How to control quality

Abstract

The present invention relates to a method for typing archaea and rapidly identifying contaminants in pure strain cultures of methanogenic archaea, thereby isolating mutant variants from the culture population.

Description

[Technical Field]

[0001] The present invention relates to a method for typing archaea and rapidly identifying contaminants in pure strain cultures of methanogenic archaea.

[0002] The project that led to this patent application was partly funded by the European Union's Horizon 2020 Research and Innovation programme under contract number 691797. [Background technology]

[0003] The search for cheaper, more efficient energy supply solutions for the environment is a challenge set out by the European Commission, US business groups, environmental organizations and governments, and climate action programs around the world, from Japan, which is attempting to replace nuclear energy for electricity supply in the wake of the tragic Fukushima accident, to seven emerging economies, and therefore a global effort by member states to address climate change and ultimately stabilize the planet's temperature, with the ultimate goal being to meet the growing need for sustainable, renewable energy while addressing climate change, towards a transition to a low-carbon, modern economy.

[0004] Methane has the highest energy density per carbon atom of any volatile hydrocarbon, and its potential for energy conversion, either directly by combustion in the presence of oxygen or using fuel cells to generate electricity, is higher than that of any other natural gas.

[0005] As such, methane, like natural gas, constitutes a sustainable and renewable energy source that is already today increasingly replacing coal and other fossil fuels. However, the secondary quest for a significant reduction in the environmental impact and the effectiveness of its production, storage and transportation is still incomplete.

[0006] One of the main technical difficulties in the large-scale development of methane, for example, relates to the high levels of contaminants remaining in fossil natural gas, i.e., the need for costly purification procedures. Furthermore, many unresolved issues, such as the need for sufficient storage plants for methane and the associated need for better pressurized containment systems, the odor caused by large gas accumulations, mostly due to sulfate-rich contaminants in natural gas, the risk of explosions, and leakage during transportation and storage, all currently hinder and delay the development of methane.

[0007] However, the potential energy production capacity of methane is becoming increasingly important in the global market. Therefore, recent research has focused on the development and improvement of methods for producing methane using methanogens, such as archaea, which can produce renewable methane from carbon dioxide and hydrogen very efficiently. Currently, the current state of the art describes several attempts to concentrate gas compositions containing methane produced by methanogenic microorganisms. For industrial methane production using archaea, for example, Methanothermobacter thermoautotrophicus DSM3590 strain (deposited at and available from the German collection of microorganisms and cell cultures (DSMZ)) is typically used.

[0008] This type of methane production, carried out in suitable bioreactors / cells, can be easily set up anywhere in the world, even in infrastructure-poor conditions, and the necessary raw materials are generally present in the atmosphere. Most importantly, this type of methane production generally produces a methane-rich gas composition with fewer contaminants, which is expected to require less effort to feed into energy research systems. Furthermore, methane is converted to energy using only carbon dioxide and water, making it the cleanest hydrocarbon fuel.

[0009] Upgrading biomethane production to a scalable, reliable, and established renewable energy source remains a challenge, particularly due to the requirement for a sustainable process.

[0010] However, due to its promising potential, large-scale utilization of biomethane is currently under careful political and economic scrutiny to make the technology viable and cost-effective, and utilization of methane is recognized as the most important short-term goal in biochemical engineering. Therefore, effective solutions to the above problems and improvements to the production process carried out by methanogenic archaea are urgently needed.

[0011] One of the key components enabling the efficient production of methane-enriched gas compositions is a distinct methanogenic microbial strain that is characterized by and exhibits uniform behavior across a number of parameters, and evidence supports that efficient methane production is linked to this uniformity of behavior, and thus to the purity of the culture medium.

[0012] Like conventional heterologous cultures, cultures initiated from pure strains may also undergo adaptation during their life cycle, for example, due to transient parameter fluctuations, resulting in the appearance of modified organisms in the culture. Although adaptation in natural systems is usually attributed to natural coping mechanisms and is intended to improve the culture and maximize the use of resources available in the surrounding environment, in the long term, the emergence of genetically modified organisms due to adaptation processes may alter global systems.

[0013] Existing methods for accurately typing archaea, using either fatty acid analysis (Daria V. Dibrova, Environ Microbiol. 2014 Apr;16(4):907-918; Villanueva L. et al., Environ Microbiol. 2017 Jan;19(1):54-69. doi:10.1111 / 1462-2920.13361. Epub 2016 Jul 7), riboprinting (Clark CG, J Eukaryot Microbiol. 1997 Jul-Aug;44(4):277-83), phenotyping, or PCR techniques, are not rigorous, accurate, widely accepted, and / or directly applicable. Furthermore, these methods only help identify the genus or family of the microorganism, but not the species. Furthermore, the ability of these methods to provide high resolution results under varying parameter conditions for pure strain cultures is neither suggested nor tested in these publications.

[0014] Furthermore, strategies for identifying methanogenic archaea in soil, feces, culture medium, or other similar substrates or ecological niches are quite recent, some of them less than 10 years old, and appear to result, among other reasons, from the growing interest in identifying microorganisms capable of fixing carbon dioxide in said substrates for the purpose of producing improved biofuels. Furthermore, in the case of microbial colonies in complex environments, the synergistic behavior of these species can lead to and persist imprecision due to the difficulty in distinguishing between species, to the extent that even identifying different species from available clone libraries can be a difficult task.

[0015] In particular, Cleland et al. (2008) examined a repeat sequence-based PCR method, primarily developed for bacteria and used in hospital and community infections, source analysis of contamination, epidemiological investigations, and genotyping and identification of archaea. Their paper focused on methanogens and extreme halophiles in cultures where growth phase and growth conditions are not systematically varied. It is well known how variations in these parameters can induce structural changes in DNA, raising questions about the reliability of any such results when applied to pure cultures where the feed and microenvironment are constantly changing, and maintaining said purity is paramount to providing an efficient methanogenic process.

[0016] Their method uses PCR amplification of short intervening repetitive DNA regions to distinguish between different archaeal species, but although the method has been shown to be effective, it has not been reported to work in a variety of environments and in cultures with little variation in their composition.

[0017] While the prediction of heterogeneity in uncontrolled / natural environments certainly facilitates methods to roughly identify and / or distinguish and / or isolate different species, it does not provide a targeted high-resolution assay for pure cultures in controlled environments, and the prediction of heterogeneity is lower.

[0018] Variation within the same species can occur throughout culture due to adaptation to culture conditions and microenvironment.

[0019] In this framework, distinguishing and / or isolating variations within the same species that arise due to adaptation and result in subtle differences would require highly accurate methods for identifying these subtle differences by precisely targeting them, but of course without prior knowledge of where such subtle differences may reside.

[0020] Thus, there is a need to provide a rapid and inexpensive quality assurance assay for monocultures using pure strains, and further methods that are specific enough to allow differentiation even within the same species. Summary of the Invention

[0021] The object of the present invention is therefore to overcome the described problems of the state of the art and in particular to provide a reliable, fast and effective method for typing archaea to ensure pure cultures. Therefore, a further object of the present invention is to establish a fast and efficient testing of pure archaeal strain cultures during their normal life cycle, which can even be optimized by a parallel cross-testing approach to enable quality control and genotyping in a dynamic environment.

[0022] In the current trend of technological advances to stabilize and intensify methanogenic processes with variable gas compositions while still ensuring high purity of the exiting methane gas composition, accurate quality control of the culture composition plays a key role.

[0023] Therefore, within this framework of technical progress, the present invention, as explicitly set forth in the claims, provides teachings on how to effectively test for the presence of the pure methanogenic microorganism Methanothermobacter thermoautotrophicus DSM 3590 (hereinafter referred to as M. thermoautotrophicus DSM 3590 or MtDSM 3590) and how to distinguish M. thermoautotrophicus DSM 3590 from other archaea. Furthermore, it teaches how to test for the quality and purity of a given composition of culture fluid of an archaeal strain involved in a continuous methanogenic process and how to effectively genotype variants in said culture fluid.

