particularly, the following steps are carried out: a) 1 gram of stool was suspended in 5 ml of a solution of 0.05 M Tris-HCl pH 7.5, and b) aliquots of the suspension were transferred to a sterile Eppendorf tube containing powder of acid-washed glass beads and agitated for mechanical lysis in a multidirectional rotating apparatus, such as a FastPrep-24 Instrumente (MP Biomedical Europe Illkirch, France) at a speed 6.5 m / s for 90 seconds, and c). The supernatant was incubated overnight at 56 ° C with a chemical lysis buffer from the EZ1® DNA Extraction Kit and an Enzymatic Lysis Buffer comprising Proteinase K, and d) after a second lysis cycle. Mechanical as described above in b), the supernatant was incubated for 10 minutes at 100 ° C and the total DNA was then extracted using the EZ1 extractor, and the EZ1 DNA extraction kit. ® (Qiagen, Courtaboeuf, France) according to the manufacturer's method.

Finally, the present invention provides a kit useful for a process according to the invention characterized in that it comprises: DNA extraction reagents comprising at least one abrasive powdery product, preferably glass powder, and chemical and / or enzymatic lysis reagents, in particular a chemical lysis buffer of the EZ1® DNA extraction kit and an enzyme lysis buffer comprising a proteinase K, DNA amplification reagents comprising primers and detection probe of said methanogenic Archaea. Preferably, real-time PCR amplification and quantification reactions are carried out using specific hydrolysis probes. The Real-Time PCR technique consists of a conventional PCR using direct and reverse sequence primers, and comprises a detection of the amplified product based on the measurement of fluorescence emission proportional to the amount of amplified genes with a so-called probe. "Hydrolysis". For this, said probe is labeled with a fluorescence emitter or 5 'fluorophore and a 3' fluorescence emission blocking agent. This blocking agent absorbs the fluorescence emitted when the fluorophore and the blocking agent are close. When the fluorophore and the blocking agent are separated, the fluorescence emission is no longer absorbed by the blocking agent. During its passage, the Taq polymerase causes a hydrolysis of the probe and thus a release of the nucleotides and the fluorophore in solution. The fluorescence emission will therefore be proportional to the number of amplifiers. The principle of real-time PCR relies on the ability of Taq polymerase during the elongation step to hydrolyze a hybridized probe on the DNA to be copied, this hydrolysis allowing fluorescence emission, which allows quantification. During the same reaction, two different targets can be quantified by introducing into the reaction mixture two primers and one probe directed against a first target, and two other primers and probe directed against the other target. Both probes are labeled with different fluorophores. More preferably, a large synthetic DNA fragment is used as a calibration standard for the quantification of DNA, said large synthetic DNA fragment comprising said specific sequences respectively of each of said bacteria or prokaryotic species whose concentrations are quantified. The presence of several molecular targets on the same nucleic fragment makes it possible to quantify different targets in the same sample and to homogenize them homogeneously, the quantification using the same calibration range of several molecular species, allowing the comparison of the effectiveness of the different PCR reactions between them and from one determination to another over time and avoids biases related to the positive control.

By "DNA fragment" is meant a DNA or oligonucleotide fragment whose sequences are written below in the 5 '-> 3' direction. "Probe" is understood here to mean an oligonucleotide, more preferably 20 to 30 nucleotides, which hybridises specifically with said specific sequence and thus makes it possible to detect and quantify it specifically by measuring the increase in bound fluorescence of the PCR reaction. The probe makes it possible to detect the amplified specific DNA and to quantify it by comparing the intensity of the signal with that of the standard of quantification. By "primer" is meant herein an oligonucleotide of preferably 15 to 25 nucleotides which specifically hybridizes with one of the two ends of the sequence that the DNA polymerase will amplify in the PCR reaction. In real-time PCR DNA quantification methods, it is important to know whether a positive reaction is not due to contamination by the recombinant plasmid used as a standard of quantification or as a positive control. To solve this problem, a restriction enzyme cleavage site is advantageously introduced into at least one of the molecular targets. This site is missing on the natural sequence. Thus, by enzymatic cleavage and analysis of the agarose gel amplified fragment, or by using a real-time PCR probe which specifically recognizes the restriction site, it is possible to detect the possible presence of the contaminating plasmid. Thus, it is possible to carry out more particularly the following steps in which: 1. a PCR-type enzymatic amplification reaction of the DNA of at least one said specific sequence of at least one of said agents is carried out, in the DNA extracted from said samples according to the invention to be tested and in the DNA of the standard calibration sample, using at least one set of primers capable of amplifying at least said sequence at least authentic specific and said modified specific sequence; 2. it is checked whether the possible amplifications of the DNA extracted from said samples to be tested, include a said specific sequence, and 3. the possible false positives are detected from possible contamination of said test samples by DNA from the test sample. standard calibration sample, by at least one of the following steps: 3a. enzymatic digestion of the PCR product obtained with an enzyme corresponding to the cleavage site and agarose gel analysis of the digest was performed in comparison with the non-digested restriction enzyme PCR product. If the digested fragment is from the amplification of the molecular target inserted into the control plasmid, it contains the restriction site, and it will be smaller in size than the undigested fragment. 3b. a real-time PCR reaction is carried out with forward and reverse primers of one of the molecular targets, and a specific probe of said exogenous sequence containing the restriction site. Only a fragment from the control plasmid and containing the exogenous sequence can be amplified. Advantageously, amplification and quantification reactions are carried out by using sets of primers and hydrolysis probes specific for each of the different bacteria known as specific sequences of each of the said bacteria to be tested, and, if appropriate, a specific sequence of human DNA in the sample to be tested, such as a specific sequence of human albumin, and said specific sequence comprises a probe sequence flanked by sequences able to serve as a primer in an amplification reaction of the type PCR of said specific sequences. Advantageously, a plurality of PCR enzyme amplification reactions, simultaneous or not, of each of said specific sequences of said bacteria are used with the same large standard synthetic DNA fragment, using a plurality of different sets of specific primers of each of the different said specific sequences of each of said bacteria, the sequences of the different primers not intersecting between the different said bacteria and said primers can be implemented in an enzymatic amplification reaction carried out according to the same protocol and especially at the same hybridization temperature. The subject of the present invention is also a diagnostic kit that is useful for implementing a method for diagnosing and monitoring the level of methanogenic Archaea in the stool samples according to the invention, characterized in that it comprises: standard calibration DNA samples at a known concentration comprising said specific sequences of each of said bacteria as defined above, and preferably a said universal bacterial sequence as defined above, and more preferably one said large synthetic DNA fragment 25 grouping said specific sequences of each of said bacteria as defined above, and preferably a said universal bacterial sequence as defined above, as well as - said sets of primers specific to said fragments of Modified synthetic DNAs specific for said bacteria and more preferably, said probes as defined above, and reagents for carrying out a PCR type DNA amplification reaction. Other features of the present invention will become apparent in the light of the following detailed description of various embodiments. FIG. 1 represents a histogram presenting the cultivation delays in days (in ordinate) of nine Archaea (in abscissa) in a medium of reference DSMZ (light gray) and medium SAB (dark gray). FIG. 2 represents a histogram presenting the cultivation delays in days (in ordinate) of nine Archaea (in abscissa) in an SAB medium of example 1 (dark gray) and in a SAB-fat medium of example 2 ( light grey). Example 1. Development of a SAB medium. To test different media compositions, the inventors cultured the following methanogens: M. smithii ATCC 35061T DSMZ 861, M. smithii DSMZ 2374, M. smithii DSMZ 2375, M. smithii DSMZ 11975, M. oralis DSMZ, M, stadtmanae ATCC 43021T DSMZ 3091, Methanobacterium beijingense DSMZ 15999 [6] and Methanosaeta conci / ii DSMZ 2139 [20], purchased from the German collection of microorganisms and DSMZ cell cultures (Brunswick, Germany). Liquid medium 119 (Table 1A below) was used to grow M. smithii and M. beijingense. The M. oralis culture was carried out on liquid medium 119 (http://www.dsmz.de) modified by the addition of 1 g of yeast extract and under an atmosphere of H2 / CO2 (80% / 20%). ) at a pressure of 2.5 bar. Medium 332 (Table 1B below) (http://www.dsmz.de) was used to grow M. stadtmanae and medium 334c (Table 1C below) (http://www.dsmz.de ) to cultivate M. conci / ii at 37 ° C in Hungate tubes (Dutscher, Issy-les-Moulineaux, France) under an atmosphere of 2 bar H2 / CO2 (80% -20%) with stirring.

