Method for determining number and / or concentration of germinable cells in sample of biological origin

Abstract

The present invention relates to a method for determining the number and / or concentration of germinatable cells in a sample of biological origin wherein the germinatable cells are bacterial spores formed in vegetative cells, comprising the following steps: a) subjecting at least a portion of a sample material of biological origin to a pretreatment involving the lysis of the vegetative cells, b) subjecting the pretreated sample material obtained from step a) to photolysis, c) isolating nucleic acids from the photolyzed sample material obtained from step b), and d) subjecting the isolated nucleic acids obtained from step c) to a quantitative polymerase chain reaction to obtain the number and / or concentration of germinable cells in the sample material of step a), the method is characterized in that the pretreatment in the step a) comprises a step of treating the sample material with an alkaline solution.

Description

Technical Field

[0001] The present invention relates to a method for determining the number and / or concentration of germinable cells in a sample of biological origin, wherein the germinable cells are bacterial spores formed within vegetative cells, and the method involves pre-treating the sample material with an alkaline solution prior to performing quantitative polymerase chain reaction. Background Art

[0002] Some food or feed products contain bacteria that are beneficial to the corresponding organism (especially for the intestinal care of the organism). For example, products of Evonik and are feed additives containing naturally fast-growing Bacillus amyloliquefaciens and / or Bacillus subtilis. These products improve the condition of animals and help producers address challenges in quality, profitability, and sustainability. The quality of such products is directly associated with the number of germinable cells contained in the product.

[0003] The total cell number includes both live and non-live cells in a specific culture. Live cells (also referred to as viable cells) are cells that are capable of surviving, germinating, and growing. In contrast, non-live cells (also referred to as dead cells) are cells that cannot grow in a cell culture to produce colonies. They are not capable of surviving, germinating, or reproducing. The group of live cells includes both vegetative cells and germinable cells. Vegetative cells are cells that grow normally. They have metabolic activity and function and have the ability to form spores. While vegetative cells are the active form of bacterial cells, spores can be considered the dormant form of the cells. It allows survival under adverse conditions, but it does not allow cell growth or reproduction. Their effects are mainly manifested in the gastrointestinal tract. However, vegetative cells are not resistant to harsh environmental conditions and cannot survive without nutrients. Germinable cells are bacterial spores formed within vegetative cells. They have the ability to germinate. Additionally, they are also resistant to physical and chemical treatments, which allows the persistence and spread of the bacterial species. In contrast to vegetative cells, germinable cells can survive without nutrients. Therefore, in any sample in any product as well as in samples of biomass, feed, feces, and digestates, germinable cells must be distinguished from all other cells (i.e., non-live cells and vegetative cells). This involves the determination of the concentration of germinable cells in the sample.

[0004] Microbial spore counting is a method for determining the concentration of germinable cells in a sample. The concentration value obtained by microbial spore counting is expressed as CFU (colony-forming units) per gram or per milliliter, which is equal to the concentration of germinable cells present in the sample. Microbial spore counting allows for the differentiation of phenotypes (colony morphology). However, it is difficult to distinguish at the strain level or even between closely related species using this method. Instead, this method can only count bacteria that can form colonies under the applied conditions. If species or strains need to be identified, each colony needs to be exposed to suitable methods such as MALDI-TOF, 16s sequencing, or specific identity PCR. Another disadvantage of microbial spore counting is its low precision, which is also matrix and organism / cell-dependent. The slow speed of this method is another obstacle: depending on the organism, the formation of visible and countable colonies takes at least 24 hours or up to 48 hours. Another significant drawback of this method is that, for example, it is time-consuming and labor-intensive, and causes waste of many disposable items (such as plastic pipettes and culture dishes). However, microbial spore counting has become the gold standard for determining the concentration of germinable cells in a sample and is still used as the standard for determining the concentration of germinable cells in a sample.

[0005] Quantitative polymerase chain reaction (qPCR) is a DNA-based method for quantifying target genes after nucleic acids are extracted from a sample. This method allows for the differentiation of species, strains, and even genotypes with several base differences.

[0006] For example, US2018 / 0037936 A1 discloses a method for quantitatively monitoring bacterial endospores in the aqueous environment of a paper mill or a board mill, which involves destroying vegetative bacteria in a first sample by suitable treatment (e.g., heating), and then using, for example, propidium monoazide (PMA) for photolysis. The heating is most preferably at least 75 °C. A method is also disclosed that further includes the steps of determining the amount of vegetative cells in a second sample and comparing the measured endospore level from the first sample with the amount of vegetative bacterial cells in the second sample.

[0007] US2011 / 0318750 A1 discloses a method that allows for the detection and quantification of live bacterial endospores from samples with low and high biomass. A pretreatment step of heating at 80 / 85 °C for about 15 minutes is performed to inactivate / kill live bacterial cells in the sample. Then the sample is labeled with PMA for photolysis, nucleic acids are extracted, and qPCR is performed.

[0008] The article "PCR-Based Method Using Propidium Monoazide to Distinguish Viable from Nonviable Bacillus subtilis Spores" (Rawsthorne, H. et al., Applied and Environmental Microbiology, 2009, Vol. 75, No. 9, 2936 - 3939) describes the determination of the number and / or concentration of viable Bacillus subtilis spores in an overnight culture. Vegetative cells were spread onto sporulation agar and exposed to aerobic conditions, resulting in 95% sporulated cells. The harvested spores were further exposed to heat (115 °C in an oil bath) to mimic the heat inactivation procedure in food processing technology (a pretreatment involving lysis of vegetative cells). The suspension was then treated (photolyzed) with PMA, DNA was isolated, and quantitative PCR was performed. The PMA-qPCR method was compared with the "gold standard" counting by the plate method and qPCR without PMA treatment. The identification of viable bacterial spores based on PMA-qPCR was discussed as an alternative to culture methods in the validation of food processing efficacy.

[0009] Depending on the extraction method, qPCR has high precision. The time to obtain results is in the range of 3 to 4 hours, which is relatively short compared to microbial counting. However, the disadvantage of this method is that it counts all target cells regardless of their viability status. In other words, qPCR cannot distinguish dead cells, vegetative cells, or germinable cells.

[0010] Therefore, there is still a need for a method that allows specific and accurate determination of the number and / or concentration of germinable cells in a sample of biological origin.

[0011] It has been found that the solution to this problem lies in an additional pretreatment of the sample material before quantitative polymerase chain reaction. The sample material to be pretreated contains a group of live cells that can be induced to germinate under specific conditions and non-live cells. Thus, the sample material contains non-live (dead) cells and live (viable or surviving) cells, and the live cells include vegetative cells and germinable cells. At least a portion of the sample material is first pretreated, and this pretreatment involves lysis of vegetative cells. This step aims to distinguish germinable cells from all other cells (i.e., vegetative cells and dead (non-live) cells). Subsequently, the pretreated sample material is photolyzed with a photo-reactive DNA-binding dye. Next, nucleic acids are isolated from the photolyzed sample material thus obtained, and then qPCR is performed to obtain the number of germinable cells in the original sample material. The effect of this procedure is that during qPCR, only DNA from intact cells is amplified. Summary of the Invention

[0012] Accordingly, an object of the present invention is a method for determining the number and / or concentration of germinable cells in a sample of biological origin, wherein the germinable cells are bacterial spores formed within vegetative cells, the method comprising the steps of:

[0013] a) pretreating at least a portion of a sample material of biological origin, the pretreatment involving lysis of vegetative cells,

[0014] b) photolyzing the pretreated sample material obtained from step a),

[0015] c) separating nucleic acids from the photolyzed sample material obtained from step b), and

[0016] d) performing quantitative polymerase chain reaction on the separated nucleic acids obtained from step c) to obtain the number and / or concentration of germinable cells in the sample material of step a),

[0017] characterized in that the pretreatment comprises the step of treating the sample material with an alkaline solution.

[0018] The method according to the present invention allows determination of the number of germinable cells in a sample, the concentration of germinable cells in a sample, or both. Determination of the cell number has the advantage that it does not require a reference basis such as specific volume or mass and is thus somewhat easier to determine. However, when the result is obtained as the cell number in step d) and will be further used to determine other parameters, see the embodiments below, the same volume or the same mass of the sample under consideration must be used in all measurements. Alternatively, it is already possible to determine the concentration of germinable cells in a sample, which has the advantage that the cell number relative to a reference basis has been determined. This facilitates further use of the result obtained in step e) in other embodiments and allows easier comparison of the results obtained thereby. Accordingly, in the context of the present invention, determination of the cell concentration is preferred. The concentration of the cells can be given as copies / g or copies / μL.

[0019] Generally, the pretreatment of step a) is intended to improve the differentiation of germinable cells from all other cells. It has been found that lysis of vegetative cells in the pretreatment of step a) results in a significant increase in the accuracy of the quantitative polymerase chain reaction of step d).

[0020] In principle, the lysis of vegetative cells in the pretreatment of step a) is not restricted by anything. On the contrary, it can be achieved by any conceivable treatment that causes the lysis of vegetative cells. However, it has been found that treating the sample material with an alkaline solution alone or in combination with elevated temperature or additional mechanical treatment results in effective lysis of vegetative cells. The alkaline solution causes the denaturation of vegetative cells. Specifically, these conditions lead to the disorder of the wall structure of vegetative cells or damage to the cell wall structure (including the cortical region and the inner membrane wall). However, due to high alkali resistance and / or high heat resistance, some cells (especially germinable cells) can survive under these harsh conditions.

[0021] In an embodiment of the method according to the invention, step a) further comprises subjecting the sample material to elevated temperature and / or additional mechanical treatment.