[0024] The object of the present application is achieved by a newly developed method, as defined in claim 2 of the present invention, using a PCR primer pair as defined in claim 1 of the present invention. Further embodiments refer to variants of this newly developed method and to a quality control kit for use in detecting the presence of the methanogenic microorganism Methanothermobacter thermoautotrophicus DSM 3590 in a given sample. Additionally, the method provides for the identification of M. thermoautotrophicus DSM 3590 variants identified by said kit and isolated as described in the subsequent dependent claims. DETAILED DESCRIPTION OF THE INVENTION

[0025] In particular, to achieve the stated objectives, the present invention provides a first polymerase chain reaction (PCR) primer selected from the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and / or a second polymerase chain reaction primer selected from the nucleotide sequences of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, wherein each of the first primers and each of the second primers are capable of hybridizing with the DNA of the methanogenic microorganism M. thermoautotrophicus DSM3590, and SEQ ID NO: 1 and SEQ ID NO: 4, SEQ ID NO: 2 and SEQ ID NO: 5, and SEQ ID NO: 3 and SEQ ID NO: 6 constitute primer pairs used for PCR amplification of a portion of the genomic DNA sequence of M. thermoautotrophicus DSM3590.

[0026] The strain "Methanothermobacter thermoautotrophicus DSM3590" used in this application is also known as M. thermoautotrophicus str. hveragerdi and is commercially available at the DSMZ (German collection of Microorganisms and cell cultures). Interestingly, it should be noted that no sequence data is available for this strain.

[0027] The inventors of the present invention set themselves the task of providing a simple method for identifying M. thermoautotrophicus DSM3590 in a given sample containing archaea, compared to current methods that are time-consuming, such as genome sequencing and / or amplicon sequencing.

[0028] First, through trial and error experiments, the inventors designed various random primers with the aim of creating primers that are suitable for amplifying the DNA sequence of M. thermoautotrophicus DSM3590 and for distinguishing the DNA sequence of M. thermoautotrophicus DSM3590 from the DNA sequences of other archaea other than MtDSM3590.

[0029] In other words, the inventors of the present invention developed a primer pair that specifically amplifies the gene sequence of the species M. thermoautotrophicus DSM3590, and the primer pair is not suitable for PCR amplification of the gene sequences of archaeal species other than M. thermoautotrophicus DSM3590, as ruled out by trial and error experiments, making it possible to distinguish M. thermoautotrophicus DSM3590 from these other archaeal species. That is, the idea was that by using these primers in a PCR reaction, it would be possible to test whether M. thermoautotrophicus DSM3590 DNA, and only M. thermoautotrophicus DSM3590 DNA, is present in a sample containing archaeal DNA.

[0030] Surprisingly, from among the large number of primers tested, the inventors have found that the primers disclosed according to the present invention are suitable not only for amplifying the DNA sequence of M. thermoautotrophicus DSM3590, but also for amplifying the DNA sequence of at least one other archaea other than Methanothermobacter thermoautotrophicus.

[0031] More interestingly, the present inventors have found that the amplicons generated by the primers of the present invention contain single-base changes within the DNA sequence (amplicon) amplified by the primers compared to other archaea other than Methanothermobacter thermoautotrophicus and compared to other variants related to M. thermoautotrophicus DSM 3590. These single-base changes have been found to make it possible to distinguish M. thermoautotrophicus DSM 3590 from other archaea other than MtDSM 3590 and from other variants related to M. thermoautotrophicus DSM 3590 using specific downstream digestion reactions, as shown herein below.

[0032] Thus, the particularly surprising advantage that makes the primers according to the invention different from other conventional primers capable of binding to nucleic acid sequences of methanogenic microorganisms lies in their ability to discriminate with high resolution within the same species, making it possible to identify sequence variations that would otherwise be assigned to the same species from which they originate.

[0033] The PCR primer pair is used in a method for detecting the presence of the methanogenic microorganism M. thermoautotrophicus DSM3590 according to the present invention, which method comprises the following steps: Obtaining a sample containing methanogenic microorganisms (cells) and applying means and techniques for releasing DNA from the microorganisms to obtain a non-purified DNA sample or to obtain a sample containing purified DNA of the methanogenic microorganisms. A step of obtaining an amplified DNA sequence (amplicon) using a non-purified or purified DNA sample according to step a. in PCR amplification using a primer pair comprising the first and second primers of claim 1, wherein the amplicon contains at least one restriction enzyme recognition sequence. purifying each of the amplicons. performing a first digestion reaction on the amplicon of step c. by incubating the amplicon in a reaction buffer containing at least one first restriction enzyme that recognizes a first recognition sequence for a time sufficient to form restriction fragments. determining the number of restriction fragments formed and their sizes (restriction pattern), for example via gel electrophoresis; genotyping the methanogenic microorganism based on its restriction pattern. Optionally, sequencing the purified amplicon or at least a portion thereof in step c. and / or the amplicon or at least a portion thereof after the digestion reaction in step d.

[0034] To test whether the primer pair of the present invention specifically amplified M. thermoautotrophicus DSM 3590, the inventors performed quality control by exposing the amplicon generated using the primers of the present invention to a digestion reaction by applying a first restriction enzyme after PCR amplification. Interestingly, the first restriction enzyme, which recognizes the first recognition sequence, did not digest the amplicon of M. thermoautotrophicus DSM 3590.

[0035] However, surprisingly, the inventors of the present invention identified a restriction pattern unrelated to the species of M. thermoautotrophicus DSM3590 by performing the above-described restriction enzyme digestion reaction on a predetermined amplicon of a sample that was thought to contain only the archaeal species M. thermoautotrophicus DSM3590 (i.e., the sample was thought to be a pure monoculture sample). This unrelated restriction pattern indicated the presence of a variant strain of DSM3590. That is, the inventors unexpectedly found that a single restriction fragment was observed using gel electrophoresis analysis.

[0036] Through further testing involving amplicon sequencing, the inventors found that the primers were also capable of amplifying DNA sequences of other M. thermoautotrophicus strains different from M. thermoautotrophicus DSM3590.

[0037] In step f), the genotyping of methanogenic microorganisms, which are M. thermoautotrophicus DSM3590 or which are not M. thermoautotrophicus DSM3590, can be assessed visually based on the restriction pattern of the primer-amplified products after digestion.

[0038] This at least one first restriction enzyme recognition sequence can therefore be used in a subsequent restriction fragment length polymorphism (RFLP) analysis, as is widely known in the state of the art, to distinguish the resulting restriction pattern of a given amplicon of M. thermoautotrophicus DSM3590 treated with a restriction enzyme from the restriction patterns of other archaeal samples other than MtDSM3590, also treated with at least one first restriction enzyme, in particular.

[0039] According to an embodiment of the present invention, the M. thermoautotrophicus DSM3590 amplicon alone will yield a restriction pattern of amplicons equivalent to the respective undigested amplicon, whereas other M. thermoautotrophicus variants will yield at least two restriction fragments of a given size, and most ideally, a single base change in the first recognition sequence of the amplicon will yield fragments of different sizes that can be clearly distinguished by appropriate means, in particular gel electrophoresis.

[0040] According to the present invention, it is first necessary to locate the respective region of genomic DNA and amplify it using the primers of the present invention so that the presence of the nucleotide sequence to be analyzed can be subsequently demonstrated. Another requirement of the method of the present invention is that the restriction enzyme recognition sequence is present only in M. thermoautotrophicus DSM3590 or, conversely, only in archaea other than M. thermoautotrophicus DSM3590 (hereinafter referred to as "non-MtDSM3590") due to a single base change.

[0041] During ongoing experiments, these single base changes in the amplicon (also referred to as "single nucleotide polymorphisms" (SNPs)) were found to be suitable for performing a digestion reaction using a selective restriction enzyme that recognizes a recognition sequence within the amplicon, the presence of which depends on the identity of each nucleotide base of this SNP contained in the recognition sequence. In other words, depending on the presence and nucleotide identity of this SNP, the recognition sequence is either recognized or not recognized by the respective restriction enzyme. That is, the recognition sequence is either present and readable or absent, i.e., destroyed. Thus, the method according to the present invention can directly provide evidence of the presence of the nucleotide change without further sequencing the amplicon. The method can also be used to identify the respective archaeal strains based on the single base change.

[0042] A "single nucleotide polymorphism (SNP)" according to the present invention refers to a nucleotide change (transversion or transition), as well as a deletion or insertion of one or more nucleotides in a nucleotide sequence, generally resulting in (at least) a single base change in the nucleotide sequence.