M. / uminyensis (DSMZ 24529) was grown using modified medium 119 by the addition of the methanol / selenite / tungstate solution with stirring under a 2 bar H 2 / CO 2 (80% / 20%) atmosphere ( Table 1D below).

The optimal culture medium derived from medium 119 contained g / I: KH 2 PO 4 (0.5), MgSO 4 x 7H 2 O (0.4), NaCl (5), NH 4 Cl (1), CaCl 2 x 2 H 2 O (0.05). , Na-acetate (1.6), cysteine-HCl (0.5), yeast extract (1), trypticase peptone (1), Widdel trace element solution (1 ml / l) (Table 6) , 2 ml / 1 solution of Selenite-Tungstate NaOH (0.4 g / l), Na2Se03.5H2O (3 mg / l) (Table 7), Na2W04.2H2O (4 mg / l), resazurin (0.001 g / l). 1) and distilled water (qsp); the pH is adjusted to 7.5 with KOH (10 M) before autoclaving. The medium is boiled under N2 gas to remove the maximum oxygen and cooled to room temperature. The medium is dispensed in 5 ml Hungate tubes or in anaerobiosis flasks obtained with N2 / CO2 (80:20, v / v) at atmospheric pressure. The addition of an N2 + CO2 mixture in microorganism cultures allows, in a known manner, the establishment of anaerobiosis and supply of CO2 to the anaerobes that metabolize it as a carbon source. The tubes and / or flasks are then autoclaved for 45 minutes at 110 ° C. After autoclaving, the medium is supplemented with NaHCO 3 (4 g / l), Na 2 S x 9H 2 O (0.5 g / l), Na-formate (2 g / l) and 10 ml / l of a solution. of Balch vitamins (Table 6) (Balch et al., 1979). Finally, the methanol is added (40 mM) with a gaseous mixture composed of H2 / CO2 (80/20) at a pressure of only 1 bar constituting the atmosphere on the surface of the culture. The gaseous mixture composed of H 2 / CO2 (80/20) at 1 bar pressure is introduced into the culture by bubbling through a sterile needle until total disappearance of bubbles, the content of the tube is then 1 bar pressure H2 / CO2 (80/20) . The inventors have furthermore developed a polyvalent original culture medium, hereinafter referred to as SAB medium, having an effect on the growth of methanogenic archaea.

To do this, the inventors have discovered that medium 119, modified with methanol and a solution of selenite / tungstate (Table 7), was advantageously supplemented with various ingredients, in particular trace metals Fe, Ni, Mo and Mg, Mn. , Cu and Zn as described in Table 1, allowing in particular to obtain faster growths of all Methanobrevibacter methanogenic archaea tested. This SAB medium was prepared as follows. The culture medium is boiled under a stream of nitrogen, in a container closed with aluminum foil until it becomes transparent (the redox indicator, in this case resazurin, is pink in color). becomes transparent after reduction by boiling under a stream of nitrogen). It is then cooled to room temperature and rinsed with N 2 or 80% N 2/20% CO 2.