[0022] In principle, the pretreatment of step a) is not restricted by any specific alkaline solution or alkaline solution strength, as long as the alkaline solution alone or in combination with additional heating and / or additional mechanical treatment causes the lysis of vegetative cells. However, it is preferred that the alkaline solution used in step a) has a pH of at least 8, at least 8.5, or at least 12. For example, the alkaline solution is an aqueous solution of sodium hydroxide or potassium hydroxide with a concentration of 0.05 mol / L. For example, the pKb of the alkaline solution is 0 or less, so as to ensure that the pretreatment of step a) alone has caused the desired lysis of vegetative cells. The preferred bases in the alkaline solution of step a) are sodium hydroxide and / or potassium hydroxide. The pretreatment of step a) is not restricted by any period of time during which the sample material is treated with the alkaline solution, and the treatment may take 10 minutes to 1 hour, 10 minutes to half an hour, or 10 minutes to 15 minutes.

[0023] In a preferred embodiment of the method according to the invention, the pKb of the base in the alkaline solution of step a) is 0 or less.

[0024] Moreover, the additional steps of heating the sample to an elevated temperature or additional mechanical treatment are not restricted by any specific temperature in principle, as long as the elevated temperature contributes to the lysis of vegetative cells. However, it is preferred that the elevated temperature of the additional heating of the sample is a temperature of at least 80 °C, because this temperature contributes to making the lysis of vegetative cells more effective. The additional mechanical treatment can be mixing or shaking in the presence of beads, such as orbital shaking, when appropriate.

[0025] In another preferred embodiment of the method according to the invention, the elevated temperature of the additional heating is a temperature of at least 80 °C.

[0026] Apply an elevated temperature of additional heating (in particular a temperature of at least 80 °C) for a period of 10 minutes to 1 hour, 10 minutes to half an hour, or 10 minutes to 15 minutes. Similarly, apply additional mechanical treatment for a period of 10 minutes to 1 hour, 10 minutes to half an hour, or 10 minutes to 15 minutes.

[0027] To improve the precision of qPCR, in step b) of the method according to the invention, a photo-reactive DNA-binding dye is added to the pretreated sample material obtained from step a). Preferably, the pretreated sample material is kept away from light before adding the photo-reactive DNA-binding dye. After adding the photo-reactive DNA-binding dye, the sample material is subjected to photolysis.

[0028] In one embodiment of the method according to the invention, step b) comprises the following steps:

[0029] b1) Keep the pretreated sample material obtained from step a) away from light,

[0030] b2) Add a photo-reactive DNA-binding dye to the sample material of step b1), and

[0031] b3) Subject the sample material obtained from step b2) to photolysis.

[0032] This procedure aims to inhibit the amplification of all cells with disrupted wall structure or damaged cell wall structure (including the cortex region and the inner membrane wall). Although dead cells already have a disrupted wall structure or a damaged cell wall structure, the pretreatment in step a) causes lysis of vegetative cells, particularly damage to the cell wall structure of vegetative cells. The photo-reactive DNA-binding dye can embed or penetrate into cells with damaged cell wall structure (including the cortex region and the inner membrane wall), and upon photolysis, the photo-reactive DNA-binding dye binds to the DNA of these cells. This process makes the DNA insoluble and causes its loss during subsequent genomic DNA extraction. Therefore, the DNA of cells with disrupted wall structure or damaged cell wall structure (including the cortex region and the inner membrane wall) will not be amplified in quantitative polymerase chain reaction. In contrast, the DNA of cells with intact cell wall structure is amplified in quantitative polymerase chain reaction.

[0033] Photolysis is preferably carried out for a period of 10 to 25 minutes, or 10 to 20 minutes, for example, 10, 11, 12, 13, 14 or 15 minutes.

[0034] The irradiation wavelength in photolysis (i.e., step b3)) depends on the maximum absorption wavelength of the photo-reactive DNA-binding dye added to the sample material. For example, the photo-reactive DNA-binding dye propidium monoazide (PMA) has a maximum absorption at a wavelength of 464 nm before reacting with the DNA of cells, while after the reaction, PMA bound to the DNA of cells has a maximum absorption at a wavelength of 510 nm and a maximum emission at a wavelength of 610 nm.

[0035] In another embodiment of the method according to the invention, the photo-reactive DNA-binding dye is embedded in or penetrates into the DNA of cells with disrupted wall structures and / or cells with damaged cell wall structures (including the cortex region and the inner membrane wall).

[0036] In principle, step b) is not restricted by any specific photo-reactive DNA-binding dye, as long as the dye is photo-reactive and binds to DNA in photolysis. However, it is preferred that the photo-reactive DNA-binding dye is propidium monoazide, ethidium monoazide, a derivative of any of these, and / or a mixture thereof, since these dyes have been shown to bind extremely effectively to the DNA of cells with damaged cell wall structures (including the cortex region and the inner membrane wall).

[0037] In another embodiment of the method according to the invention, the photo-reactive DNA-binding dye is propidium monoazide, ethidium monoazide, a derivative of any of these, and / or a mixture thereof.

[0038] Preferably, the photo-reactive DNA-binding dye is propidium monoazide (PMA). PMA is a fluorescent and photo-reactive DNA-binding dye that preferentially binds to double-stranded DNA. Cells are mixed with PMA such that the dye enters the cell membrane. Visible light (a high-power halogen lamp or a specific LED device) induces the photo-reaction of the chemical, which results in the covalent bond of PMA to dsDNA. Specifically, when exposed to light, the photolysis of PMA causes the azide group of PMA to be converted into a nitrene radical, which covalently binds to any adjacent carbon (including double-stranded DNA). In theory, dead microorganisms lose the ability to maintain the integrity of their membranes, which makes the "naked" DNA in the cytosol ready to react with PMA. The DNA of living organisms is not exposed to PMA because they have intact cell membranes. After treatment with the chemical, only the DNA from live bacteria is available for qPCR, thus allowing only the amplified DNA of living organisms to be obtained.

[0039] In principle, the method according to the invention is not subject to any restrictions regarding the concentration of the photo-reactive DNA-binding dye added to the same material in step b2). However, it has been found that when the photo-reactive DNA-binding dye is used at a relatively low or even very low concentration (such as 50 or even only 20 micromoles (μM)), the method according to the invention even allows for a quite precise determination of germinable cells. Therefore, it is preferred to use a photo-reactive DNA-binding dye at a concentration of at most 50 micromoles (μM) or at a concentration of at least 20 micromoles (μM). More preferably, a photo-reactive DNA-binding dye at a concentration of 20 to 50 micromoles (μM) is used.

[0040] According to the invention, nucleic acids are separated from the photo-lysed sample material obtained in step b) and then subjected to quantitative polymerase chain reaction to obtain the number of germinable cells in the sample of step a). The photo-reactive DNA-binding reaction with the DNA of cells with disrupted wall structure or damaged cell wall structure (including the cortical region and the inner membrane wall) renders this DNA insoluble. This facilitates the separation of the DNA of germinable cells for subsequent quantitative polymerase chain reaction.

[0041] In principle, the separation in step c) is not subject to any restrictions and in principle can be carried out in any conceivable way as long as it allows for the separation of the DNA of germinable cells as completely as possible.

[0042] For nucleic acid separation, the nucleic acids must be released from the germinable cells as completely as possible, which is preferably achieved by lysing the photo-lysed sample material obtained in step b) to release proteins, DNA, phospholipids, etc. from the cells. The remaining tissue is discarded. The lysis is chemical and / or mechanical lysis. As already mentioned above, the photo-lysis in step b) above results in the reaction of the photo-reactive DNA-binding dye with the DNA of cells with disrupted wall structure or damaged cell wall structure (including the cortical region and the inner membrane wall), thereby causing the insolubility of the DNA of non-viable cells. Therefore, it can be expected that the DNA of non-viable cells is discarded together with the tissue. Further separation of the nucleic acids of germinable cells involves binding the nucleic acids released and obtained from the lysis to beads to obtain loaded beads. The beads can be made of silica or zirconia. To remove any potential contaminants that may still be attached to the beads, it is preferred to wash the loaded beads. Next, the nucleic acids are preferably released again from the washed beads by treatment with an elution buffer, which gives a solution containing the released nucleic acids.

[0043] In yet another embodiment of the method according to the invention, step c) comprises the following steps:

[0044] c1) subjecting the photo-lysed sample material obtained in step b) to chemical and / or mechanical lysis to release nucleic acids from the sample material,

[0045] c2) Bind the released nucleic acid obtained from step c1) to beads to obtain loaded beads.

[0046] c3) Wash the loaded beads obtained from step c2), and

[0047] c4) Release the nucleic acid from the washed beads obtained from step c3) by treatment with an elution buffer or water to obtain a solution containing the released nucleic acid.

[0048] In the context of the present invention, it is preferred that the beads are magnetic, which facilitates the separation of the loaded beads from the solution, suspension or dispersion obtained from the chemical and / or mechanical lysis of the photolyzed sample material, and their transfer to different containers for washing and releasing the nucleic acid.

[0049] In principle, the chemical lysis of the photolyzed cells in step c1) is not subject to any limitation. On the contrary, it can be achieved by any conceivable chemical treatment that causes cell lysis. However, it has been found that the chemical lysis of the photolyzed sample material under chaotropic conditions, either alone or in combination with mechanical lysis, results in effective lysis of the cells. Therefore, it is preferred that the chemical lysis in step c1) is carried out under chaotropic conditions.