[0043] One advantage of the method underlying the present invention (also known as the PCR-RFLP or CAPS assay) is that specific fragment patterns can be easily visualized by gel electrophoresis after digestion of PCR products. For example, this may be the case for a mixed archaeal culture of two archaeal variants (different from each other by SNP11, SNP19, or SNP20), where the SNP is located within a recognition sequence and differs between the two different archaea. By applying the present invention to the restriction patterns of these cultures using at least one specific restriction enzyme that recognizes the restriction sequence due to the SNP, a specific, distinct band can be seen in the agarose gel after gel electrophoresis. Thus, applying this method to the present invention allows for easy and informative visual identification of M. thermoautotrophicus DSM3590 from other archaea and for determining whether a pure seed culture or a mixed culture is present.

[0044] Advantageously, the inventors of the present invention have found that the method makes it possible to clearly identify whether a given starting sample contains a pure M. thermoautotrophicus DSM3590 culture medium containing only M. thermoautotrophicus DSM3590 as archaea, or whether a given starting sample also contains non-MtDSM3590 archaea or only non-MtDSM3590 archaea.

[0045] Notably, the methods of the present invention are easy to perform and provide reliable results for identifying whether a given sample containing methanogenic microorganisms contains only M. thermoautotrophicus DSM3590 or other non-MtDSM3590 methanogenic archaea, avoiding time-consuming and expensive methods. Furthermore, the methods of the present invention advantageously allow for the identification of M. thermoautotrophicus DSM3590 and the differentiation of M. thermoautotrophicus DSM3590 from other methanogenic archaea, as well as the identification of other M. thermoautotrophicus-based strains, avoiding time-consuming and expensive sequencing methods, such as whole genome sequencing. Given the accuracy of the methods of the present invention, sequencing of amplicons generated with PCR primer pairs is not even necessary, although sequencing may, of course, be performed for further quality control reasons.

[0046] Amplification of a desired region of DNA is accomplished by polymerase chain reaction (PCR). "Amplification," according to the present invention, is understood as the creation of additional copies of a nucleic acid sequence. Amplification is generally accomplished using PCR techniques, which are well known in the art. "Polymerase chain reaction," or "PCR," is a method that uses PCR primers to increase the concentration of a segment of a target sequence in a mixture of genomic DNA.

[0047] According to the present invention, a "primer" is understood to mean an oligonucleotide (whether naturally occurring, as in a purified restriction digest, or synthetically produced) that can act as a point of initiation of synthesis when placed under conditions that induce the synthesis of a primer extension product that is complementary to a nucleic acid strand (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase, and at a suitable temperature and pH). The primer is preferably single-stranded to maximize amplification efficiency, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of an extension product in the presence of an inducing agent. The exact length of the primer will depend on many factors, including temperature, source of primer, and the use of the method. PCR primers are preferably at least about 10 nucleotides in length, most preferably at least about 20 nucleotides in length. Examples of PCR primers that may be used in the methods of the present invention include primers such as those found in SEQ ID NOS: 1-6.

[0048] As used herein, the terms "restriction endonuclease" and "restriction enzyme" refer to bacterial enzymes that cleave double-stranded DNA at or near specific nucleotide sequences known as "recognition sequences." They are "sequence-specific DNA endonucleases" that can create double-stranded breaks in double-stranded DNA in a sequence-specific manner at one or more recognition sequences. The DNA cleavage can result in blunt DNA ends or so-called "sticky" ends (with 5' or 3' overhangs). The cleavage site can be located within or outside the recognition sequence. Various types of endonucleases can be used. Restriction enzymes are well known in the art and can be readily obtained, for example, from various suppliers (e.g., New England Biolabs, Inc., Beverly, Massachusetts). Similarly, methods for using restriction enzymes are generally well known and routine in the art. Preferred restriction enzymes are those that generate at least two DNA fragments when cleaving an amplicon. The DNA fragments obtained using restriction enzymes can be detected as bands, for example, by gel electrophoresis. Restriction enzymes can be used to generate restriction fragment length polymorphisms (RFLPs). An "RFLP" is essentially a snapshot of the unique fingerprint of a piece of DNA (whether an entire chromosome (genome) or a portion thereof (such as a genomic region)) that contains the SNP locus disclosed in the present invention.

[0049] RFLPs are produced by cutting ("restricting") DNA molecules with restriction endonucleases. Hundreds of such enzymes have been isolated, as they are naturally produced by bacteria. Hundreds of different restriction enzymes are known to cut (i.e., "cleave" or "restrict") DNA at different sequences of the four basic nucleotides (A, T, G, and C) that make up all DNA molecules; for example, one enzyme can specifically recognize only the sequence A-AT-GAC, another can specifically recognize only the sequence GTACTA, and so on. Depending on the particular enzyme involved, such recognition sequences can vary in length from as short as four nucleotides to as long as 21 nucleotides. The longer the recognition sequence, the fewer restriction fragments will be produced; the larger the recognition site, the lower the probability that the recognition site will be repeated throughout the DNA.

[0050] After digestion, the resulting individual fragments are separated from one another based on their size.

[0051] Any method suitable for separating DNA is encompassed by the methods of the present invention, including, but not limited to, gel electrophoresis, high performance liquid chromatography (HPLC), mass spectrometry, and the use of microfluidic devices.

[0052] In one embodiment, DNA fragments are separated by agarose gel electrophoresis. Gel electrophoresis separates charged molecules of different sizes according to the speed at which they migrate through a stationary gel under the influence of an electric current. These separated DNA fragments can be easily visualized, for example, by staining with ethidium bromide and viewing the gel under UV illumination. The banding pattern reflects the size of the restriction-digested DNA.

[0053] Other methods using the novel SNPs of the present invention to detect or genotype M. thermoautotrophicus DSM3590 or to distinguish M. thermoautotrophicus DSM3590 from variants of M. thermoautotrophicus DSM3590 are also taught herein. These methods include hybridization methods using the nucleic acid molecules of the present invention as probes or nucleic acid molecules capable of hybridizing to the disclosed nucleotide sequences of the present invention. See, for example, Sambrook et al. (1989) Molecular Cloning: Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York). Such methods using hybridization techniques are well known to those of skill in the art and are incorporated herein by reference. These methods include, but are not limited to, techniques well known to those of skill in the art, such as Southern blots, mobility shift assays, and fluorescent in situ hybridization (FISH).

[0054] In hybridization techniques, the hybridization probe(s) may be genomic DNA fragments, PCR amplification products, or other oligonucleotides, and may contain all or part of the known nucleotide sequences disclosed herein. 32 The probe may be labeled with a detectable group such as P, or any other detectable marker such as other radioisotopes, fluorescent compounds, enzymes, or enzyme cofactors. With respect to probes, the term "labeling" is intended to include directly labeling the probe by attaching (i.e., physically linking) a detectable substrate to the probe, and indirectly labeling the probe by reactivity with another reagent that is directly labeled. An example of indirect labeling is biotin labeling the end of a DNA probe so that it can be detected with fluorescently labeled streptavidin.

[0055] Methods including any hybridization techniques as disclosed above may be used to isolate the respective M. thermoautotrophicus DSM3590 or also to isolate the respective variants.

[0056] By "sample" herein is meant a liquid or solid source of methanogenic archaea, e.g., derived from a deposited microbial population and / or alternatively obtained from a number of environmental sources, as reported in the state of the art. Generally, examples of environmental sources of methanogenic microorganisms include anaerobic soils and sands, marshes, swamps, swamps, estuaries, dense algal reefs, both terrestrial and marine muds and sediments, e.g., subsurface tidal flat sediments, deep ocean and deep well bottoms, sewage, organic waste dumps and treatment facilities, and animal intestines and feces. Furthermore, a "sample" containing methanogenic archaea can be a sample obtained directly from the liquid phase of a running bioreactor, i.e., a bioprocess fermentation.

[0057] In the context of the present invention, a bioreactor refers to a biological reactor, as contemplated in the state of the art, and is either a biological reactor vessel, a biological reactor enclosure or tank, and / or at least a biological reactor chamber and / or cell, or a combination thereof, capable of withstanding variations, e.g., temperature and / or pressure, and / or maintaining, e.g., any given temperature and / or pressure, before, after, or during the reaction process, even if the temperature and / or pressure is assigned or must be maintained, and capable of carrying out the target reactions suitable for carrying out the present invention. Such reactions are understood as biological reactions because they relate to the range of reactions involving microorganisms, and herein refer to normal physiological processes, such as metabolic fermentation, aerobic digestion, or anaerobic digestion, which require a suitable environment, a suitable culture of microorganisms, a suitable culture medium, and suitable reaction substrates for the reaction to take place. A bioreactor, in the sense of the present invention, is expected to operate reliably within the tolerances of each variable and reliably perform the enumerated steps over time, in order to enable the disclosed method.