All solutions must be prepared under a strictly anaerobic atmosphere, especially under a N2 / CO2 flow (replacing oxygen) [9,21]. The pH should be adjusted with 10 M KOH to a final pH of 7.5. The medium is distributed in Hungate tubes, then it is autoclaved at 120 ° C. for 40 minutes under a pressure of 2 bar and then cooled to room temperature. The incomplete culture medium comprised the following elements of Table 1: Quantities per liter of H 2 O NiCl 2 x 6H 2 O 0.07 mg Na 2 MoO 4 x 2H 2 O 0.02 mg FeSO 4 x 7H 2 O mg MgSO 4 x 7H 2 O 0.01 g K 2 HPO 4 0.5 g KH21304 0.5 g KCI 0.05 g CaCl2 0.05 g NaCl 1.5 g NH4Cl 1 g Na-Acetate 1 g Yeast extract 1 g Biotrypticase 1 g L-Cysteine, HCI 0.5 g Trace micro-nutrient solution 2 ml Widdel (see Table 5) 10 ml micronutrient solution Balch (see Table 4) Resazurin 1 mg Na2Se203.5H20 0.015 mg Na2W04.2H20 0.02 mg NiCl2.6H20 0.07 mg After autoclaving, the medium is supplemented by the following elements: NaHCO3 4 g / I Na2S.9H20 0.5 g / I Na-Formate 0.4 g / I Methanol 40 mM Vitamins Table 6) Balch (cf 10 m1 / 1 The culture is incubated under an atmosphere using the mixture of 80% H2 + 20% CO2 at 2 bars The culture was carried out in Hungate tubes (Dutscher, Issy-les-Moulineaux, France) incubated at 37 ° C. with stirring.

The growth of methanogens was verified daily by optical microscopic observation and parallel measurement of methane production using a GC-8A gas chromatograph (Shimadzu, Champs-sur-Marne, France) equipped with a thermal conductivity detector. , an injector and a column of the SP100 mesh of Chromosorb WAW 80/100 (Alltech, Carquefou, France). N2 at a pressure of 100 kPa was used as the carrier gas. The temperature of the detector and the injector were 200 ° C and the column temperature 150 ° C. The inventors used the growth of each strain of methanogen in the appropriate DSMZ culture medium as a positive control and tubes containing the same non-inoculated culture medium as negative controls. They compared the growth time of each strain of methanogen in standard reference media and in BSA-medium. The experiments were done in triplicate. The negative controls have all remained sterile. All the methanogenic strains tested cultivated in their reference medium without exception. All the methanogenic strains tested were grown in SAB medium without exception. The results obtained are presented on the histogram of FIG. 1 and show that the culture time is always shorter in the SAB medium than in the reference medium. The growth time to reach the exponential growth phase of M. smithii was different depending on the phylotype observed.

Indeed, M. smithii DSMZ 2374 and M. smithii DSMZ 2375 arrive in the exponential growth phase after 24 hours of incubation in SAB medium against three days in DSMZ 119. The strain M. smithii ATCC 35061T cultivates after 3 days incubation in SAB medium against 7 days in DSMZ 119. The strain M. smithii DSMZ 11975 cultivated after 7 days of incubation in SAB-medium against 10 days in the medium of 119 DSMZ. The strain M. oralis 7256 cultivates after 5 days of incubation in SAB medium against 10 days in the medium of 119 DSMZ modified from Table 1D. The M. stadtmanae ATCC 43021T strain cultivates after 24 hours of incubation in SAB medium against 3 days in 322 DSMZ medium. Strain M. / uminyensis DSMZ 24529 cultivates after 5 days of incubation in SAB medium against 7 days in modified DSMZ 119 medium. The strain M. beijingense DSMZ 15999 develops after 24 hours of incubation in SAB medium against 3 days in 119 DSMZ medium. The M. conci / ii strain DSMZ 2139 cultivates after 3 days of incubation in SAB medium against 7 days in 334c DSMZ medium. These results show that the inventors have developed a medium that meets the two objectives they have set for themselves, namely a growth medium that supports the growth of all methanogens and, secondly, a medium that makes it possible to shorten the time to culture of these methanogens. Since the preparation time of such a medium is shorter than the preparation time of each of the reference media separately, such a medium can be introduced for the routine cultivation of archaea methanogens in the microbiology laboratory. Media 119, 332 and 334 are described in Tables 1A, 1B and 1C hereinafter. Example 2. Development of a SAB + fat medium.

The SAB liquid medium previously described in Example 1, (Table 1) was further completed to cultivate all the strains mentioned above, by developing an original polyvalent culture medium, hereinafter referred to as SAB + fat medium having an effect. on the growth of methanogenic Archaea. The inventors have, in fact, discovered that the SAB medium of Table 1 of Example 1 was advantageously supplemented with a mixture of volatile fatty acids as described in Table 2 below, allowing in particular to obtain higher growths. of all Methanobrevibacter Methanogenic Archaea tested. This SAB + fat medium was prepared as follows. The culture medium is boiled under a stream of nitrogen, in a container closed with aluminum foil until it becomes transparent (the redox indicator, in this case resazurin, is pink in color). becomes transparent after reduction by boiling under a stream of nitrogen). It is then cooled to room temperature and rinsed with N 2 or 80% N 2/20% CO 2. All solutions must be prepared under a strictly anaerobic atmosphere, especially under a N2 / CO2 flow (replacing oxygen) [9,21]. The pH should be adjusted with 10 M KOH to a final pH of 7.5. The medium is distributed in Hungate tubes, then it is autoclaved at 120 ° C. for 40 minutes under a pressure of 2 bar and then cooled to room temperature. Incomplete SAB + fat culture medium included the following elements of Table 2: Quantities per liter of H 2 O NiCl 2 x 6H 2 O 0.07 mg Na 2 MoO 4 x 2H 2 O 2 mg FeSO 4 x 7H 2 O mg MgSO 4 x 7H 2 O 0.01 g K 2 HPO 4 , 5 g KH2PO4 0.5 g KCl 0.05 g CaCl2 0.05 g NaCl 1.5 g NH4Cl 1 g Na-Acetate 1 g Yeast extract 1 g Biotrypticase 1 g L-Cysteine, HCI 0.5 g trace elements of 2 ml Widdel (see table 5) 10 ml micronutrient solution Balch (see table 4) Resazurin 1 mg Na2Se203.5H20 0.015mg Na2WO4.2H20 0.02mg NiCl2.6H20 0.07mg After autoclaving, the medium is supplemented with: NaHCO3 4 g / I Na2S.9H20 0.5 g / I Na-Formate 0.4 g / I Methanol 40 mM Vitamins (see Table 6) 10 m1 / 1 Valeric acid 5.88 mM (0.6 g / l) Isovaleric acid 5.88 mM (0.6 g / l) 2-8.8 mM (0.6 g / l) 2-methylbutyric acid 6.81 mM isobutyric acid (0.6 g) / 1) Methylvaleric acid 5, 16 mM (0.6 g / 1) The culture is incubated under an atmosphere using the mixture 80% of H2 + 20% of CO2 at 2 bars. The culture was carried out in Hungate tubes (Dutscher, Issy-les-Moulineaux, France) incubated at 37 ° C. with stirring. The growth of methanogens was verified daily by optical microscopic observation and parallel measurement of methane production using a GC-8A gas chromatograph (Shimadzu, Champs-sur-Marne, France) equipped with a thermal conductivity detector. , an injector and a column of the SP100 mesh of Chromosorb WAW 80/100 (Alltech, Carquefou, France). N2 at a pressure of 100 kPa was used as the carrier gas. The temperature of the detector and the injector were 200 ° C and the column temperature 150 ° C.