[0050] Chaotropic conditions lead to the structural disruption and denaturation of macromolecules such as proteins and nucleic acids (e.g., DNA and RNA). Chaotropic solutes increase the entropy of the system by interfering with the intermolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. The structure and function of macromolecules depend on the net effect of these forces. Therefore, it can be seen that an increase in chaotropic solutes in a biological system will denature macromolecules, reduce enzyme activity, and induce stress on the cells (i.e., the cells will have to synthesize stress protectants). Tertiary protein folding depends on the hydrophobic forces of the amino acids in the protein sequence. Because of the disordering of the water molecules adjacent to the protein, chaotropic solutes reduce the net hydrophobic effect of the hydrophobic regions. This solubilizes the hydrophobic regions in the solution, thereby denaturing the protein. This also applies directly to the hydrophobic regions in the lipid bilayer; if the critical concentration of the chaotropic solute (in the hydrophobic region of the bilayer) is reached, the membrane integrity will be compromised and the cells will lyse.

[0051] Common chaotropic agents include n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea.

[0052] Chaotropic salts that dissociate in solution exert their chaotropic effects via different mechanisms. While chaotropic compounds such as ethanol interfere with non-covalent intramolecular forces as outlined above, salts can have chaotropic properties by shielding charges and hindering the stabilization of salt bridges. Hydrogen bonds are stronger in non-polar media, so salts that increase the chemical polarity of the solvent can also destabilize hydrogen bonds. Mechanistically, this is because there are not enough water molecules to effectively solvate the ions. This can lead to ion-dipole interactions between the salt and the hydrogen-bonded species, which are more favorable than normal hydrogen bonds. Therefore, it is preferred that the chemical cleavage in step c1) is carried out under chaotropic conditions, especially in the presence of high concentrations of chaotropic salts.

[0053] In a preferred embodiment of the method according to the invention, the chemical cleavage in step c1) is carried out under chaotropic conditions.

[0054] In principle, the chemical cleavage of the photolyzed cells in step c1) is not subject to any restrictions. On the contrary, it can be achieved by any conceivable chemical treatment that causes cell lysis. Typically, in mechanical lysis, the cell membrane is physically disrupted by using shear forces. Because it combines high throughput and higher lysis efficiency, this method is the most popular and commercially available. Examples of mechanical lysis techniques include the use of high-pressure homogenizers and bead mills.

[0055] High-pressure homogenizers (HPH) are one of the most widely used large-scale microbial cell disruption devices. In this method, high pressure is used to force the cells in the medium through an orifice valve. When the cells are compressed upon entering the orifice and expanded upon discharge, the membrane ruptures due to the high shear forces at the orifice.

[0056] Bead milling (also known as bead beating) is a widely used laboratory-scale mechanical cell lysis method. Cells are disrupted by microbeads made of glass, steel, or ceramic that are mixed with the cell suspension by high-speed agitation. The beads collide with the cells, thereby breaking the cell membrane by shear forces and releasing the intracellular components. This process is affected by many parameters such as bead diameter and density, cell concentration, and agitator speed. Smaller beads in the range of 0.25 - 0.5 mm in diameter are more effective and are therefore recommended for lysis.

[0057] According to the invention, the released nucleic acids obtained from step c1) are bound to beads to obtain bead-loaded beads. Therefore, it is preferred that the mechanical lysis in step c1) has been carried out using beads (i.e., using the same beads as in step c2).

[0058] The advantage of using beads in the mechanical lysis of step c1) and the combination of step c2) is that these two steps can be carried out simultaneously or at least with a high degree of temporal overlap. For example, when silica-based beads are used in step c1), nucleic acid separation can be carried out by silica adsorption. This DNA separation method is based on the binding of DNA molecules to the silica surface in the presence of a specific salt and under specific pH conditions. To separate DNA by silica adsorption, a sample (which can be anything from purified cells to tissue samples) is lysed to release proteins, DNA, phospholipids, etc. from the cells. The remaining tissue is discarded. Then the supernatant containing DNA is exposed to silica in a solution with a high ionic strength. The highest DNA adsorption efficiency occurs in the presence of a buffer solution with a pH equal to or lower than the pKa of the surface silanol groups.

[0059] The mechanism behind the adsorption of DNA to silica is not fully understood; one possible explanation involves the reduction of the negative charge on the silica surface due to the high ionic strength of the buffer. This reduction in surface charge leads to a decrease in the electrostatic repulsion between the negatively charged DNA and the negatively charged silica. At the same time, the buffer also reduces the water activity by forming hydrated ions. This causes the silica surface and the DNA to become dehydrated. These conditions result in an energetically favorable situation for the adsorption of DNA to the silica surface.

[0060] A further explanation of how DNA binds to silica is based on the role of guanidinium chloride (GuHCl) acting as a chaotropic agent. Chaotropic agents denature biomolecules by disrupting the hydration shell around them. This allows positively charged ions to form salt bridges between the negatively charged silica and the negatively charged DNA backbone at high salt concentrations. The DNA can then be washed with high salt and ethanol, and finally eluted with low salt.

[0061] After the DNA has bound to the silica, it is then washed to remove contaminants and finally eluted using an elution buffer or distilled water. The elution buffer can be any suitable buffer that allows the DNA to be dissolved while protecting it from degradation. A suitable elution buffer is, for example, TE buffer, which contains Tris (a common pH buffer) as well as EDTA (a molecule that chelates cations such as Mg 2+ ). For example, EDTA further inactivates the enzyme by binding to the metal cations required by the DNA enzyme, thus protecting the DNA from degradation.

[0062] According to the present invention, quantitative polymerase chain reaction (qPCR) is carried out on the isolated nucleic acid obtained from step d) to obtain the number of germinable cells in the sample of step a).

[0063] The qPCR of the method according to the invention uses nucleotides (i.e., forward primer, reverse primer, probe, and quantification vector) that are synthesized for quantifying the target of interest and are thus specific for the bacterial strain under consideration.

[0064] In principle, the quantitative polymerase chain reaction of step d) is not subject to any restrictions as long as it allows the number and / or concentration of germinable cells to be obtained. The term quantitative polymerase chain reaction (or qPCR for short) is used as known to those skilled in the art and denotes a method widely used to rapidly prepare millions to billions of copies (complete or partial) of a specific DNA sample, thus allowing scientists to take a very small DNA sample and amplify it (or a part of it) to a large enough amount for detailed study. In principle, qPCR is a DNA amplification reaction of the target DNA (also called template DNA in this context). A typical amplification reaction includes target DNA, a heat-stable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (NTPs), a reaction buffer, and magnesium. Once the reaction mixture is mixed, it is placed in a so-called cycler, which exposes the reaction mixture to a series of different temperatures for a set amount of time. This series of temperature and time adjustments is called an amplification cycle. Each qPCR cycle theoretically doubles the amount of the target (amplicon) in the reaction. Ten cycles theoretically multiply the amplicon by about 1020 cycles, multiplying it by more than a million times within a few hours.

[0065] Each cycle of qPCR includes steps of template denaturation, primer annealing, and primer extension. The initial step denatures the target DNA by heating it to an elevated temperature (e.g., to a temperature of about 95 °C) for 15 seconds to two minutes. In the denaturation step, the two intertwined strands of double-stranded DNA (dsDNA) separate from each other, thus generating the single-stranded DNA required for replication by the heat-stable DNA polymerase. In the next step of the cycle (annealing step), the temperature is lowered to a temperature in the range of 40 to 60 °C. At a temperature in this range, the oligonucleotide primers can form a stable association with the denatured target DNA; in other words, they anneal to the single-stranded DNA and act as primers for the DNA polymerase. This step lasts for about 15 to 60 seconds. The third step of the qPCR cycle (extension step) begins as the reaction temperature is raised again to a temperature in the range of 70 to 74 °C. This is the optimal temperature range for most heat-stable DNA polymerases. The extension step lasts for about 1 to 2 minutes. The next cycle begins with a return to a temperature of about 95 °C for denaturation.

[0066] Each step of the cycle should be optimized for each template and primer pair combination. If the temperatures during the annealing and extension steps are similar, these two steps can be combined into one step in which both primer annealing and extension occur. In the context of the present invention, the amount of amplification product is analyzed after 20 to 40 cycles.

[0067] The specific temperature and duration of each step of the cycle in qPCR depend on the target DNA under consideration. Thus, the qPCR in step d) of the method according to the present invention is not subject to any limitations. However, it is preferred that denaturation is carried out at a temperature of at most 95 °C for a duration of 3 minutes + / - 1 minute, the annealing step is carried out at a temperature of about 55 °C for a duration of 30 seconds + / - 10 seconds, and the amplification step is carried out at a temperature of at most 95 °C for a duration of about 15 seconds + / - 10 seconds. Preferably, the total cycle time is at most about 1 hour 18 minutes + / - 10 minutes.

[0068] Because quantitative polymerase chain reaction (qPCR) measures concentration while the synthesis process is taking place, it allows for real-time quantification and detection of specific DNA sequences.

[0069] In quantitative polymerase chain reaction, relative fluorescence unit (RFU) measurements are used for DNA analysis. Photolysis in step b) results in a DNA probe consisting of nucleic acids labeled with a fluorescent dye, which allows for detection after hybridization with complementary DNA targets. A thermal cycler equipped with a fluorescence detection unit is used to measure fluorescence in real time during target gene amplification. The amount of fluorescence measured (expressed as RFU) reflects the amount of amplification product in each cycle. For quantification of the target, the quantification cycle (Cq) at which the fluorescence reaches a detectable signal is determined. Cq is proportional to the initial concentration of the target gene at the start of amplification. A computer program measures the results and determines the number or size of the fragments at each data point based on the fluorescence intensity level. Thus, the quantitative polymerase chain reaction in step d) of the method according to the present invention gives the concentration of germinable cells in the sample of step a).

[0070] Based on this result, it is also possible to determine the number and / or concentration of other cells (i.e., non-viable cells and vegetative cells) in the sample of step a). To this end, the number and / or concentration of all cells in a portion of the sample material of step a) is determined, and then the difference between the number and / or concentration of all cells thus obtained and the number and / or concentration of germinable cells in the sample material of step a) is taken. Thus, the parameter obtained thereby is a sum parameter.