[0058] In an embodiment of the invention, the method further comprises sequencing the purified amplicon or at least a portion thereof in step d. and / or the amplicon or at least a portion thereof after the digestion reaction in step e. This step can optionally be performed to further verify the results of the method of the invention, as described above.

[0059] According to an embodiment of the present invention, the method further comprises subjecting the sample containing methanogenic microorganisms to at least one freezing and thawing cycle according to step a. to release DNA from the methanogenic microorganisms. That is, it is also possible to apply means and techniques for releasing DNA from the methanogenic microorganisms before performing a PCR reaction according to step b. of the method. Other means and techniques for releasing DNA from cells, including unicellular methanogenic microorganisms, are well known to those skilled in the art and are included in the present invention. Using unpurified DNA generated from this approach for further PCR reactions according to the method of the present invention has the advantage of eliminating time-consuming and expensive purification. However, obstacles and risks in subsequent reactions may still be that the amount of DNA may not be calculated in the unpurified DNA sample and that DNAse may be present, actively digesting the genomic DNA of the sample. However, these factors can be controlled and minimized by using a fresh cell sample and always preparing DNA on ice. Of course, it is also possible to add a DNAse inhibitor to these samples that does not interfere with the components of the subsequent reaction (e.g. PCR) and / or to calculate the amount of DNA using appropriate state-of-the-art methods known to those skilled in the art.

[0060] According to another embodiment of the method of the present invention, the method further comprises the following steps: performing a quality control of the genotyping of step f. by performing a second digestion reaction on the amplicon of step b. or step c. by incubating the amplicon in a reaction buffer containing at least one second restriction enzyme that recognizes a second recognition sequence that partially overlaps the first recognition sequence for a time sufficient to form restriction fragments; determining the number of restriction fragments formed and their sizes (restriction pattern), e.g., via gel electrophoresis; genotyping the methanogenic microorganism based on its restriction pattern.

[0061] The second recognition sequence is present in at least an amplicon of the archaeal strain that was not cleaved by the first restriction enzyme in the first digestion reaction due to a single nucleotide polymorphism (SNP) in the second recognition sequence of the amplicon, i.e., the second recognition sequence is present in at least an amplicon of M. thermoautotrophicus DSM3590 or at least an amplicon of a non-MtDSM3590 archaea.

[0062] According to an embodiment of the invention, the second recognition sequence is present in both the M. thermoautotrophicus DSM3590 amplicon and the non-MtDSM3590 archaeal amplicon.

[0063] In another embodiment, the second recognition sequence is present only in the M. thermoautotrophicus DSM3590 amplicon due to a single nucleotide polymorphism (SNP) in the second recognition sequence of the M. thermoautotrophicus DSM3590 amplicon, and therefore the second restriction enzyme is selected based on the fact that it digests only amplicons derived from M. thermoautotrophicus DSM3590.

[0064] Surprisingly, the inventors have found that, after identifying a restriction pattern that is unrelated to the restriction pattern of M. thermoautotrophicus DSM3590, performing the second digestion reaction provides a restriction pattern that is useful for further distinguishing the found variant from M. thermoautotrophicus DSM3590.

[0065] Optionally, a genotyping step based on the amplified DNA (amplicon) can then be applied, eg determined by sequencing at least part of the amplicon or variant amplicons.

[0066] Each of the found variants related to M. thermoautotrophicus DSM3590 can be unambiguously identified and / or characterized by optionally genotyping the variant strain based on the amplified DNA (amplicon) and then confirming the genotyping of the methanogenic microorganism based on the restriction pattern according to step i.) of the method of the present invention by comparing it with sequences found in public sequence databases. Furthermore, publicly available programs that allow prediction of restriction sites in a given DNA sequence with a given restriction enzyme may be applied to the sequenced amplicons to advantageously further verify the previously observed size-dependent restriction patterns found by the inventors, i.e., allow another quality control.

[0067] "Quality control" or "quality assay" in the sense of the present invention refers to carefully and systematically controlling or confirming the identity of a species / strain in a culture to assess the purity of the species / strain in the culture. According to the present invention, species / strain purity means that the genomes of all individuals of the culture are relatively identical across individuals of the same single species / strain culture. Generally, "relatively identical" means that the nucleotide sequence of the genome is at least 95%, 96%, 97%, or 98% identical, or at least 98% or 99% identical. Methods for sequencing small portions of DNA as amplicons are well known to those skilled in the art, and include, for example, Sanger sequencing.

[0068] Surprisingly, according to another embodiment, the inventors are even able to isolate, in a further step, said variant strains found using common methods of the state of the art, said variants optionally being genotyped in a further step based on comparison of the genomic DNA of the variants, assessed for example by sequencing the genomic DNA or at least part of it.

[0069] That is, the method of the present invention enabled the inventors to identify M. thermoautotrophicus, which was designated as strain UC120910. The strain "Methanothermobacter thermoautotrophicus strain UC120910" used in this application is also synonymously referred to as ECH0100 (laboratory name).

[0070] According to a further embodiment, the method of the invention is used to distinguish between the M. thermoautotrophicus DSM3590 strain and other M. thermoautotrophicus strains or strains from other archaea, for example from the group consisting of Methanothermobacterium, Methanobrevibacter, Methanothermobacter, Methanococcus, Methanosarcina, Methanopyrus, Methanospirillium, Methanosaeta, Methanogenium, Methanoculleus and Methanothermococcus, or mixtures thereof.

[0071] In some cases, archaeal cultures may spontaneously modify in response to the specific conditions they are cultivated in. Culture conditions are influenced by several parameters, such as temperature, pH, pressure, cell density, volume, humidity, salt content, conductivity, carbon content, nitrogen flux, vitamin content, amino acid content, mineral content, or a combination thereof, and depending on each of these conditions, any number of species may undergo certain adaptation processes within the reactor environment.

[0072] The advantage of using a particular pure strain depends on increasing methanogenic efficiency and therefore on monitoring its identity over time to ensure its purity is maintained. The methods provided herein are well suited to identifying strains, i.e., ensuring a pure culture and therefore maintaining high methanogenic efficiency.

[0073] According to one embodiment, the provided method distinguishes between different strains based on at least one SNP in the amplicon, which SNP is part of the overlapping sequence of a first recognition sequence of a first restriction enzyme and a second recognition sequence of a second restriction enzyme, and which SNP is selected from the group selected from SNP19, SNP11, and SNP20.

[0074] In the context of the present application, "SNP19" is understood as a single nucleotide polymorphism at position 418 in a PCR fragment (amplicon) of the genomic nucleotide sequence of a non-MtDSM3590 archaeon, PCR-amplified with the primer pair of SEQ ID NO: 3 (forward primer) and SEQ ID NO: 6 (reverse primer), when compared to a PCR fragment (amplicon) of the genomic nucleotide sequence of M. thermoautotrophicus DSM3590 at position 418, amplified with the same primer pair.

[0075] In one embodiment of the present invention, the non-MtDSM3590 archaea is M. thermoautotrophicus UC120910, and the single nucleotide polymorphism is a G to A transition mutation in the corresponding amplicon.

[0076] In the context of the present application, "SNP11" is intended to mean a single nucleotide polymorphism at position 105 in a PCR fragment (amplicon) of the genomic nucleotide sequence of a non-MtDSM3590 archaeon, PCR-amplified with the primer pair, i.e., SEQ ID NO: 1 (forward primer) and SEQ ID NO: 4 (reverse primer), when compared to a PCR fragment (amplicon) of the genomic nucleotide sequence of M. thermoautotrophicus DSM3590, PCR-amplified with the primer pair, i.e., SEQ ID NO: 1 (forward primer) and SEQ ID NO: 4 (reverse primer).

[0077] In the context of the present application, "SNP20" is intended to mean a single nucleotide polymorphism at position 168 in a PCR fragment (amplicon) of the genomic nucleotide sequence of a non-MtDSM3590 archaeon, PCR-amplified with the primer pair, i.e., SEQ ID NO: 2 (forward primer) and SEQ ID NO: 5 (reverse primer), when compared to a PCR fragment (amplicon) of the genomic nucleotide sequence of M. thermoautotrophicus DSM3590, PCR-amplified with the primer pair, i.e., SEQ ID NO: 2 (forward primer) and SEQ ID NO: 5 (reverse primer).