The inventors used the growth of each strain of methanogen in the SAB-medium culture medium and tubes containing the same non-inoculated culture medium as negative controls. They compared the growth time of each strain of methanogen in standard reference media and in SAB + fat-medium. The experiments were done in triplicate. The negative controls have all remained sterile. All the methanogenic strains tested were grown in SAB medium without exception. All the methanogenic strains tested cultivated in SAB + fat medium without exception. The results obtained are presented on the histogram of FIG. 2 and show that the culture time is comparable or shorter in the SAB + fat medium than in the SAB medium. The growth time to reach the exponential growth phase of M. smithii was different depending on the phylotype observed. Indeed, M. smithii DSMZ 2374 and M. smithii DSMZ 2375 arrive in the exponential phase of growth after 24 hours of incubation in the two millies tested. M. smithii strain ATCC 35061T is cultured after 2 days of incubation in SAB + fat medium against 3 days in SAB medium. The strain M. smithii DSMZ 11975 cultivates after 5 days of incubation in SAB-fat against 7 days in SAB medium. The strain M. oralis 7256 cultivates after 4 days of incubation in SAB + fat medium against 5 days in SAB medium. The M. stadtmanae ATCC 43021T strain cultivates after 24 hours of incubation in both culture media tested. Strain M. / uminyensis DSMZ 24529 cultivates after 3 days of incubation in SAB-fat medium against 5 days in SAB medium. The strain M. beifingenseDSMZ 15999 develops after 24 hours of incubation in both culture media tested. The strain M. conci / ii DSMZ 2139 cultivates after 3 days of incubation in both culture media tested.

These results show that the inventors have developed a medium that meets the two objectives they have set for themselves, namely a growth medium that supports the growth of all methanogens and, moreover, a medium that makes it possible to shorten the culture time. of these methanogens. In addition, since the preparation time of such a medium is shorter than the preparation time of each of the reference media separately, such a medium can be introduced for the routine cultivation of Archaea methanogens in the microbiology laboratory. Example 3. Semi-automated extraction of DNA from methanogens. The inventors have previously developed a technique for extracting DNA from methanogens from clinical specimens, in particular stool samples (34), 2009). However, this protocol relies solely on manual steps, making this protocol difficult to use in routine laboratory. Here, the inventors have attempted to automate all or part of this protocol in order to reduce the working time and to set up a protocol compatible with routine use. For this purpose, the inventors compared automated, semi-automated and manual protocols for DNA extraction from Archaea. In this prospective study, the inventors included 110 stool specimens collected from 110 individuals, Marseille, France, between July and August 2011. This study was approved by the local IFR48 Ethics Committee. Three different DNA extraction protocols were performed in parallel. A manual protocol using NucleoSpin® kit (Macherey Nagel, Hoerdt, France) was performed as previously described [34]. An automated protocol used the DNA extraction technique using the EZ1 extractor, and the EZ1C DNA extraction kit (Qiagen, Courtaboeuf, France) according to the manufacturer's method. A semi-automated protocol was performed as follows: approximately 1 gram of stool was suspended in 5 ml of a solution of 0.05 M Tris-HCl pH 7.5. 250 μl aliquots of the suspension were transferred to a sterile Eppendorf tube containing 0.3 g of 106-mm acid-washed glass beads; Sigma, Saint-Quentin Fallavier, France) and shaken to achieve mechanical lysis in a FastPrep-24 Instrumente apparatus, (MP Biomedical Europe Illkirch, France) at a speed of 6.5 m / s for 90 seconds. This apparatus operates a multidirectional rotation agitation. The supernatant was incubated overnight at 56 ° C with 180 ml lysis buffer (Tissue DNA Kit, Qiagen EZ1®) and 25 μl Proteinase K (20 mg / ml). After a second cycle of mechanical lysis as described above, the supernatant was incubated for 10 minutes at 100 ° C and the total DNA was then extracted using the tissue DNA kit, Qiagen EZ1® in the EZ1 extractor, Qiagen. Negative controls consisting of sterile water without DNA were introduced in all the manipulations and underwent the same extraction process as the stool samples. The DNA extracted by the three extraction protocols, were incorporated into a detection of the 165 M. smithii methanogens by real-time PCR. The PCR primers Smit.16S-740F: 5'-CCGGGTATCTAATCCGGTTC-3 and Smit.16S-862R: 5'-CTCCCAGGGTAGAGGTGAAA-3 and the Smit.16S FAM probe: 5'-CCGTCAGAATCGTTCCAGTCAG-3 were adapted from a protocol previously described [14]. The PCR products were purified and sequenced using the BigDye terminator 1.1 sequencing kit and the 3130 sequencer (Applied Biosystems). Negative controls were incorporated into each analysis. After the first step, (Proteinase K digestion and overnight lysis buffer), the semi-automated protocol takes 15 to 30 minutes depending on the operator, compared with 2.5 to 3 hours for the manual technique. The automated protocol is performed in two steps, digestion of proteinase K at 70 ° C for 10 minutes and the automated step for 15 minutes. Results and Discussion: Negative controls introduced in all experiments remained negative and no Archaea DNA was extracted in these negative controls. The automated protocol yielded 35/110 (32%) positive samples with an average value of Ct (number of real-time PCR cycles to obtain a positive signal) of 36.1. The semi-automated protocol yielded 90/110 (87%) positive samples with an average Ct value of 27.4. In comparison with the automated protocol, this semi-automated protocol reported a significantly higher number of positive samples (P <0.001) with significantly lower Ct values (P <0.001). The manual protocol as described in reference [14] reported 100/110 (91%) positive samples with an average Ct value of 30.33. In comparison with the automated protocol, this manual protocol reported a significantly higher number of positive samples (P <0.001) with significantly lower values of Ct (P <0.001). However, in comparison with the semi-automated protocol, this manual protocol reported reported a non-significantly higher number of positive samples (P = 0.1) with non-significantly lower Ct values (P = 0.1). These data have indicated to the inventors that the combination of mechanical lysis by mechanical stirring in the presence of glass beads with enzymatic and chemical lysis has significantly increased the extraction efficiency of the DNA of the methanogens, amplifiable by PCR. According to the present invention, however, it is possible to combine a manual portion of the extraction process with an automated part so as to reduce the working time in view of a routine implementation. Similar results were obtained by PCR quantization of M. ummyensis and M. stadtrnanae DNA with the primers of the M / uminyensis and M stadtrnanae rDNA genes of Table 8 and the attached DNA sequence listing.