[0071] In another embodiment of the method according to the present invention, it further comprises the following steps:

[0072] e) determining the number and / or concentration of non-viable cells and vegetative cells in the sample of step a), which comprises the following steps:

[0073] e1) Determine the number and / or concentration of all cells in the sample material of step a), and

[0074] e2) Take the difference between the number and / or concentration of all cells obtained in step e1) and the number and / or concentration of germinable cells obtained in step d) to give the number and / or concentration of non-viable cells and vegetative cells in the sample material of step a).

[0075] In principle, step e1) is not subject to any restrictions on the procedure for determining the number and / or concentration of all cells, as long as the procedure considered allows for a reliable and accurate determination of the number and / or concentration of all cells in a sample of biological origin. For example, microbial counting or qPCR is suitable for the determination in step e1). In any case, the determination of the number and / or concentration of all cells is carried out in the absence of DNA-binding dyes in the sample material. In the context of the present invention, it is preferred to use qPCR for step e1), since it has already been used in step d) and it allows for the determination of the value of interest with the desired precision.

[0076] When determining the number of germinable cells in step d), step e1) uses the same volume and / or mass of sample material as used in step d). When determining the concentration of germinable cells in step d), step e1) uses the same type of concentration (meaning copy number / g or copy number / μL) as used in step d).

[0077] In cases of interest, the method according to the invention also allows for the determination of the percentage of the number of germinable cells obtained in step d) relative to the number of all cells obtained in step e1), and / or the percentage of the number of non-viable cells and vegetative cells obtained in step e2) relative to the number of all cells obtained in step e1).

[0078] In a preferred embodiment of the method according to the invention, it further comprises the following steps:

[0079] f) Determine the number and / or concentration of vegetative cells in the sample of step a), which comprises the following steps:

[0080] f1) Determine the number and / or concentration of viable cells in a portion of the sample material of step a), and

[0081] f2) Take the difference between the number and / or concentration of viable cells obtained in step f1) and the number and / or concentration of germinable cells obtained in step d) to give the number and / or concentration of vegetative cells in the sample material of step a).

[0082] In principle, step f1) is not restricted by any procedure for determining the number and / or concentration of viable cells, as long as the procedure under consideration allows for a reliable and accurate determination of the number and / or concentration of viable cells in a sample of biological origin. In the context of the present invention, it is preferred to perform step f1) using qPCR, wherein the sample material is treated with a DNA-binding dye prior to qPCR.

[0083] When determining the number of germinable cells in step d), step f1) uses the same volume and / or mass of sample material as used in step d). When determining the concentration of germinable cells in step d), step f1) uses the same type of concentration (meaning copy number / g or copy number / μL) as used in step d).

[0084] In addition, the method according to the present invention also allows for determining the change in the number and / or concentration of germinable cells in the sample under consideration relative to the number or concentration of all cells, based on the results obtained in steps d) and e1). The measured value of this change describes the change between the "before" and "after" values of the quantity. Here, the percentage of relative change is used. The percentage change is equal to the quotient of the absolute change and the baseline value multiplied by 100%. Thus, the percentage change is a relative change in percentage terms without physical units. The base value y1 is also the 100% value. The percentage change describes, in percentage terms, how much a given reference value has changed (i.e., increased or decreased). The number or concentration of cells in the sample of interest does not change over time; however, the number of each type of cell has changed. Specifically, the number of germinable cells has decreased, and thus the negative value of the percentage relative change reflects the degree of decrease in the number and / or concentration of germinable cells.

[0085] In another preferred embodiment of the method according to the present invention, it further comprises the following steps:

[0086] g) Determining the relative change in the number and / or concentration of germinable cells relative to the number and / or concentration of all cells, by means of the following formula:

[0087] and / or

[0088] where

[0089] rel.(%) is the relative change in the number and / or concentration of germinable cells in percentage terms,

[0090] n g is the number of germinable cells obtained in step d),

[0091] c g is the concentration of germinable cells obtained in step d),

[0092] n 所有 is the number of all the cells obtained in step e1), and

[0093] c 所有 is the concentration of all the cells obtained in step e1).

[0094] In principle, the method according to the present invention is not subject to any restrictions regarding a specific sample of biological origin or a sample regarding a specific biological origin. However, the method according to the present invention is particularly applicable to biomass, feed, feces, or digestate samples.

[0095] In another embodiment of the method according to the present invention, the sample in step a) is a biomass, feed, feces, or digestate sample.

[0096] In a preferred embodiment of the method according to the present invention, the sample in step a) is a probiotic. Preferably, the sample is a probiotic containing Bacillus spp., such as Bacillus amyloliquefaciens and / or Bacillus subtilis, such as those of Evonik's and / or

[0097] The qPCR of the method according to the present invention uses nucleotides (i.e., forward primer, reverse primer, probe, and quantification vector) that are sequenced for quantifying the target of interest and thus are specific for the bacterial strain under consideration. Therefore, it is preferred to sequence the nucleotides (i.e., forward primer, reverse primer, probe, and quantification vector) used in this qPCR for quantifying Bacillus spp., such as Bacillus amyloliquefaciens and / or Bacillus subtilis. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1a shows the correlation of microbial spore counts between Laboratory 1 (external laboratory) and Laboratory 2 (Evonik's QA laboratory).

[0099] Figure 1b is a boxplot representation (N = 13) of the number of colony forming units (CFU) of Bacillus spp. determined in an inter-laboratory ring trial test among participating laboratories, where the identifiers of the participating laboratories are randomized to anonymize each laboratory.

[0100] Figure 2a shows the linear regression between the values measured by the method according to the present invention and the values measured by microbial spore counts, where the measurement is related to Laboratory 1.

[0101] Figure 2bShows the linear regression between the values measured by the method according to the invention and the values measured by microbial spore counting, where the measurement is related to Laboratory 2.

[0102] Figure 3a Shows the evaluation of different sample pretreatments without adding the DNA-binding dye propidium monoazide (PMA) to the feed sample DE22-006 with the Bacillus subtilis DSM 32315 strain. Feed sample DE22-006 was evaluated for different sample pretreatments without adding the DNA-binding dye propidium monoazide (PMA).

[0103] Figure 3b Shows the linearity in the range of 0 μM to 120 μM of the PMA concentration.

[0104] Figure 4 Illustrates for Feed sample DE22-006, the number of germinable cells of the Bacillus subtilis DSM 32315 strain determined by the method according to the invention under different pretreatments compared to the microbial spore count.

[0105] Figure 5 Illustrates for Feed sample DE22-007, the number of germinable cells of the Bacillus subtilis DSM 32540 strain determined by the method according to the invention under different pretreatments compared to the microbial spore count.

[0106] Figure 6 Illustrates for Feed sample DE22-008, the number of germinable cells of the Bacillus amyloliquefaciens CECT 5940 strain determined by the method according to the invention under different pretreatments compared to the microbial spore count.

[0107] Figure 7 Illustrates for the DE22-001 feed sample, the number of germinable cells of the Bacillus subtilis DSM 32315 strain determined by the method according to the invention under different pretreatments compared to the microbial spore count and the Bacillus subtilis DSM32540 strain in combination.

[0108] Figure 8 Illustrates for the DE22-004 feed sample, the number of germinable cells of the Bacillus subtilis DSM 32315 strain determined by the method according to the invention under different pretreatments compared to the microbial spore count and the Bacillus amyloliquefaciens CECT5940 strain in combination.

[0109] Figure 9Shows, for feed sample DE22 - 003, the number of germinable cells of the combination of Bacillus subtilis DSM 32540 strain determined by the method according to the invention under different pretreatments compared to the microbial spore count and Bacillus amyloliquefaciens CECT5940 strain .

[0110] Figure 10 Shows the results for feed sample DE22 - 008 with a single strain of Bacillus amyloliquefaciens CECT 5940 determined by the method according to the invention when pretreated with pure water, without using PMA and with different PMA concentrations .

[0111] Figure 11 Shows the results for feed sample DE22 - 008 with a single strain of Bacillus amyloliquefaciens CECT 5940 determined by the method according to the invention when pretreated with NaOH, without using PMA and with different PMA concentrations .

[0112] Figure 12 Shows the results for feed sample DE22 - 008 with a single strain of Bacillus amyloliquefaciens CECT 5940 determined by the method according to the invention when pretreated with heat, without using PMA and with different PMA concentrations .

[0113] Figure 13 Shows the linear regression between the values determined by the method according to the invention and the values determined by microbial spore count when applying 50 μM PMA, where the measured values are related to CFU laboratory 2

[0114] Figure 14 Shows the linear regression between the values measured by the method according to the invention and the values determined by microbial spore count when applying 20 μM PMA, where the measured values are related to CFU laboratory 2 Examples

[0115] Experiments

[0116] 1. Pretreatment and Photolysis

[0117] 1.1 Required Consumables and Materials

[0118] - 100 - 1000 μl pipette

[0119] - Sterile filter tips for 100 - 1000 μl pipette

[0120] - 10 - 100 μl pipette

[0121] - Sterile filter tips for 10 - 100 μl pipettes

[0122] - 0.5 - 10 μl pipettes

[0123] - Sterile filter tips for 0.5 - 10 μl pipettes

[0124] - 1.5 ml tubes

[0125] - Holder for 1.5 ml tubes

[0126] - Disposable gloves

[0127] - Waste bags

[0128] - Propidium monoazide bromide (PMA)

[0129] - Zirconia glass beads

[0130] - Aluminum foil

[0131] - 0.9% NaCl

[0132] - Ultra - pure water

[0133] - qPCR stabilizer buffer

[0134] 1.2 Required equipment

[0135] - BagMixer (Interscience)

[0136] - PMA LiteTM LED photolysis device (E90002, Biotium)

[0137] - Centrifuge for 1.5 ml centrifuge tubes, with a speed up to 5000 rcf (relative centrifugal force)

[0138] - MillMix20

[0139] - Eppendorf thermostatic mixer (heating bath)

[0140] 1.3 Pretreatment of sample materials

[0141] 1.3.1 Pretreatment of sample materials with sodium hydroxide

[0142] - Prepare a sodium hydroxide stock solution (c(NaOH) = 0.05 mol / L) by dissolving 2 g of sodium hydroxide in 1 L of ultra - pure water.