[0078] In an embodiment of the present invention, the identified non-MtDSM3590 archaea have a single-nucleotide deletion mutation as a single-nucleotide polymorphism.

[0079] According to another embodiment, three SNPs, namely SNP19, SNP11 and SNP20, may also be used in a complementary manner for such a discrimination, and thus the method according to the invention may use SNP19, SNP11 and SNP20 in combination for said discrimination. In case of conflicting results, where one result for each SNP differs from the other, the corresponding amplicons may be sequenced in order to genotype the archaeal strain contained in the sample to be analyzed.

[0080] According to another embodiment, if the SNP is SNP19, the first restriction enzyme is BamHI and the second restriction enzyme is AvaII. In yet another embodiment, if the SNP is SNP11, the first restriction enzyme is SfcI and the second restriction enzyme is BstNI or ECORII, or if the SNP is SNP20, the first restriction enzyme is NdeI.

[0081] According to a further embodiment, the non-MtDSM3590 variant identified by the methods of the present invention is designated M. thermoautotrophicus UC120910.

[0082] In a further aspect of the invention there is provided a quality control kit for use in detecting the presence of the methanogenic microorganism M. thermoautotrophicus DSM3590 in a given sample, comprising: at least one container; a first primer according to claim 1 which acts as a forward primer; a second primer according to claim 1 which acts as a reverse primer; - (The first and second primers constitute a primer pair capable of PCR amplifying the DNA sequence of the methanogenic microorganism M. thermoautotrophicus DSM3590) at least one first restriction enzyme according to the invention, or at least one first restriction enzyme and at least one second restriction enzyme, optionally at least one buffer, such as an elution buffer, a storage buffer and / or a reaction buffer, and optionally -Instructions for use and A kit comprising:

[0083] In particular, the quality control kit is intended for use in probing and genotyping cultures of methanogenic microorganisms.

[0084] Restriction enzymes such as those included in the kit are best used in RFLP analysis.

[0085] The kit may also include a control sample or a series of control samples (positive and negative controls) that can be assayed and compared to the included test sample. Each component of the kit is typically contained in an individual container, with all of the various containers being within a single package along with instructions for use. The kit may also include a preservative or protein stabilizing agent.

[0086] The first and second restriction enzymes are selected from the group consisting of AvaII, BamHI, BstNI, NdeI, SfcI and EcoRII.

[0087] Taken together, the three SNP tests as provided by the kit according to the invention may be used complementary and therefore may be performed in parallel. If conflicting results occur, where one assay differs from the other, it is necessary in each case to sequence the corresponding amplicon to genotype the archaeal strain contained in the cell sample being analyzed.

[0088] In another aspect, the present invention relates to the methanogenic microorganism M. thermoautotrophicus DSM3590 variant identified and isolated by the kit according to the present invention.

[0089] In this respect, the method according to the invention has also proven suitable for identifying one particular variant, designated M. thermoautotrophicus UC120910.

[0090] References Dibrova DV, Galperin MY, Mulkidjanian AY..Phylogenomic reconstruction of Archaeal fatty acid metabolism.Environ Microbiol.2014 Apr;16(4):907-18.doi:10.1111 / 1462-2920.12359.Environ Microbiol.2017 Jan;19(l):54-69. Villanueva L, Schouten S, Damste JS.Phylogenomic analysis of lipid biosynthetic genes of Archaea shed light on the 'lipid divide'.Environ Microbiol.2017 Jan;19(l):54-69. Clark CG.Riboprinting: a tool for the study of genetic diversity in microorganisms.J Eukaryot Microbiol.1997 Jul-Aug;44(4):277-83. Cleland D, Krader P, Emerson D. Use of the DiversiLab repetitive sequence-based PCR system for genotyping and identification of Archaea.J Microbiol Methods.2008 May;73(2):172-8. Huang, Q., Baum, L., Fu, W.-L. (2010). Simple and practical staining of DNA with GelRed in agarose gel electrophoresis. Clinical laboratory 56 / 3-4, 149-152. [Brief explanation of the drawings]

[0091] [Figure 1] FIG. 1 is an agarose gel showing the results of electrophoresis of PCR products generated via temperature gradient PCR using test annealing temperatures of 55°C to 70°C for SNP11 and SNP20 amplicons. [Figure 2] Agarose gel showing electrophoresis of PCR products generated via temperature gradient PCR to determine the optimal annealing temperature for SNP19 primers (lanes 9-15). SNP19 amplicon results are plotted from left to right with increasing annealing temperatures. For space reasons, a negative control for SNP19 primers was included on a separate gel. [Figure 3]To examine the sensitivity of the assay, PCR was performed with all primers amplifying SNP11, SNP19, and SNP20 using various dilutions (1:10, 1:50, and 1:100) of 547 ng / μl of starting DNA solution as PCR template, along with their respective negative controls ("NTC" lanes). [Figure 4] This is an agarose gel after gel electrophoresis, loaded with samples derived from the digestion of the SNP19 amplicon using purified genomic DNA samples as the initial template for PCR. The DSM3590 amplicon and non-MtDSM3590 archaeal amplicon were incubated with BamHI restriction enzyme for 2 hours and then loaded onto a 2.5% agarose gel along with the NTC (N) and untreated PCR amplicon. The marker (M) used was the GeneRuler 50-bp DNA Ladder from Thermo Fisher. The use of NEBuffer in the digestion reaction did not result in the expected digestion of the amplicon derived from the non-MtDSM3590 archaeal sample, and the amplicon was not cleaved. However, switching from NEBuffer to CutSmart buffer yielded the expected results: the amplicon derived from the non-MtDSM3590 archaeal sample was cleaved and clearly visible on the gel. [Figure 5] Agarose gel after gel electrophoresis loaded with samples from digestion reactions of SNP11 amplicons using purified genomic DNA samples as the initial template for PCR. DSM3590 amplicons and non-MtDSM3590 archaeal amplicons were incubated with SfcI restriction enzyme for 2 hours and then loaded onto a 2.5% agarose gel along with NTC and untreated PCR amplicons. The marker used was the GeneRuler 50 bp DNA Ladder from Thermo Fisher. [Figure 6]Agarose gel after gel electrophoresis loaded with samples from digestion reactions of SNP20 amplicons using purified genomic DNA samples as the initial template for PCR. DSM3590 amplicons and non-MtDSM3590 archaeal amplicons were incubated with NdeI restriction enzyme for 2 hours and then loaded onto a 2.5% agarose gel along with NTC (N) and untreated PCR amplicons. The marker used was the GeneRuler 50 bp DNA Ladder from Thermo Fisher. [Figure 7] Agarose gels after gel electrophoresis were loaded with samples from the first and second digestion reactions of the SNP19 amplicon, using unpurified DNA from cell samples as the test template and purified DNA samples as the positive control. The UC120910 and DSM3590 amplicons were incubated with BamHI or AvaII, respectively, for 2 hours and then loaded onto a 2.5% agarose gel along with a no-template control (NTC, containing water instead of DNA sample) and samples of each untreated PCR amplicon. The molecular marker used was the GeneRuler 50-bp DNA Ladder from Thermo Fisher.

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[0092] The following examples illustrate practical ways of carrying out the described methods as intended, and are not intended to limit the invention to those examples.

[0093] general part The inventors of the present invention aim to provide a simple method for identifying M. thermoautotrophicus DSM3590 in a given sample containing archaea, compared to time-consuming state-of-the-art methods, such as genome sequencing and / or amplicon sequencing.

[0094] That is, the present inventors designed many primers that were suitable for amplifying the DNA sequence of M. thermoautotrophicus DSM3590 and for distinguishing it from the DNA sequences of other archaea other than Methanothermobacter thermoautotrophicus, and analyzed them through trial and error experiments.

[0095] Surprisingly, the present inventors found that the primers of the present invention not only amplify the DNA sequence of M. thermoautotrophicus DSM3590, but also amplify several other archaea besides Methanothermobacter thermoautotrophicus. Even more interestingly, the present inventors found that the amplicons generated by the primers of the present invention contain single-base changes relative to other archaea besides Methanothermobacter thermoautotrophicus and other M. thermoautotrophicus DSM3590 variants. These so-called SNPs in the amplicon are then suitable for digestion using a selective restriction enzyme that recognizes a recognition sequence within the amplicon. The presence of a recognition sequence depends on the identity of each nucleotide base of the SNP contained in the recognition sequence. In other words, depending on the presence of each specific nucleotide base at the SNP position, the recognition sequence is either recognized or not recognized by the respective restriction enzyme. That is, depending on the presence of each SNP, the recognition sequence is either present and readable by the restriction enzyme or unreadable (destroyed). Therefore, the method according to the invention makes it possible to provide direct evidence of the presence of a nucleotide change without further sequencing of the amplicon, and the method can even be used to identify the respective archaeal strain by means of said single base change.