In the following tables, the weight concentrations of the salts in the form of hydrates do not take into account the weight of the water molecules involved in the hydrates.

Table 1: SAB medium Quantities per liter of H2O NiCl2 x 6H20 0.07 mg Na2MoO4 x 2H20 0.02 mg FeSO4 x 7H20 0.2 mg MgSO4 x 7H20 0.01 g K2HPO4 0.5 g KH2R04 0.5 g KCl 0, 05 g CaCl2 0.05 g NaCl 1.5 g NH4Cl 1 g Na-Acetate 1 g Yeast extract 1 g Biotrypcase 1 g L-Cysteine, HCI 0.5 g Trace elements (Table 5) from Widdel 2 ml Olig- elements (Table 4) from Balch 10 ml Resazurin 1 mg solution of 5 ml Selenite / tungstate from Table 7 After autoclave: NaHCO3 4 g / I Na2S.9F-120 0.5 g / I Na-Formate 0.4 g / I Methanol 40 mM 6 ) Balch Vitamins (Table 10 m1 / 1) Table 1A: Medium 119 Amount per liter of H2O KH21304 0.5 g MgSO4 x 7H20 0.4 g NaCl 5 g NH4Cl 1 g CaCl2 x 2H20 0.05 g Na-acetate 1, 6 g Cysteine-HCl 0.5 g Yeast extract 1 g Trypticase peptone 1 g Resazurin 0.001 g Widdel trace elements 1 ml (see Table 5) NaHCO3 4 g Na2S x 9H20 0.5 g Na-formate 2 g Vitamins of Balch (cf 10 ml table 6) Table 1B: Medium 332 Quantity per liter of H2O Resazurin 1 mg Rumen fluid e, clarified 100 ml Trypticase 2 g Yeast extract 2 g Na-acetate 0.5 g Na-formate 0.5 g 6) Balch vitamin (Table 1 ml Balch trace elements (Table 4) 10 ml Na2SeO4 1, 9 mg NiCl2 x 6H2O 0.7 mg FeSO4 x 7H20 3 mg K2HPO4 0.6 g KH2PO4 2.8 g (NH4) 2504 0.3 g NH4Cl 1 g NaCl 0.6 g MgSO4 x 7H20 0.15 g CaCl2 x 2H20 0.076 g Methanol 5 ml Cysteine-HCl x H2O 0.875 g Na2S x 9H20 0.375 g Table 1C: Medium 334 Amount per liter of H2O KH2PO4 0.3 g NaCl 0.6 g MgCl2 x 6H20 0.1 g CaCl2 x 2H20 0.08 g KHCO3 0.4 g Na2S x 9H2O 0.3 g NH4Cl 1 g Resazurin 1 mg Cysteine-HCl x H2O 0.3 g Balch vitamin (cf. 10 ml Table 6) Balch trace elements (see Table 10, Table 10) Table 1D: Quantity per liter of H2O KH2R04 0.5 g MgSO4 x 7H20 0.4 g NaCl 5 g NH4Cl 1 g CaCl2 x 2H20 0.05 g Na -acetate 1.6 g Cysteine-HCl 0.5 g Yeast extract 1 g Trypticase peptone 1 g Resazurin 0.001 g Widdel trace elements 1 ml (see Table 5) 2 ml solution selenite / tungstate (Table 7) After autoclave: Quantity per liter of H2O NaHCO3 4 g Na2S x 9H20 0.5 g Na-formate 2 g Vitamins Table 6) Balch (10 ml Methanol 40 mM Table 2: Composition of Balch medium (a) (b) ( c) (d) (e) (f) (g) (h) (i) (j) (k) (I) 50 50 10 10 0.002 5 g 2.5 2.5 2 g 2 g 0.5 0 The ingredients are added to distilled water to have a final volume of 1 liter.Cysteine and Na2S are added after bubbling the medium with a gaseous mixture 80% N2-20% CO2, the Final gas phase 5 of the tube is composed of a mixture of 80% H2-20% CO2. (a): Contains 6 g of KH2PO4 per liter of water distilled (b): Contains in grams per liter of distilled water: KH2PO4 (6), (NH4) 2504 (6), NaCl (12), MgSO4.7H2O (2.6), CaCl2.2H2O (0.16 ). (c): Contains in grams per liter of distilled water (pH adjusted to 7 with KOH): nitriloacetic acid (1.5), MgSO4.7H2O (3), MnSO4.2H2O (0.5), NaCl (1) ), FeSO4.7H2O (0.1), CoSO4 or CaCl2 (0.1), CaCl2H2O (0.1), ZnSO4 (0.1), CuSO4.5H2O (0.01), Na2MoO4.2H2O (0. , 01); Dissolve the nitriloacetic acid with KOH (pH 6.5) and add the other minerals. (D): Contains in milligram per liter of distilled water the following vitamins: biotin (2), folic acid (2), pyridoxine hydrochloride (10), thiamine hydrochloride (5), riboflavin (5), nicotinic acid (5), DL-calcium pantothenate (5), vitamin B12 (0.1), p-aminobenzoic acid (5), lipoic acid (5). (E) FeSO4 7H2O; (f): NaHCO3; (g): Na acetate; (h): Na formate; (i): DIFCO yeast extract; (j): trypticase BBL; (k): L-cysteine hydrochloride; (I): Na2S, 9H2O.