[0143] - Weigh 5 g of sample material (e.g., feed containing bacillus spores) into a stomacher bag and add the sodium hydroxide stock solution to a final weight of 100 g.

[0144] The filled stomacher bag was clamped in a stomacher apparatus (BagMixer, Interscience) and shaken at speed level 3 for 5 minutes. Shaking resulted in a 5-minute homogenization step in the stomacher apparatus. A pH of at least 8.5 in the initial solution ensured that the absorption of the feed by the bacillus spores was kept to a minimum. Dilution with a sodium hydroxide stock solution destroyed the vegetative cells in the bag.

[0145] - After the homogenization step, the suspension thus obtained is transferred to In the bottle.

[0146] - 10 mL of the sample solution thus obtained was pipetted into a 50 mL centrifuge tube, and the tube was sealed.

[0147] 1.3.2 Pretreatment of sample materials by heat treatment

[0148] - 5 g of sample material (eg feed containing Bacillus spores) were weighed into a stomacher bag and filled with 100 mL of ultrapure water.

[0149] - The filled stomacher bag was clamped in a stomacher apparatus (BagMixer, Interscience) and shaken for 5 minutes at speed level 3. This resulted in a 5 minute homogenization step in the stomacher apparatus.

[0150] - After the homogenization step, the suspension thus obtained is transferred to In the bottle.

[0151] - 10 mL of the sample solution thus obtained was pipetted into a 50 mL Falcon tube, and the tube was sealed.

[0152] - Incubate the sample tubes in a pre-heated water bath at 80°C for 10 minutes.

[0153] - Following incubation, cool the tubes in an ice bath for a few minutes.

[0154] 1.4 Preparation for PMA staining and extraction process

[0155] - Aliquot the suspensions obtained in 1.3.1 and / or 1.3.2. To do this, take four 1 mL aliquots from each suspension and dispense them into 4 mL tubes.

[0156] - Protect the aliquots thus obtained from light and maintain this protection throughout the PMA staining procedure.

[0157] - PMA stock solution is provided at a concentration of 20 mM.

[0158] - Add 2.5 μl or 1 μl of the stock solution to the light-protected fractionated sample to give a concentration of 50 μM (2.5 μl) or 20 μM (1 μl).

[0159] - Incubate the light-protected fractionated sample containing PMA for 10 minutes with shaking at 10 °C and 1200 rpm (20 Hz).

[0160] - Next, incubate the sample in PMA Lite (photolysis) at an LED output wavelength of 465 to 475 nm for 15 minutes.

[0161] - During the light exposure phase, remove the tubes from the PMA lite every 4 minutes and briefly shake or mix to ensure good light exposure for each point.

[0162] - Centrifuge all sample tubes at 10,000 rpm for 10 minutes.

[0163] - Remove the supernatant from each tube with a pipette and discard it.

[0164] - Add 1 mL of lysis buffer and 5 zirconia beads from the sample collection tube to each tube.

[0165] - Next, mix the contents of the tubes in a Millmix at 20 Hz for 10 minutes.

[0166] - After mixing, temper the tubes in a hot bath at 85 °C for 20 minutes.

[0167] - After tempering, mix the contents of the tubes again in a Millmix at 20 Hz for 10 minutes.

[0168] - Then, perform an extraction and qPCR procedure on the contents of the tubes.

[0169] 2. Extraction

[0170] ]>2.1 Required consumables and materials

[0171] - 100 - 1000 μl pipette

[0172] - Sterile filtered tips for 100 - 1000 μl pipettes

[0173] - E3

[0174] - Eppendorf Combitips 0.2 ml, light blue, biopure grade (single blistered)

[0175] - Eppendorf Combitips 10 ml, light blue, biopure grade (single blister)

[0176] - KingFisher deep well plates

[0177] - KingFisher microplates

[0178] - KingFisher Tip Combs

[0179] - Foil for sealing DNA elution plates (should be easy to remove)

[0180] - Racks for 1.5 ml and 2 ml tubes

[0181] - Disinfectant for workbench and KingFisher cleaning

[0182] - Waste liquid disposal tank

[0183] - Funnel for liquid handling from KingFisher plates

[0184] - Waste bags

[0185] - Disposable gloves

[0186] - Nucleic acid extraction kit

[0187] - Internal DNA extraction control kit VIC-channel (PrimerDesign)

[0188] 2.2 Required equipment

[0189] - KingFisher Flex (ThermoFisher)

[0190] - Vortex mixer

[0191] - Centrifuge 5425 (Eppendorf) with rotor FA-24x2

[0192] 2.3 Methods

[0193] · Select the " _AnsExtraction" protocol on the KingFisher Flex and press "Start". Follow the instructions on the KingFisher display.

[0194] · The "Comb plate" is prepared by adding 96 pipette tips combs to an empty 96 deep well plate, placing the plate in the loading position in the instrument, and pressing start to become continue loading.

[0195] · The "Elution Plate" (KingFisher TM 96-well plate, 200 μl) is prepared by adding 80 μl of elution buffer to each well used. Place the plate in the loading position in the instrument and press Start to change to continue loading.

[0196] · The "Wash 3 Plate" (KingFisher TM 96-deep well plate) is prepared by adding 500 μl of Wash Buffer 3 to each well used. Place the plate in the loading position in the instrument and press Start to change to continue loading.

[0197] · The "Wash 2 Plate" (KingFisher TM 96-deep well plate) is prepared by adding 500 μl of Wash Buffer 2 to each well used. Place the plate in the loading position in the instrument and press Start to change to continue loading.

[0198] · The "Wash 1 Plate" (KingFisher TM 96-deep well plate) is prepared by adding 500 μl of Wash Buffer 1 to each well used. Place the plate in the loading position in the instrument and press Start to change to continue loading.

[0199] · The "Sample Plate" is prepared by adding 850 μl of sample (from PMA staining), 25 μl of magnetic beads, 20 μl of enhancer, and 4 μl of resuspended internal control (lyophilized internal control DNA template, from PrimerDesign) to each well. Place the plate in the loading position in the instrument and press Start to start nucleic acid extraction.

[0200] When reloading Fisher TM Flex, follow the instructions on the display. When qPCR is not performed directly, seal the elution plate and store it short-term at 4 °C and long-term at 20 °C. Avoid repeated thawing and freezing of the elution plate.

[0201] 3. qPCR Detection

[0202] 3.1 Required Consumables and Materials

[0203] - 100 - 1000 μl pipette

[0204] - Sterile filtered tips for 100 - 1000 μl pipette

[0205] - 10 - 100 μl pipette

[0206] - Sterile filtered tips for 10 - 100 μl pipette

[0207] - 0.5 - 10 μl pipette

[0208] - Sterile filter tips for 0.5 - 10 μl pipettes

[0209] - E3

[0210] - Eppendorf Combitips 0.2 ml, light blue, biopure grade (single blister)

[0211] - 8 - channel pipette 1 - 10 μl

[0212] - 96 - well qPCR plate

[0213] - qPCR plate foil

[0214] - Roller for sealing adhesive film

[0215] - DNA removal reagent

[0216] - Arm sleeves for working under qPCR hood

[0217] - Rack for 1.5 and 2 ml tubes

[0218] - Eppendorf 2 ml tubes, qPCR clean grade

[0219] - Eppendorf 1.5 ml tubes, qPCR clean grade

[0220] - Forceps for picking up sterile tubes

[0221] - Waste bag

[0222] - Disposable gloves

[0223] - Luna Universal Probe qPCR Premix (M3004X)

[0224] - PCR - grade nuclease - free water

[0225] - Internal DNA extraction control kit with VIC fluorophore (PrimerDesign)

[0226] 3.2 Required equipment

[0227] - CFX96 Touch qPCR Cycler (BioRad)

[0228] - UV cabinet / qPCR hood

[0229] - Vortex mixer

[0230] - Centrifuge for microtubes, 2x

[0231] - Centrifuge for plates

[0232] 3.3 Nucleotides required

[0233] The qPCR method uses nucleotides, namely, forward primer, reverse primer, probe, and quantification vector, which are sequenced and synthesized for quantifying the target of interest and are thus specific for the bacterial strain under consideration. Here, as shown in the example of Bacillus amyloliquefaciens. All other products of the genus Bacillus are carried out under the same measurement conditions and standards described in this chapter.

[0234] Long-term storage of primer and probe stock solutions is carried out at -20 °C. Working stock solutions of primers and probes at the following concentrations are stored at 4 °C for up to 2 months:

[0235] - Primer: 20 μmol, 1:5 diluted stock solution

[0236] - Probe: 10 μmol, 1:10 diluted stock solution

[0237] A large number of freeze-thaw cycles are avoided.

[0238] For QS, dissolve the lyophilized vector in the elution buffer of the nucleic acid extraction kit, and calculate the copy number according to the following formula:

[0239]

[0240] Based on the calculated copy number / μl, for the corresponding target of interest (i.e., the ID gene for quantifying Bacillus amyloliquefaciens strain CECT 5940), prepare the following quantitative standards by serial dilution in the elution buffer:

[0241] Standard Concentration QS 4 1.00E+04 QS 3 1.00E+03 QS2 1.00E+02 QS1 1.00E+01

[0242] Table 1: Quantitative standards (QS) required for the assay method

[0243] For long-term storage, store the quantitative standards at -20 °C. For short-term storage at 4 °C for up to 5 weeks. A large number of freeze-thaw cycles of the stock solution and dilution vector are avoided. This component contains a high copy number of templates and has a very significant risk of contamination.