[0096] All agarose gels tested were stained using 100x concentrated GelRed (Biotium, Fremont, USA) according to the method of Huang et al., 2010. GelRed was added to the gel loading buffer and then mixed with each sample and the size marker used (Gene Ruler 50 bp DNA ladder, Fisher Scientific, Schwerte, Germany).

[0097] The nucleotide sequences of the primers designed according to the present invention are shown in Table 1. [Table 1]

[0098] We first attempted to create a common protocol with identical experimental conditions for all PCR reactions using the primer pairs of the present invention so that all assays could be performed simultaneously. First, purified genomic DNA was extracted from pure M. thermoautotrophicus DSM3590 and non-MtDSM3590 archaea. The DNA concentrations of the resulting preparations were determined photometrically. As a first step, we experimentally determined the amount of template that would yield optimal PCR results. Best results were obtained using 10–20 ng of DNA, although template amplification was sometimes successful at significantly lower or higher concentrations. All primer combinations yielded fragments of the predicted size (see Table 1).

[0099] The DNA polymerase used was AccuPOL polymerase. Its 3' to 5' exonuclease activity allows screening of inserted nucleotides. The proofreading activity reduces the error rate to 1.1 x 10 per base per amplified copy, as reported in the state of the art. -6 Its accuracy is higher than that of Taq DNA polymerase, which has an error rate of 8.0 × 10 as reported in the current state of the art.-6 Based on the AccuPOL polymerase protocol, a master mix formulation according to Table 2 is used. [Table 2]

[0100] To avoid experimental artifacts, the extension time was shortened as much as possible without affecting the amplification of the longest amplicon. To minimize the total time required for amplification, the denaturation times per cycle and during initial denaturation were also shortened, resulting in significant time savings. To improve PCR yield and stringency, temperature gradient PCR was performed for all primers. For example, Figure 1 shows the results for SNP11 and SNP20, and Figure 2 shows the results for SNP19. For primers amplifying SNP11 and SNP19, PCR at an annealing temperature of 57.5°C yielded the strongest band. Notably, for primers amplifying SNP19, only a weak band was obtained at the previous annealing temperature of 55.1°C.

[0101] Primers amplifying SNP20 worked best at the highest temperature, 70.1°C. There was a clear amount of PCR product / temperature gradient. At 55.1°C, no bands were visible on the gel. As the annealing temperature increased, so did the band intensity.

[0102] The experimentally determined ideal reaction conditions were then applied to all of the respective primers using the standard protocol: the annealing temperature was set to 57.1°C for the primers amplifying SNP11 and SNP19, and 70.1°C for the primers amplifying SNP20.

[0103] To further improve the specificity of the PCR reaction, a touchdown program was established for all assays, as long as the ideal annealing temperature was allowed (which was not the case for SNP20), in which the annealing temperature was decreased by 1°C per cycle, followed by further amplification at the final extension temperature over several cycles.

[0104] The touchdown PCR parameters for the primer pair for amplifying SNP11 (sequence number 1 (forward primer), sequence number 4 (reverse primer)) and the primer pair for amplifying SNP19 (sequence number 2 (forward primer), sequence number 5 (reverse primer)) are shown in Table 3. [Table 3]

[0105] The primer pair for amplifying SNP20 (SEQ ID NO: 3 (forward primer), SEQ ID NO: 6 (reverse primer)) has an optimal annealing temperature of 70°C. Therefore, touchdown PCR is not feasible. Therefore, the new annealing temperature was incorporated into the improved PCR program in Table 4. The duration and number of cycles of each step remained unchanged. The PCR program for amplifying SNP20 is listed in Table 4. [Table 4]

[0106] The sensitivity of the assay was examined by preparing a dilution series of the DNA template using the PCR protocol established above. The original concentration of the undiluted DNA template was determined photometrically to be 547 ng / μl. From this DNA solution, 1:10, 1:50, and 1:100 dilutions were prepared, and each of these solutions was used as a template in a PCR approach using different primer combinations (Figure 3).

[0107] Consistent and reliable results were obtained for the two different assays (SNP11 and SNP19), regardless of the amount of template used. For the SNP19 assay only, increasing template dilution resulted in decreased amplicon yield. Based on these results, in all further experiments, approximately 5 ng of genomic DNA was used as the template for the positive control by default, and approximately 50 ng of template was used for the SNP19 PCR only by default.

[0108] PCR amplicon digestion: testing and optimization All restriction enzymes used were purchased from New England BioLabs (Frankfurt am Main). After optimizing the amplification protocol, selected restriction enzymes were tested in the first digestion reaction and did not specifically cut the amplicon generated with M. thermoautotrophicus DSM3590 DNA, which should remain undigested.

[0109] A series of preliminary experiments revealed that PCR amplicon purification was necessary for reliable digestion (data not shown). Amplicon purification is necessary to remove salts, primers, nucleotides, and other inhibitors prior to restriction digestion. To this end, we first tested various purification kits (e.g., QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and Roti-Prep PCR Purification Kit (Carl Roth, Karlsruhe, Germany)). Comparison of these kits showed that all of the tested products produced relatively good purified final products, so we subsequently used the kit with the lowest initial cost (GF-1 PCR Clean-up Kit (GeneOn, Ludwigshafen, Germany)).

[0110] In a first step, all digestion reactions were tested and optimized with amplicons prepared using purified genomic DNA as PCR template. In a second step, established digestion reactions were assayed in triplicate (n=3) with amplicons generated using non-purified cell samples (see below).

[0111] Digestion reaction performance The amplicons generated by the designed primers of the present invention were used in the first digestion reaction. The test samples were the same as above: one was purified genomic DNA extracted from M. thermoautotrophicus DSM3590 (which does not have the recognition sequence of the applied restriction enzyme(s) within the DNA sequence to be amplified by the predetermined primers of the present invention), and the other was purified genomic DNA extracted from archaea (non-MtDSM3590 archaea) (which has the recognition sequence of the applied restriction enzyme(s) within the DNA sequence to be amplified). Each no-template control (NTC) (also referred to as a negative control) contained the respective SNP-specific master mix, but contained water (HO) instead of the DNA template.

[0112] Each amplicon template was digested with each of the amplicon-specific enzymes. Restriction reactions were performed according to the manufacturer's instructions. According to the manufacturer (New England Biolabs), samples can be incubated for 5 to 15 minutes (although overnight is acceptable) without inducing DNA degradation. An incubation period of 2 hours was initially chosen. Preferably, the digestion reactions are performed in a thermocycler or incubator (37°C).

[0113] SNP19 First, digestion of the SNP19 PCR product with BamHI was performed in NEBuffer3.1 as recommended by the manufacturer. However, both amplicons derived from M. thermoautotrophicus DSM3590 and non-MtDSM3590 archaeal genomic DNA remained undigested after the digestion reaction. Increasing the incubation time to 4 hours or even overnight did not improve the results. In further tests, CutSmart buffer (New England Biolabs, Frankfurt am Main, Germany) was used in addition to NEBuffer3.1, according to the manufacturer's instructions, resulting in 100% enzyme activity. In the case of non-MtDSM3590 archaeal genomic DNA, this alternative buffer hydrolyzed the corresponding PCR product after only 2 hours (see Figure 4). Therefore, CutSmart buffer was used in further experiments using SNP19 and the restriction enzyme BamHI.

[0114] SNP11 As expected, in the case of the amplicon generated from purified genomic DNA of M. thermoautotrophicus DSM3590 (positive control), no restriction reaction of the amplicon occurred, and the length of the fragment detected on the agarose gel corresponded to the size of the full-length PCR product first applied in the digestion reaction. However, when the sample was a given purified non-MtDSM3590 archaea, as expected, digestion of the genomic DNA of the purified SNP11 PCR product with SfcI revealed that the non-MtDSM3590 archaeal amplicon was cleaved into two fragments of different lengths (approximately 200 bp and approximately 100 bp). These fragments were clearly visible on the applied agarose gel after gel electrophoresis (see Figure 5).