Table 3: Medium El Quantity per liter of H2O KH2PO4 0.3 g K2HPO4 0.3 g NaCl 1 g NH4Cl 1 g CaCl2 x2H20 0.1 g KCl 0.1 g MgCl2 x 6H20 0.5 g Yeast extract 1 g Cysteine-HCI 0.1 g resazurin 0.001 g Balch trace elements (Table 4) 2.5 ml NaHCO3 4 g Na2S x 9H20 0.5 g Na-formate 2 g Balch vitamins 10 ml (Table 6) 40 mM methanol Table 4: Solution of trace elements of Balch Quantity per liter of H2O Nitrilotriacetic acid 1.5 g MgSO 4 x 7H 2 O 3 g MnSO 4 x H 2 O 0.5 g NaCl 1, g FeSO 4 x 7H 2 O 0.1 g CoSO 4 x 7H 2 0, 18 g CaCl 2 x 2H 2 O 0.1 g ZnSO 4 x 7H 2 O 0.18 g KAI (SO 4) 2 x 12H 2 O 0.02 g H 3 BO 3 0.01 g Na 2 MoO 4 x 2H 2 O 0.01 g NiCl 2 x 6H 2 O 0.03 g Na 2 SiO 3 x 5H 2 0 Table 5: Widdel Micronutrient Solution Amount per 1 liter of distilled water 25% HCI 10ml FeCl2 x 4H20 1.5g CoCl2 x 6H20 190mg MnCl2 x 4H20 100mg ZnCl2 70mg H3B03 62mg Na2MoO4 x 2H20 36mg NiCl2 x 6H20 24mg CuCl2 x 2H20 17mg Table 6: Balch Vitamin Solution Amount per liter Biotin H2O 2m g Folic acid 2 mg Pyridoxine -HCl 10 mg Thiamine-HCl x 2H20 5 mg Riboflavin 5 mg Nicotinic acid 5 mg D-Ca-Pantothenate 5 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5 mg Lipoic acid 5 mg Table 7: Selenite / tungstate solution Amount per liter of H2O NaOH 0.5 g Na2Se03 x 5H20 3 mg Na2WO4 x 2H20 4 mg Table 8: Organism (a) Target Sequence (5'-3 ') of primers and probes SEQ. ID. M. smithii 16S rDNA Smit.16S-740F, 5'-CCGGGTATCTAATCCGGTTC-3 '4 Smit.16S-862R, 5'-CTCCCAGGGTAGAGGTGAAA-3' Smit.16S FAM, 5'-CCGTCAGAATCGTTCCAGTCAG-3 '6 FAM ( MGB) M. stadtmanae 16S rDNA Stadt_16SF, 5'-AGGAGCGACAGCAGAATGAT-3 '7 Stadt_16SR, 5'-CAGGACGCTTCACAGTACGA-3' 8 Stadt_16SFAM, 5'-TGAGAGGAGGTGCATGGCCG-3 '9 FAM (Taqman) M. / uminyensis 16S rDNA B10-dir , 5'-GTTCGTAGCCGGTCTGGTAA-3 '1 B10-rev, 5'-CCAAGTCTGGCAGTCTCCTC-3' 2 B10-VIC, 5'-TCGGGAAGCTTAACTTTCCGACTTCC-3 '3 VIC (Taqman) References [1] Hungate RE: Roll-tube method for the cultivation of strict anaerobes. Methods in Microbiology 1969, 3B: 117-132. [2] Miller T.L.and Wolin M.J .: A serum bottle modification of technical Hungate for cultivation obligate anaerobes. Applied Microbiology 1974, 27: 985-987. [3] Dermoumi HL, Ansorg RAM: Isolation and Antimicrobial Susceptibility Testing of Fetal Strains of Archaeon Methanobrevibacter srnithii. Chemotherapy 2001, 47: 177-183. 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[9] Wolfe RS, Metcalf WW: A vacuum-vortex technique for the preparation of anoxic solutions or liquid culture in small volumes for cultivating methanogens or other strict anaerobes. Anaerobe 2010, 16: 216-219. [10] Murray PA, Zinder SH: Nutritional Requirements of Methanosarcina sp. Strain TM-1. Applied and Environmental Microbiology 1985, 50: 49-55. [11] Dridi B, Raoult D, Drancourt M .: Archaea as emerging organisms in complex human microbiomes. Anaerobe 2011, 17: 56-63. [12] Bryant MP, TZENG SF, IM ROBINSON, Joyner AE: Nutrient Requirements of Methanogenic Bacteria. In Anaerobic Biological Treatment Processes. 