[0244] 3.4 Method

[0245] 3.4.1 Premix setup

[0246] All reagents and samples were completely thawed, mixed (by pipetting or gentle vortexing), and briefly centrifuged before use. This workflow contains a heterologous internal control (IC) that is used as a control for the sample preparation procedure (nucleic acid extraction) and a qPCR inhibition control.

[0247] The premix contains Luna Universal Probe qPCR Premix (M3004X), forward primer, reverse primer, probe, primer-designed IC mixture (heterologous internal control), premix volume, and pretreated sample. The heterologous internal control (IC) is used as a control for the sample preparation procedure (nucleic acid extraction) and a qPCR inhibition control.

[0248] For the required reaction volume, the composition of the premix was calculated as indicated in the following table. Consider loading each quantitative standard and non-template control (NTC) twice, and also consider an additional approximately 10% compensation for pipetting losses.

[0249] Following steps A and B listed below, and carefully considering the area to be prepared, the premix was set up.

[0250] 3.4.2 Cycler Program

[0251] Under the following settings, qPCR was performed on the target of interest:

[0252]

[0253]

[0254] Table 2: CFX96 Settings for qPCR

[0255] The cycling program was set as follows:

[0256]

[0257] Table 3: Cycling Program for qPCR

[0258] The cycling run time was 1 hour and 18 minutes.

[0259] The target was detected via the following fluorescent dyes:

[0260]

[0261] Table 4: Detectors and Channels for qPCR of the Target

[0262] 4. Result Interpretation

[0263] Before calculating the copy number of ID - gene in the original sample, check the following points:

[0264] 1. The baseline threshold is set to a specific RFU value for a specific CFX96 cycler setting.

[0265] 2. Fluorescence drift correction is activated.

[0266] 3. Check that the no-template control has an internal control (IC) and no other targets (if failed, the run is invalid).

[0267] 4. Check the presence and Cq of the IC for all negative samples (if failed, the sample is invalid).

[0268] 5. Check the slope of the standard curve (R^2 > 0.95) and qPCR efficiency (95 - 105%). If there are deviations, the run must be reviewed, and

[0269] 6. Check that the endpoint fluorescence is within a specific range (the range depends on the cycler and fluorophore).

[0270]

[0271] Table 5: Result Interpretation

[0272] *FAM TM Detection channel or 5 Detection channel. Positive results do not require VIC TM Detection of the internal control in the detection channel. High loading in the sample can cause a decrease or absence of the internal control signal.

[0273] ** Retest the eluate / original sample, or collect a new sample and retest.

[0274] For positive samples, the standard curve of the qPCR assay can quantify the target gene in the form of the starting quantity (SQ) per μl of eluate. The standard curve is automatically created by the CFX Manager software (when the appropriate wells are assigned as standards) or calculated by the following formula:

[0275] (1) Cq = m × log(N0) + b

[0276] Where

[0277] Cq = quantification cycle

[0278] m = slope

[0279] N0 = starting concentration (SQ)

[0280] b = intercept.

[0281] To determine the concentration in the original sample, apply the following formula:

[0282]

[0283] 5. Technical Specifications

[0284] 5.1 Specifications of the qPCR Method

[0285] 5.1.1 Limit of Detection (LOD)

[0286] The analytical sensitivity of the assay was determined as the 95% limit of detection (LOD), which represents the lowest amount of DNA molecules detected with a 95% probability. The LOD of the optimized qPCR assay was determined by performing serial tests on highly diluted quantitative standards. Twenty replicate tests were performed on the dilution series.

[0287] The 95% limits of detection for the following were calculated and obtained:

[0288] - (Bacillus amyloliquefaciens CECT 5940), 1.57 ± 0.20 copies / μl, and the confidence interval was 1.28 to 2.15 copies / μl

[0289] - (Bacillus subtilis DSM 32315), 1.16 ± 0.16 copies / μl, and the confidence interval was 0.92 to 1.65 copies / μl.

[0290] - (Bacillus subtilis DSM 32540), 1.72 ± 0.23 copies / μl, and the confidence interval was 1.38 to 2.41 copies / μl.

[0291] 6. Results

[0292] Multiple products and feed products containing different bacterial strains were applied to the microbial spore counting and the method according to the present invention. The data of the products are summarized in Table 6.

[0293]

[0294] Table 6: Data of the probiotic products used, the strains contained therein, and data of the feed products used

[0295] Samples with different combinations of the products in Table 6 were prepared. Additionally, the expected microbial spore counts in colony forming units (CFU) and the expected concentrations of the strains of the individual products in the resulting combinations were estimated.

[0296]

[0297] Table 7: Composition and microbial spore counts of the samples.

[0298] A total of 9 different samples with the composition of Table 7 were measured using the method according to the present invention and microbial spore counting (i.e., not according to the method of the present invention). The measurement results obtained thereby are shown in Figure 1a ) to 14, and these results will be discussed in detail below.

[0299] Figure 1a ) shows the correlation of microbial spore counts between Laboratory 1 (external laboratory) and Laboratory 2 (Evonik's QA laboratory). Linear regression fitting was performed on the measured samples and determined as the decimal logarithm (Log10) of colony forming units per gram (CFU / g), and a good linearity (coefficient of determination R 2 > 0.90) was obtained. The formula for the regression curve is f(x) = 0.7864 * x + 1.29.

[0300] Figure 1b ) is a box plot representation of the number of colony forming units (CFU) within the scope of the inter-laboratory test of microbial spore counts between the participating laboratories (N = 13). The evaluation of the results was reported by VDLUFA (Verband Deutscher Landwirtschaftlicher Untersuchungs-und Forschungsanstalten). The box shows the median and interquartile range. Based on this information, the average value is 3.70E+07 CFU / g, resulting in a tolerance range between 2.43E+07 and 5.24E+07 CFU / g. The inaccuracy of CFU determination is defined as a relative variance of 18.64%.

[0301] Figure 2a ) and 2b) show the linear regression between the values measured by the method according to quantitative PCR and the values measured by microbial spore counting (CFU), where the measurement is related to Laboratory 1 ( Figure 2a ) and Laboratory 2 ( Figure 2b ). Both curves can be established by plotting the starting quantity (SQ) concentration represented as a decimal logarithm (logarithmic scale) on the x-axis against the decimal logarithm of colony forming units per gram (CFU / g). In summary, 9 feed samples from broilers (DE22-001 to DE22-009, known spore count range of 1.0E+06 to 2.0E+07 spores / g) were analyzed and calculated with 3 valid replicates. The linear correlation of the corresponding regression curves was calculated, and a moderate positive linear correlation between the considered laboratories was indicated, where R 2 微生物孢子计数_实验室1vs.qPCR = 0.701 and R 2 微生物孢子计数_实验室2vs.qPCR= 0.864. The difference between the two methods is caused by the different detection of cell types quantified by each method: while microbial spore counting only quantifies spores that are capable of germinating under specific growth conditions, the molecular method measures all bacterial cells containing the target ID fragment, regardless of whether they are present as viable cells, dead cells, vegetative cells, or germinable cells.

[0302] Figure 3a ) shows the evaluation of different sample pretreatments without adding the DNA-binding dye propidium monoazide (PMA) to the feed sample DE22-006 with Bacillus subtilis strain DSM 32315. To evaluate the feasibility and impact of different pretreatments, the test feed samples were pretreated in lysis buffer, pure water (H2O), sodium hydroxide (NaOH) solution, and alternatively by heat pretreatment. Both sodium hydroxide pretreatment and heating at 80 °C were used in microbial spore counting. The concentrations [SQ / g] for the corresponding treatments were as follows: lysis buffer pretreatment was 2.22E+07; pure water pretreatment was 2.18E+07; sodium hydroxide pretreatment was 1.25E+07; heat pretreatment was 9.65E+07.

[0303] Figure 3b ) shows the linearity of the PMA concentration and covers the range from 120 μM to 0 μM. Both curves can be established by plotting the PMA concentration (in micromoles) on the x-axis against the quantification cycle (Cq) on the y-axis. For the broiler feed sample with, the linearity of sodium hydroxide pretreatment and pure water pretreatment was obtained. To measure the germinable cells in the feed sample, sodium hydroxide was used as part of the sample pretreatment to lyse all vegetative cells and thus had an impact on quantifying only germinable cells when using microbial spore counting. Compared with the samples pretreated with water, the linear curve of sodium hydroxide shifted upward along the y-axis, which means the number of cycles increased due to sodium hydroxide, and the cell concentration decreased due to the selection of vegetative cells and non-viable cells. The regression curve shows an obvious upward trend of Cq with the increase in PMA concentration. The higher the dye concentration, the easier PMA can penetrate intact cells. At a PMA concentration of 50 μM, the two curves were out of sync. At higher dye concentrations, an interaction occurred between the suspension with the pretreatment solution and the DNA-binding dye propidium monoazide. PMA concentrations of 20 μM and 50 μM were used for these measurements.

[0304] Figure 4 shows for Feed sample DE22 - 006, the number of germinable cells of Bacillus subtilis strain DSM 32315 determined by different pretreatment methods using the method according to the invention, compared to the microbial spore count. Sodium hydroxide pretreatment and a PMA concentration of 20 μM showed a slightly relative deviation between the method according to the invention and the microbial spore count, while at a PMA concentration of 50 μM, the number of germinable cells approached the expected concentration of 1E+07 CFU / g. Thermal pretreatment showed the largest deviation, which may be due to the denaturing effect of heating on DNA. The following concentrations of germinable cells were obtained by the respective determination methods: microbial spore count in laboratory 1, 1.50E+07 CFU / g; microbial spore count in laboratory 2, 1.14E+07 CFU / g; the method according to the invention using NaOH pretreatment and 50 μM PMA, 1.01E+07 SQ / g; the method according to the invention using NaOH pretreatment and 20 μM PMA, 1.15E+07 SQ / g; the method according to the invention using thermal pretreatment and 50 μM PMA, 4.75E+06 SQ / g; the method according to the invention using thermal pretreatment and 20 μM PMA, 4.03E+06 SQ / g.