[0115] SNP20 Similar to the digestion of the SNP11 amplicon, the SNP20 PCR product was digested with the restriction enzyme NdeI (Figure 6). The M. thermoautotrophicus DSM3590 amplicon remained undigested after incubation with the restriction enzyme NdeI; that is, it was the same size as the untreated PCR product (approximately 325 bp). However, when the sample was purified genomic DNA from a given non-MtDSM3590 archaea, digestion of the purified SNP20 PCR product with NdeI revealed that the DNA-generated amplicon was cleaved into two fragments of different lengths. These fragments were clearly visible on the agarose gel used after gel electrophoresis (see Figure 6).

[0116] Testing the designed primers with fresh archaeal cell samples from a running bioreactor without prior DNA purification After establishing the protocol for the detection reaction of the three SNPs, the next step was to prepare fresh viable cell DNA from a cell sample from a running bioreactor as a template for PCR amplification, according to the method of the present invention. The cells in the bioreactor cell sample were simply mechanically disrupted by repeated freezing (-80°C) and thawing cycles, thereby releasing the DNA. The amount of released genomic DNA (unpurified DNA) was sufficient to obtain PCR product yields comparable to those obtained using a previously prepared protocol for purified DNA (data not shown). The method of the present invention advantageously eliminated the time-consuming isolation of genomic DNA, as in current state-of-the-art methods, in this experiment. Subsequent digestion of the generated amplicons was similar to that of previously examined samples. Only a slightly greater tendency for incomplete digestion was observed when using DNA from a fresh bioreactor cell sample (not purified before PCR) than when using purified DNA for PCR.

[0117] Assessment of assay reliability After optimizing the test protocol, the reliability of the strain identification test was examined. For this purpose, all PCR amplification and digestion reactions of the three SNP amplicons were tested independently on three consecutive days. Archaeal cells from an active bioreactor experiment were used as identification samples; these cells were considered pure M. thermoautotrophicus DSM3590. All primer pairs were tested under the conditions described above. The positive controls were, on the one hand, extracted and purified DNA of M. thermoautotrophicus DSM3590 (which does not contain the recognition sequence of the applied restriction enzyme within the DNA sequence amplified by the selected primers of the present invention), and, on the other hand, extracted and purified DNA of a selected non-MtDSM3590 archaea (which contains the recognition sequence of the applied restriction enzyme within the DNA sequence to be amplified). The negative control (no template control, NTC) contained water instead of the template.

[0118] SNP19 Surprisingly, the inventors found that amplicons derived from archaeal cell samples harvested from a running bioreactor were cleaved into two fragments, at least to some extent, in the applied BamHI digestion reaction (see Figure 7, lane 3), whereas amplicons derived from purified genomic DNA of M. thermoautotrophicus DSM3590 remained undigested, as expected (see Figure 7, lane 10). To rule out this as an experimental artifact, the experiment was repeated several times with freshly prepared reaction components and freshly prepared genomic DNA harvested from another sample of the bioreactor, as well as tested in purified form (data not shown). Nevertheless, in all cases, the results were largely the same as before. That is, after the digestion reaction, the amplicon was clearly, at least to some extent, cleaved into two distinct fragments of approximately 400 bp and approximately 100 bp, although some of the applied amplicon remained undigested (approximately 500 bp) as seen on the agarose gel (see Figure 7, lane 3). These two restriction fragments were also obtained after the digestion reaction when purified DNA samples recovered from the archaeal cell samples in the operating bioreactor were used instead of the unpurified samples described above, further ruling out experimental artifacts (data not shown).

[0119] To further analyze this unexpected result, we performed another parallel digestion reaction using the same amplicon sample previously obtained from the non-purified DNA approach for the BamHI experiment, except that a different restriction enzyme, AvaII, was used instead of BamHI. This revealed the restriction pattern seen in lanes 2, 6, and 9 of Figure 7. The SNP19 amplicon (approximately 500 bp) from the bioreactor cell sample was cleaved to yield three clearly distinguishable restriction fragments of different sizes (approximately 320 bp, approximately 130 bp, and approximately 50 bp; see lane 2 of Figure 7). Furthermore, as expected, the amplicon (full-length, approximately 500 bp) from purified genomic DNA of M. thermoautotrophicus DSM3590 yielded four different restriction fragment sizes (approximately 230 bp, approximately 130 bp, approximately 100 bp, and approximately 50 bp; see lane 9 of Figure 7).

[0120] However, in the first round, as shown in Figure 7, digestion with AvaII in lane 6 (the non-MtDSM3590 archaeal positive control) revealed a weak but specific band in addition to the expected band. This band had not been observed previously. In a separate experiment, the non-MtDSM3590 archaeal positive control was cleaved with AvaII as expected (data not shown), suggesting that the positive control from the first test run may have been an experimental artifact.

[0121] Subsequent amplicon sequencing revealed that the methanogenic organism in the cell sample from the bioreactor was a pure culture of M. thermoautotrophicus UC120910. Applying the method according to the present invention with a verified purified genomic DNA sample of M. thermoautotrophicus UC120910 showed a similar restriction pattern compared to the above cell sample of M. thermoautotrophicus UC120910. More specifically, digestion of a PCR amplicon generated using the SNP19 primers from a purified genomic DNA sample of M. thermoautotrophicus UC120910 (Fig. 7, lane 8) with BamFII and AvaII yielded the same restriction pattern on an agarose gel as that obtained from the DNA from the test cell sample described above, confirming the identity of M. thermoautotrophicus UC120910 and confirming the results of amplicon sequencing (see Fig. 7, lanes 4 and 8 (PCR product), lanes 3 and 7 (BamFII digestion), and lanes 2 and 9 (AvaII digestion)). The restriction efficiencies of the unpurified DNA sample derived from the UC120910 cell sample and the UC120910 purified DNA control were similar in both cases, but the reason for the failure to achieve complete digestion with BamFII in both the unpurified DNA from the cell sample and the purified DNA of M. thermoautotrophicus UC120910 (see Fig. 7, lanes 3 and 7) remains unclear.

[0122] In the next step, the method according to the present invention was tested using a cell sample of a known source of M. thermoautotrophicus DSM3590. The PCR amplicons from the cell sample and the purified DNA sample were of the expected size (see lanes 14 and 17 in Figure 8). The results of the first digestion reaction with BamFII were also as expected: the DSM3590 PCR amplicon was not digested at all (see lanes 13 and 16 in Figure 8 for the cell sample and the purified DNA sample). However, unexpectedly, the second digestion reaction with AvaII yielded a less specific restriction pattern, i.e., more than the four expected fragments (approximately 230 bp, approximately 130 bp, approximately 100 bp, and approximately 50 bp).

[0123] Therefore, definitive identification of M. thermoautotrophicus DSM 3590 could not be made based on the AvaII digestion. However, initial testing of the primer pair for SNP19 showed that a second digestion with AvaII also worked as expected, at least with purified DNA from M. thermoautotrophicus DSM 3590, and we therefore argue that the assay works in principle.

[0124] That is, the method of the present invention can be used to test for the presence of M. thermoautotrophicus DSM3590 and distinguish its presence from the presence of M. thermoautotrophicus DSM3590 variants, such as M. thermoautotrophicus UC120910. The characteristic restriction patterns (number and size of the restriction fragments) of the resulting variants, revealed in digestion assays of DNA from both M. thermoautotrophicus DSM3590 and M. thermoautotrophicus UC120910 bioreactor cell samples, can be used to genotype the underlying methanogens. Furthermore, the method of the present invention can also be used to isolate the genotyped methanogens in the background.

[0125] The restriction efficiency of the UC120910 cell samples and the UC120910-purified DNA control was the same in all cases. However, given the finding that BamHI does not digest M. thermoautotrophicus DSM3590 DNA at all, the distinction between M. thermoautotrophicus DSM3590 and M. thermoautotrophicus UC120910 (which show a specific restriction pattern of at least two restriction fragments after digestion with BamHI) allows for unambiguous differentiation of both strains.

[0126] Based on the essentially positive experimental results with the cell samples from the operating bioreactor containing pure M. thermoautotrophicus UC120910, these were also used in further analyses using the SNP11 and SNP20 primer pair according to the invention. The above-described purified DNA of the validated M. thermoautotrophicus UC120910 was also used as a positive control in all further experiments.