105 edition AMERICAN CHEMICAL SOCIETY; 1971: 23-40. [13] Taylor CD, McBride BC, Wolfe RS, Bryant MP: Coenzyme M, Essential for Growth of a Rumen Strain of Methanobacterium ruminantium. Journal of Bacteriology 1974, 120: 974-975. [14] Fricke WF, Seedorf H, Henne A, Kr ++ er M, Liesegang H, Hedderich R et al .: The Genome Sequence of Methanosphaera Stadtmanae Reveals Why This Human Intestinal Archaeon Is Restricted to Methanol and H2 for Methane Formation and ATP Synthesis. Journal of Bacteriology 2006, 188: 642-658. [15] Scherer P, Sahm H: Effect of trace elements and vitamins on the growth of Methanosarcina barkeri. Acta Biotechnol 1981, 1: 5765. [16] Tanner RS, Wolfe RS: Nutritional Requirements of Methanomicrobium Mobile. Applied and Environmental Microbiology 1988, 54: 625-628. [17] Diekert G, Konheiser U, Piechulla K, Thauer RK: Nickel requirement and factor F430 content of methanogenic bacteria. Journal of Bacteriology 1981, 148: 459-464. [18] [19] [20] [21] [22] [23] Schonheit P, Moll J, Thauer RK: Nickel, cobalt, and molybdenum requirement for growth of Methanobacterium thermoautotrophicum. Archives of Microbiology 1979, 123: 105107. KE Hammel, Cornwell KL, Diekert GB, Thauer RK: Evidence for a nickel-containing carbon monoxide dehydrogenase in Methanobrevibacter arboriphilicus. Journal of Bacteriology 1984, 157: 975-978. Girishandra B.Patel and G.Dennis sprott: Methanosaeta concilii gen. Nov., sp. nov. (Methanothrix concilil) and Methanosaeta thermoacetophila noun. rev., comb. Nov. International Journal of Systematic Bacteriology 1990, 40: 79-82. Bryant MP: Commentary on the technical Hungate for culture of anaerobic bacteria. The American Journal of Clinical Nutrition 1972, 25: 1324-1328. Hartzell PL, Wolfe RS: Requirement of the nickel tetrapyrrole F430 for in vitro methanogenesis: reconstitution of methylreductase component C from its dissociated subunits. Proceedings of the National Academy of Sciences 1986, 83: 67266730. Hensgens CMH, Nienhuis-Kuiper ME, TA Hansen: Effects of tungstate on the growth of 8%; Desulfovibrio gigas8ilt; / i8igt; NCIMB 9332 and other sulfate-reducing bacteria with ethanol as a substrate. Archives of Microbiology 1994, 162: 143-147. [24] Jarrell KF, Kalmokoff ML: Nutritional requirements of the methanogenic archaebacteria. Can J Microbiol 1988, 34: 557-576. [25] Scanlan PD, Shanahan F, Marchesi, JR (2008) Human methanogenesis and the effects of diabetes mellitus. BMC Microbiol 8: 79; [26] Rieu-Lesme F, Delbes C, Sollelis L (2005) Recovery of partial 16S rDNA sequences suggests the presence of CrenArchaeaota in the human digestive ecosystem. Curr Microbiol 51: 317-321. [27] Oxley AP, Lanfranconi MP, Wurdemann D, Ott S, Schreiber S, McGenity TJ, KN Timmis, Nogales B (2010) Halophilic Archaea in the intestinal mucosa human. About Microbiol .12: 2398-2410. [28] Nam YD, Chang HW, Kim KH, SW Roh, Kim MS, Jung MJ, SW Lee, JY Kim, Yoon JH, Bae JW (2008) Bacterial, Archaeal, and eukaryal diversity in the intestines of Korean people. J Microbiol 46: 491-501 [29] Mihajlovski A, Alric M, Brugere JF (2008) A putative new order of methanogenic Archaea inhabiting the human gut, as revealed by molecular analyzes of the mcrA gene. Res Microbiol 159: 516-521 [30] Mihajlovski A, Dore J, Levenez F, Alric M, Brugere JF (2010) Molecular evaluation of the human methamytic gut Archaeal microbiota reveals an age-associated increase of the diversity. Envir Microbiol Rep 2: 272-280. [31] Miller TL et al. Appl. Environ Microbiol. 1982; 43: 227-232. [32] Miller TL and Wolin MJ Arch Microbiol. 1985; 141: 116-122. [33] Balch WE et al. Microbiol Rev. 1979; 43: 260-296. [34] Dridi B, Henry M, El Khéchine A, Raoult D, Drancourt M. 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Claims (6)