[0305] Table 8 shows the results of the conversion from Figure 4 the relative deviation between the Log10 SQ / g determined by the method according to the invention and the Log10 CFU / g determined by microbial spore count in two laboratories. Overall, all the results of the pretreatment showed a relative difference of less than 8% compared to the microbial spore count. For PMA concentrations of 20 μM and 50 μM, the method according to the invention using sodium hydroxide NaOH pretreatment showed the smallest deviation from the microbial spore count in laboratory 2.

[0306]

[0307] Table 8: Relative deviation (results of conversion from Figure 4 to ) between the results (Log10 SQ / g) obtained by the method according to the invention and the results (Log10 CFU / g) obtained by microbial spore count in two laboratories.

[0308] Figure 5 shows for Feed sample DE22 - 007, number of germinable cells of Bacillus subtilis strain DSM 32540 determined by different pretreatment methods using the method according to the invention, compared to microbial spore counts. Sodium hydroxide pretreatment and a PMA concentration of 50 μM showed a slightly relative deviation between the method according to the invention and microbial spore counts, while a PMA concentration of 20 μM was closer to the expected concentration of 1E+07 CFU / g. Thermal pretreatment showed the largest deviation, which may be due to the denaturing effect of heating on DNA. The following concentrations of germinable cells were obtained by the respective determination methods: microbial spore count in laboratory 1, 1.70E+07 CFU / g; microbial spore count in laboratory 2, 6.93E+06 CFU / g; method according to the invention with NaOH pretreatment and 50 μM PMA, 7.97E+06 SQ / g; method according to the invention with NaOH pretreatment and 20 μM PMA, 9.35E+06 SQ / g; method according to the invention with thermal pretreatment and 50 μM PMA, 4.04E+06 SQ / g; method according to the invention with thermal pretreatment and 20 μM PMA, 6.51E+06 SQ / g.

[0309] Table 9 shows the results of the conversion from Figure 5 the relative deviation between Log10 SQ / g obtained by the method according to the invention and the Log10 CFU / g of microbial spore counts determined in two laboratories. Overall, all results of the pretreatment showed a relative difference of less than 9% compared to microbial spore counts. For PMA concentrations of 20 μM and 50 μM, the method according to the invention with sodium hydroxide NaOH pretreatment showed the smallest deviation from the microbial spore counts in laboratory 2.

[0310]

[0311]

[0312] Table 9: Relative deviation (results of the conversion from Figure 5 of ) between the results (Log10 SQ / g) obtained by the method according to the invention and the results (Log10 CFU / g) obtained by microbial spore counts in two laboratories.

[0313] Figure 6 Shows the results compared to the determinations in two laboratories The number of germinable cells of Bacillus amyloliquefaciens strain CECT 5940 determined by the method according to the invention using different pretreatment methods, compared to the microbial spore count of feed sample DE22-008. Sodium hydroxide pretreatment and a PMA concentration of 50 μM showed a slightly relative deviation between the results determined by the method according to the invention and those determined by microbial spore count, and were closer to the expected concentration of 1E+07 CFU / g. Thermal pretreatment showed the largest deviation, which may be due to the denaturing effect of heating on DNA. The following concentrations of germinable cells were obtained by the corresponding determination methods: microbial spore count in laboratory 1, 1.60E+07 CFU / g; microbial spore count in laboratory 2, 1.07E+07 CFU / g; the method according to the invention using NaOH pretreatment and 50 μM PMA, 9.91E+06 SQ / g; the method according to the invention using NaOH pretreatment and 20 μM PMA, 1.39E+07 SQ / g; the method according to the invention using thermal pretreatment and 50 μM PMA, 5.96E+06 SQ / g; the method according to the invention using thermal pretreatment and 20 μM PMA, 7.07E+06 SQ / g.

[0314] Table 10 shows the results of the conversion from Figure 6 the relative deviation between Log10 SQ / g determined by the method according to the invention and Log10 CFU / g of the microbial spore count in two laboratories. Overall, all results of the pretreatment showed a relative difference of less than 6% from the microbial spore count. For PMA concentrations of 20 μM and 50 μM, the method according to the invention using sodium hydroxide NaOH pretreatment showed the smallest deviation from the microbial spore count in laboratory 2.

[0315]

[0316] Table 10: Relative deviation (results of the conversion from Figure 6 to ) between the results obtained by the method according to the invention (Log10 SQ / g) and the results obtained by microbial spore count in two laboratories (Log10 CFU / g).

[0317] Figure 7 Shows the number of germinable cells of the combined strains of Bacillus subtilis DSM 32315 and Bacillus subtilis DSM 32540 determined by the method according to the invention using different pretreatment methods, compared to the microbial spore count of feed sample DE22-001 determined by two laboratories.

[0318] Laboratory 1 (Lab1) could not distinguish between the two strains, such thatFigure 7 Only the microbial spore count as the sum of two products is indicated. The following concentrations of germinable cells of the corresponding strains and determination methods were determined: Microbial spore count Laboratory 1 (microbial spore count as the sum of two products), 2.80E+07 CFU / g; Microbial spore count, for was 6.17E+06 CFU / g and was 1.04E+07 CFU / g; For the method of the present invention using NaOH pretreatment and 50 μM PMA, for was 5.43E+06 SQ / g and was 8.38E+06 SQ / g; For the method of the present invention using NaOH pretreatment and 20 μM PMA, for was 7.45E+06 SQ / g and was 9.07E+06 SQ / g; For the method of the present invention using heat pretreatment and 50 μM PMA, for was 5.81E+06 SQ / g and was 5.90E+06 SQ / g, and; For the method of the present invention using heat pretreatment and 20 μM PMA, for was 6.63E+06 SQ / g and was 7.82E+06 SQ / g.

[0319] Tables 11, 12, and 13 show the conversion results from Figure 7 the relative deviation of Log10 SQ / g determined by the method according to the present invention to the Log10 CFU / g of the microbial spore count determined by two laboratories for the corresponding target strains. Overall, all results of the pretreatment showed a relative difference of less than 1.3% in Table 11, less than 3.5% in Table 12, and less than 4.1% in Table 13 compared to the microbial spore count. For PMA concentrations of 20 μM and 50 μM, the method according to the present invention using sodium hydroxide NaOH pretreatment showed the smallest deviation from the microbial spore count of Laboratory 2.

[0320]

[0321] Table 11: Relative deviation between the results obtained by the method according to the present invention (Log10 SQ / g) and the results obtained by two laboratories using microbial spore count (Log10 CFU / g) (conversion results from Figure 7 of ).

[0322]

[0323] Table 12: Relative deviation between the results obtained by the method according to the invention (Log10 SQ / g) and the results obtained by microbial spore counting in two laboratories (Log10 CFU / g) (from Figure 7 of conversion result).

[0324]

[0325] Table 13: Relative deviation between the results obtained by the method according to the invention (Log10 SQ / g) and the results obtained by microbial spore counting in two laboratories (Log10 CFU / g) (from Figure 7 of and conversion result of the combination).

[0326] Figure 8 Shows the number of germinable bacterial cells of the combined strains of Bacillus subtilis DSM 32315 and Bacillus amyloliquefaciens CECT 5940 determined by the method according to the invention using different pretreatment methods compared to the microbial spore count of the DE22 - 004 feed sample determined in two laboratories.

[0327] Laboratory 2 (Lab2) cannot distinguish between the two strains, such that Figure 8 only the microbial spore count as the sum of the two products is indicated in is 1.20E+06 CFU / g for and 2.10E+07 CFU / g for is 1.09E+06 SQ / g for and 9.82E+06 SQ / g for is 1.50E+06 SQ / g for and 1.01E+07 for is 6.13E+05 for and 6.67E+06 SQ / g for is 8.65E+05 for and 8.02E+06 SQ / g for.

[0328] Tables 14, 15, and 16 show the conversion results from Figure 8 the relative deviation of Log10 SQ / g determined by the method according to the invention to the Log10 CFU / g of the microbial spore count determined by two laboratories for the corresponding target strains. Overall, all results of the pretreatment showed a relative difference from the microbial spore count of less than 6.8% in Table 14, less than 4.8% in Table 15, and less than 6.6% in Table 16. For PMA concentrations of 20 μM and 50 μM, the method according to the invention pretreated with sodium hydroxide NaOH showed the smallest deviation from the microbial spore count of Laboratory 2.

[0329]

[0330] Table 14: Relative deviation between the results obtained by the method according to the invention (Log10 SQ / g) and the results obtained by two laboratories using microbial spore count (Log10 CFU / g) (conversion results from Figure 8 of ).

[0331]

[0332] Table 15: Relative deviation between the results obtained by the method according to the invention (Log10 SQ / g) and the results obtained by two laboratories using microbial spore count (Log10 CFU / g) (conversion results from Figure 8 of ).

[0333]

[0334] Table 16: Relative deviation between the results obtained by the method according to the invention (Log10 SQ / g) and the results obtained by two laboratories using microbial spore count (Log10 CFU / g) (conversion results from Figure 8 of and ).

[0335] Figure 9 shows, for the DE22 - 003 feed sample, the number of germinable bacterial cells of the combined strains of Bacillus subtilis DSM 32540 and Bacillus amyloliquefaciens CECT5940 determined by different pretreatment methods using the method according to the invention compared to the microbial spore count.