[0127] SNP11 The first digestion reaction with the primary restriction enzyme SfcI yielded the expected results for all runs using the positive controls. The amplicon was either not digested at all in the case of purified DNA from M. thermoautotrophicus DSM3590 or, in the case of purified DNA from M. thermoautotrophicus UC120910, was digested to yield two fragments of different but expected sizes. PCR was successful in all cases, but the bands produced were weak (data not shown). Analysis of fresh cell samples from a running bioreactor, i.e., pure M. thermoautotrophicus UC120910 cell samples (which should yield two fragments of different but expected sizes as the corresponding positive control), did not work because the amplicons of these test samples were not digested at all with the primary restriction enzyme SfcI specific for M. thermoautotrophicus UC120910 (see Figure 10).

[0128] A second digestion reaction with the second restriction enzyme, BstNI, yielded positive results in all runs: the amplicon was either not digested at all in the case of purified DNA from M. thermoautotrophicus UC120910 or, in the case of purified DNA from M. thermoautotrophicus DSM3590, was digested to yield two fragments of different and expected sizes. PCR was successful in all cases (data not shown).

[0129] Even more interestingly, digestion of the SNP11 amplicon from M. thermoautotrophicus UC120910 with a second restriction enzyme, BstNI, yielded a restriction pattern related to DSM 3590. However, BstNI should be specific for DSM 3590 and should not digest the amplicon from strain UC120910 due to the SNP in the enzyme's recognition sequence.

[0130] SNP20 Analysis of the SNP20 amplicon yielded reproducible results. Restriction of the UC120910 cell samples with the primary restriction enzyme NdeI was successful in all cases. However, partial digestion was also observed in some UC120910 positive control samples, clearly distinguishing them from the DSM3590 strain, since the corresponding DSM3590 amplicon remained undigested in both cases. More specifically, hydrolysis of purified UC120910 DNA (positive control) and unpurified DNA samples derived from fresh cell samples was correct in both cases; the DSM3590 control was not hydrolyzed. However, digestion of both the unpurified and purified DNA templates was incomplete, as shown in Figure 9 (see lanes 2 and 5). However, regular bands typical of this primer pair appeared in each run. Nevertheless, it was possible to unambiguously assign the restriction pattern to UC120910 (see Figure 9).

[0131] Table 5 shows the parameters and characteristics of the first digestion reaction, and Table 6 shows the parameters and characteristics of the second digestion reaction, respectively, for each SNP tested and for the different archaeal variants, i.e., Methanothermobacter thermoautotrophicus (Mt) strains DSM3590 and MtUC12091. The restriction enzymes used, as well as the number and size of the final restriction fragments generated after the digestion reaction, are also shown. [Table 5] [Table 6]

[0132] The results of independent reliability tests on all four SNP PCR products (amplicons) are summarized in Table 7. Amplification of the examined genomic regions from unpurified DNA derived from cell samples and from purified DNA was consistently successful using established testing protocols. [Table 7]

[0133] Taking this into account, the three SNP tests used can be considered complementary and can be performed together in parallel. If contradictory results are obtained and one of the assays differs from the others, it is necessary to sequence the corresponding amplicon in each case to genotype the archaeal strain contained in the analyzed cell sample.

Claims

1. 1. A method for detecting the presence of the methanogenic microorganism Methanothermobacter thermoautotrophicus DSM 3590, comprising: a. Obtaining a sample containing methanogenic microorganisms (cells) and applying means and techniques for releasing DNA from said microorganisms to obtain a non-purified DNA sample or to obtain a sample containing purified DNA of said methanogenic microorganisms; b. A step of obtaining an amplified DNA sequence (amplicon) using the unpurified DNA sample or the purified DNA sample obtained in step a. in polymerase chain reaction (PCR) amplification using a primer pair comprising a first and a second primer, wherein the first PCR primer is a first polymerase chain reaction (PCR) primer having a nucleotide sequence selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and is combined with a second polymerase chain reaction primer having a nucleotide sequence selected from the group comprising SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, each of the first primers and each of the second primers can hybridize to the DNA of the methanogenic microorganism Methanothermobacter thermoautotrophicus DSM3590, and SEQ ID NO: 1 and SEQ ID NO: 4, SEQ ID NO: 2 and SEQ ID NO: 5, and SEQ ID NO: 3 and SEQ ID NO: 6 constitute the primer pairs used to PCR amplify the DNA; wherein the amplicon comprises at least one restriction enzyme recognition sequence; c. purifying each of said amplicons; d. performing a first digestion reaction on the amplicon of step c. by incubating the amplicon in a reaction buffer containing at least one first restriction enzyme that recognizes a first recognition sequence for a time sufficient to form restriction fragments; e. Determining the number of restriction fragments formed and their sizes (restriction pattern); f. genotyping the methanogenic microorganism based on the restriction pattern; or in addition to steps a. to f., further comprising: g. performing a second digestion reaction on the amplicon by incubating the amplicon from step b. or step c. in a reaction buffer containing at least one second restriction enzyme that recognizes a second recognition sequence that partially overlaps with the first recognition sequence for a time sufficient to form restriction fragments (digested amplicons); h. determining the number of restriction fragments formed and their sizes (restriction pattern); i. genotyping the methanogenic microorganism based on the restriction pattern; and the detection of the presence of Methanothermoautotrophicus DSM3590 is based on at least one single nucleotide polymorphism (SNP) in the amplicon obtained from step b or step c, the SNP being part of an overlapping sequence of the first and second recognition sequences, the SNP being selected from the group consisting of an SNP at a position in a PCR fragment (amplicon) of the archaeal genomic nucleotide sequence of M. thermoautotrophicus UC120910 amplified by PCR with a primer pair (when compared with a PCR fragment (amplicon) of the genomic nucleotide sequence of M. thermoautotrophicus DSM3590 at that position amplified by the same primer pair); a.) The position of the SNP is position 418 (SNP19), and the primer pair is SEQ ID NO: 3 (forward primer) and SEQ ID NO: 6 (reverse primer), or b.) The SNP position is position 105 (SNP11), and the primer pair is SEQ ID NO: 1 (forward primer) and SEQ ID NO: 4 (reverse primer), or c.) The position of the SNP is position 168 (SNP20), and the primer pair is SEQ ID NO: 2 (forward primer) and SEQ ID NO: 5 (reverse primer), and a detection method in which the SNP is SNP19, the first restriction enzyme is BamHI, and the second restriction enzyme is AvaII, or the SNP is SNP11, the first restriction enzyme is SfcI, and the second restriction enzyme is BstNI or ECORII, or the SNP is SNP20, and the first restriction enzyme is NdeI.

2. Sequencing the purified amplicon or at least a portion thereof in step c., and / or the digested amplicon or at least a portion thereof in step d., and / or the amplicon or at least a portion thereof after the digestion reaction in step d.; The method for detecting the presence of the methanogenic microorganism Methanothermobacter thermoautotrophicus DSM3590 according to claim 1, further comprising:

3. The method according to claim 2, wherein the strain of Methanothermobacter thermoautotrophicus UC120910 detected in step f. or i. is isolated as a variant of Methanothermobacter thermoautotrophicus DSM3590.

4. The method further comprises the steps of:

4. The method of claim 1, wherein the method is for distinguishing strains UC120910 from each other and from other archaea selected from the group consisting of Methanothermobacterium, Methanobrevibacter, Methanothermobacter, Methanococcus, Methanosarcina, Methanopyrus, Methanospirillium, Methanosaeta, Methanogenium, Methanoculeus, and Methanothermococcus, and mixtures thereof.

5. 1. A quality control kit for use in detecting the presence of the methanogenic microorganisms Methanothermobacter thermoautotrophicus DSM3590 and / or Methanothermobacter thermoautotrophicus UC120910 in a given sample, comprising: at least one container, a first primer which acts as a forward primer and whose nucleotide sequence is selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; a second primer which acts as a reverse primer and whose nucleotide sequence is selected from the group comprising SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; at least one primary restriction enzyme, or at least one primary restriction enzyme and at least one secondary restriction enzyme, at least one buffer solution, Including, the SEQ ID NO: 1 and the SEQ ID NO: 4, the SEQ ID NO: 2 and the SEQ ID NO: 5, and the SEQ ID NO: 3 and the SEQ ID NO: 6 constitute a primer pair; The above kit, wherein the first restriction enzyme is BamHI and the second restriction enzyme is AvaII, or the first restriction enzyme is SfcI and the second restriction enzyme is BstNI or ECORII, or the first restriction enzyme is NdeI.

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