REVENDICATIONS1. Sterilized culture medium useful for the cultivation of a methanogenic archaea comprising a methanogenic Archaea culture base medium characterized in that it further comprises the following components: a-Selenite salt at a concentration of 5.10-3 mM at 0.5 mM, and b- tungstate salt at a concentration of 5.10-3 mM to 0.5 mM, and c- molybdate salt at a concentration of 10-3 to 10-2 mM, and d- salt. nickel at a concentration of 10-3 to 10-2 mM, and 10 e-methanol at a concentration of at least 5 mM, preferably 10 to 100 mM. Culture medium according to claim 1, characterized in that it comprises a concentration of 10 to 50 mM of methanol. 3. Culture medium according to one of claims 1 or 2, characterized in that it further comprises the following components: a-selenite salt Na2Se03, at a concentration of 0.001 mg / l to 0.1 mg / I, preferably 0.015 mg / l, more preferably in the form of hydrate Na2Se0.5H2O, and b- tungstate salt Na2W04, at a concentration of 0.001 mg / l to 0.1 mg / l, preferably 0, 02 mg / l, more preferably in the form of hydrate Na2W04.2H20, c- molybdate salt Na2MoO4, at a concentration of 0.01 to 0.1 mg / l, preferably 0.02 mg / l, preferably still in the form of hydrate Na2MoO4.2H20, and 25 e-nickel salt NiCl2 at a concentration of 0.01 to 0.1 mg / l, preferably 0.7 mg / l, preferably in the form of hydrate NiCl2 .6H20.4. Culture medium according to one of claims 1 to 3, characterized in that it further comprises: salt formate, at a molar concentration of 1 mM to 10 mM, preferably 6 mM. 5. Culture medium according to one of claims 1 to 4, characterized in that it comprises a formate salt NaHCO2 at a concentration of 0.1 to 1 g / l, preferably 0.4 g / l. 6. Culture medium according to one of claims 1 to 5, characterized in that it further comprises volatile fatty acids, comprising a short carbon chain of less than 6 carbon atoms, at a concentration of 1 to 10 mM. preferably from 5 to 7 mM for each of said volatile fatty acids. 7. Culture medium according to claim 6, characterized in that said volatile fatty acids comprise the following acids: valeric acid, isovaleric acid, acid 2-methylbutyric acid, isobutyric acid and 2-methylvaleric acid. 8. Culture medium according to one of claims 1 to 7, characterized in that said base medium comprises at least: - several sources of carbon and nitrogen, preferably selected from a yeast extract, a salt of acetate and a tryptic peptone, - a source of phosphorus, preferably a phosphate salt, - a source of nitrogen, preferably an ammonium salt, - at least one salt of each of the metals K, Mg, Na and Ca, preferably NaCl, - a reducing substance, more particularly selected from cysteine-HCl and Na2S, - a pH regulating buffer substance to adjust the pH of 7 to 8, preferably NaHCO3 to adjust the pH to 7.5 - trace elements, preferably salts selected from salts of B, Fe, Cu, Zn, W, Se, Cu, Mn, Co, Ni and Mo, more preferably trace elements of Widdel's medium, - vitamins, preferably the vitamins of Balch's medium, and preferably a redox control indicator of anaerobic, preferably resazurin. 9. Culture medium according to one of claims 1 to 6, characterized in that said medium comprises the following species: KH2PO4, K2PO4, MgSO4.7H20, NaCl, KCl, KOH, NH4Cl, CaCIZ.2H20, KOH, NaCOOCH3 cysteine yeast extract, trypticase peptone, NaOH, NaHCO2, CH3OH, Na2S, NaHCO3, NaHCO2, Na2Se035H2O, Na2WO4H2O, Na2MoO4.2H2O, NiC12.6H2O, FeSO4.7H2O, widdel oligoelements, Balch vitamins and preferably resazurin; the pH being adjusted to 7.5. 10. Process for the preparation of a sterilized culture medium according to one of claims 1 to 9, characterized in that the following successive steps are carried out in which: a culture medium is placed which does not contain the components likely to to be degraded by heating comprising said methanol, said vitamins, labile sodium salts comprising Na HCO2, Na HCO3 and Na2S and, where appropriate, said volatile fatty acids (VFA), hereinafter referred to as "incomplete culture medium" under anaerobic atmosphere, preferably under a stream of nitrogen in at least one sealed tube, and said tube is sealed, and 2 preferably the gaseous atmosphere of said sealed tube is replaced by a N 2 / CO 2 mixture at 80/20%, and 3-is sterilized by heating said incomplete culture medium, preferably in an autoclave, at a temperature above 100 ° C for at least 15 minutes, and Said incomplete culture medium is cooled to ambient temperature and said components, which may be degraded by sterilization in an autoclave comprising methanol, said vitamins, said labile sodium salts comprising Na HCO 3, are added to said incomplete culture medium; NaHCO2 and Na2S. 11. A method of culturing an archaea with a culture medium according to one of claims 1 to 9, wherein the following successive steps are carried out: 1-is inoculated a biological sample capable of containing a said archaea in said medium of in anaerobic culture, and 2- placing said culture medium under an anaerobic atmosphere containing at least 50% hydrogen at a pressure of less than or equal to 2 atm, and incubating at a temperature of 20. at 40 ° C, preferably at 37 ° C, to reach an exponential growth phase with evolution of methane in at least 1 day, preferably not more than 15 days, more preferably not more than 7 days. 12. Process according to claim 11, characterized in that a culture of a methanogenic archaea selected from Methanobrevibacter smithii, Methanobrevibacter oralis, Methanobrevibacter stadtmanae, Methanobrevibacter luminyensis and Methanobrevibacter is carried out. beijingense and Methanobrevibacter. concilii, Methanosarcina acetivorans. 13. A method for determining the presence of an archaea in a biological sample, preferably in a sample of animal or human stool, said archaea being cultured according to the method of one of claims 11 to 12, characterized in that the DNA is extracted from said biological sample and the DNA of said Archaea is detected and, preferably, specifically quantified by PCR type enzyme amplification, preferably of the PCR type in real time, with a pair of primers and preferably a probe, sequences derived from the gene 16S rDNA. 14. Method according to claim 13, characterized in that for carrying out the extraction of the prokaryotic DNA from said stool sample, the steps are carried out in which: 1) a homogeneous suspension of said stool sample is carried out in a buffer solution, at a dilution of 0.01 to 1 g / l, preferably at a dilution of 0.2 g / l, and 2) automated mechanical lysis is carried out by mixing and stirring said suspension of step 1) with a abrasive spraying product, preferably preferably acid-washed glass powder, in a device of the type performing a strong stirring, preferably by multidirectional rotation, and 3) more preferably, the resulting suspension is heated to minus 100 ° C. for at least 10 minutes, and 4) chemical and / or enzymatic lysis is carried out by mixing and stirring the supernatant of the suspension obtained in step 2) or 3) with one or more chemical lysis buffers and / or enzymatic, then 5) the step 2) of mechanical lysis is repeated, but with the mixture obtained in step 4), and 6) preferably, the chemical and / or enzymatic lysis step 4) is repeated with the mixture obtained in step 5). 15. Kit useful for a method according to one of claims 13 to 14, characterized in that it comprises: - DNA extraction reagents comprising at least one abrasive powder product, preferably glass powder and a chemical and / or enzymatic lysis reagent; and DNA amplification reagents comprising amplification primers and detection probes of said methanogenic Archaea.