[0336] Both of the two laboratories that determined the microbial spore counts were able to distinguish between the two strains. The following concentrations of germinable cells of the corresponding strains were determined by different methods: for Microbial Spore Counting Laboratory 1, for it was 1.80E+07 CFU / g and it was 1.50E+07 CFU / g; for Microbial Spore Counting Laboratory 2, for it was 1.11E+07 CFU / g and it was 7.28E+06 SQ / g CFU / g; for the method of the present invention using NaOH pretreatment and 50 μM PMA, for it was 9.92E+06 SQ / g and it was 8.62E+06 SQ / g; for the method of the present invention using NaOH pretreatment and 20 μM PMA, for it was 1.00E+07 and it was 8.67E+06 SQ / g; for the method of the present invention using heat pretreatment and 50 μM PMA, for it was 8.01E+06 SQ / g and it was 3.15E+06 SQ / g, and for the method of the present invention using heat pretreatment and 20 μM PMA, for it was 8.55E+06 SQ / g and it was 3.20E+06 SQ / g.

[0337] Tables 17, 18, and 19 show the conversion results of the relative deviation from Figure 9 Log10 SQ / g determined by the method according to the present invention to the corresponding target strains between Log10 CFU / g determined by microbial spore counting in the two laboratories. Overall, all the results of the pretreatment showed a relative difference from the microbial spore counting of less than 5.0% in Table 17, less than 9.5% in Table 18, and less than 6.3% in Table 19. For PMA concentrations of 20 μM and 50 μM, the method according to the present invention using sodium hydroxide NaOH pretreatment showed the smallest deviation from the microbial spore counting in Laboratory 2.

[0338]

[0339] Table 17: Relative deviation (conversion result from Figure 9 of of) between the results obtained by the method according to the present invention (Log10 SQ / g) and the results obtained by microbial spore counting in the two laboratories (Log10 CFU / g).

[0340]

[0341] Table 18: Relative deviation between the results obtained by the method according to the invention (Log10 SQ / g) and the results obtained by microbial spore counting in two laboratories (Log10 CFU / g) (conversion results from Figure 9 of .

[0342]

[0343] Table 19: Relative deviation between the results obtained by the method according to the invention (Log10 SQ / g) and the results obtained by microbial spore counting in two laboratories (Log10 CFU / g) (conversion results from the combination of Figure 9 of and ).

[0344] Figure 10 , 11 and 12 represent the feed sample DE22 - 008 with a single strain of Bacillus amyloliquefaciens CECT 5940 measured, and the definition of the bacterial cell structure groups obtained for three pretreatments at PMA concentrations of 20 μM and 50 μM.

[0345] Figure 10 Shows the results measured by the method according to the invention under pretreatment involving pure water. The bar chart refers to the total concentration, which presents live and non - live cells and gives consideration to the distinction between live cells (germinable cells and vegetative cells) and non - live cells (expressed as SQ / g). Without the addition of PMA, it was determined to be 1.95E+07 SQ / g, including both live and non - live cells. The addition of PMA aims to distinguish between live and non - live cells. At a PMA concentration of 50 μM, the number of live cells was determined to be 1.70E+07 SQ / g and the number of non - live cells was 2.49E+06 SQ / g, and in the case of 20 μM, the number of live cells was 1.88E+07 SQ / g and the non - live cells were 7.04E+05 SQ / g respectively.

[0346] Figure 11 Shows the results of sample pretreatment with sodium hydroxide NaOH, and Figure 12 shows the results of sample pretreatment with heat, which were measured by the method according to the invention under the corresponding pretreatments. The bar chart refers to the total concentration, which presents live and non - live cells and gives consideration to the distinction of the group of live cells (including germinable cells and vegetative cells). Both pretreatment methods aim to lyse the vegetative cells in the sample to ensure the qualitative and quantitative determination of germinable cells. All results are expressed as SQ / g.

[0347] At Figure 11In the absence of PMA, the concentration obtained was calculated to be 1.83E+07 SQ / g, which includes all viable and non-viable cells. Addition of 50 μM PMA resulted in a concentration of germinable cells of 9.91E+06 SQ / g and 20 μM PMA of 7.07E+06 SQ / g, respectively.

[0348] exist Figure 12 The concentration obtained without PMA was calculated to be 8.52E+06 SQ / g. At 50 μM PMA, 5.96E+06 SQ / g germinable cells were determined, and at 20 μM PMA, 7.07E+06 SQ / g germinable cells were determined.

[0349] In the case of sodium hydroxide pretreatment Figure 13 (50 μM PMA applied) and Figure 14 (20 μM PMA applied) shows the linear regression between the values obtained by the method according to the invention and the values obtained for the microbial spore count determined in Laboratory 2. Both curves can be constructed by plotting the starting quantity (SQ) concentration expressed as a decimal logarithm (logarithmic scale) on the x-axis relative to the decimal logarithm of the colony forming units per gram (CFU / g). In summary, nine broiler feed samples (with a known microbial spore count ranging from 1.0E+06 to 2.0E+07 CFU / g) were analyzed and calculated with three valid replicates. The linear correlation of the corresponding regression curves was calculated and indicated an excellent linear correlation between the methods considered, with R 2 微生物孢子计数 _ 实验室2vs.本发明方法_PMA50μM =0.921 and R 2 微生物孢子计数 _ 实验室2vs.本发明方法_PMA20μM =0.958. Figure 1a 、 2a Compared to the correlation in Figure 2b, the values determined according to the method of the present invention show an excellent linear correlation with the values determined using microbial spore counting. These figures demonstrate the consistency of the method of the present invention with microbial spore counting, which allows for the determination of only germinable cells, depending on whether the gene originates from living or dead cells. However, compared to microbial spore counting, the method of the present invention is robust and allows for simple, rapid manual determination and high sensitivity.

Claims

1. A method for determining the number and / or concentration of germinable cells in a sample of biological origin, wherein the germinable cells are bacterial spores formed within vegetative cells, the method comprising the following steps: a) Pretreating at least a portion of the sample material of biological origin, said pretreatment involving lysis of the vegetative cells, b) Photolyzing the pretreated sample material obtained from step a), c) Isolating nucleic acids from the photolyzed sample material obtained from step b), and d) Performing quantitative polymerase chain reaction on the isolated nucleic acids obtained from step c) to obtain the number and / or concentration of germinable cells in the sample material of step a), characterized in that the pretreatment of step a) comprises the step of treating the sample material with an alkaline solution.

2. The method according to claim 1, wherein said step a) further comprises subjecting the sample material to an elevated temperature and / or additional mechanical treatment.

3. The method according to claim 1 or 2, wherein the base in the alkaline solution of step a) has a pKb of 0 or less.

4. The method according to claim 2 or 3, wherein the elevated temperature of step a) is a temperature of at least 80 °C.

5. The method according to any one of claims 1 to 4, wherein said step b) comprises the following steps: b1) Keeping the pretreated sample material obtained from step a) away from light, b2) Adding a photoreactive DNA-binding dye to the sample material of step b1), and b3) Photolyzing the sample material obtained from step b2).

6. The method according to any one of claims 1 to 5, wherein the photoreactive DNA-binding dye embeds or penetrates into the DNA of cells having a disrupted wall structure and / or into the DNA of cells having a damaged cell wall structure including a cortex region and an inner membrane wall.

7. The method according to any one of claims 1 to 6, wherein the photoreactive DNA-binding dye is propidium azide bromide, ethidium azide bromide, a derivative of any of these, and / or a mixture thereof.

8. The method according to any one of claims 1 to 7, wherein said step c) comprises the following steps: c1) Subjecting the photolyzed sample material obtained from step b) to chemical and / or mechanical lysis to release nucleic acids from the sample material, c2) Binding the released nucleic acids obtained from step c1) to beads to obtain loaded beads, c3) Washing the loaded beads obtained from step c2), and c4) Releasing the nucleic acids from the washed beads obtained from step c3) by treatment with an elution buffer or water to obtain a solution containing the released nucleic acids.

9. The method according to claim 8, wherein the chemical lysis of step c1) is carried out under chaotropic conditions.

10. The method according to any one of claims 8 to 9, wherein the chemical lysis of step c1) is carried out under high chaotropic salt conditions.

11. The method according to any one of claims 1 to 10, further comprising the following steps: e) Determining the number and / or concentration of non-viable cells and vegetative cells in the sample of step a), which comprises the following steps: e1) Determine the number and / or concentration of all cells in the part of the sample material of step a), and e2) Take the difference between the number and / or concentration of all cells obtained in step e1) and the number and / or concentration of germinable cells obtained in step e) to give the number and / or concentration of the non-viable cells and the vegetative cells in the sample material of step a).

12. The method according to any one of claims 1 to 11, further comprising the following steps: f) Determine the number and / or concentration of the vegetative cells in the sample of step a), comprising the following steps: f1) Determine the number and / or concentration of the viable cells in the part of the sample material of step a), and f2) Take the difference between the number and / or concentration of the viable cells obtained in step f1) and the number and / or concentration of germinable cells obtained in step d) to give the number and / or concentration of the vegetative cells in the sample material of step a).

13. The method according to any one of claims 11 to 12, further comprising the following steps: g) Determine the relative change of the number and / or concentration of germinable cells relative to the number and / or concentration of all cells by means of the following formula: and / or where rel.(%) is the relative change of the number and / or concentration of germinable cells in percentage, n g is the number of germinable cells obtained in step d), c g is the concentration of germinable cells obtained in step d) n 所有 is the number of all cells obtained in step e1), and c 所有 is the concentration of all the cells obtained in step e1).

14. The method according to any one of claims 1 to 13, wherein the sample of step a) is a biomass, feed, manure, or digestate sample.

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