Method for isolating nucleic acids from an inhibitor rich biological sample

Abstract

The present invention provides a method for isolating nucleic acids from a sample, preferably a stool or gut sample, the method comprising (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibiting agent, (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removing agent, (b) clearing the lysate; (c) isolating nucleic acids from the liquid fraction of the cleared lysate. The method is particularly useful for isolating total nucleic acids, DNA or RNA from inhibitor- rich samples, such as stool samples. In contrast to prior art method, it advantageously omits a second inhibitor removal step after lysate clearance step (b) and prior to performing nucleic acid isolation step (b).

Description

[0001] “Method for isolating nucleic acids from an inhibitor rich biological sample”

[0002] FIELD OF THE INVENTION

[0003] The present invention provides methods and kits for isolating nucleic acids, such as bacterial and other microbial nucleic acids, from inhibitor rich samples, in particular fecal samples such as stool samples. The technology of the invention advantageously reduces the necessary steps for inhibitor depletion and is suitable for automation. It provides high yields of pure DNA and / or RNA.

[0004] BACKGROUND OF THE INVENTION

[0005] Biological samples, such as gut derived samples and in particular stool samples, represent a rich source of information on the microbiome and many microbiome studies investigate these sample types. Microbes include bacteria, fungi, and viruses. There is a growing interest in the isolation of genetic information from these sample types which requires accurate and easy to use methods for recovering nucleic acids from these types of samples, e.g. to support microbiome studies or for diagnostic and / or medical purposes. The primary focus is often on the isolation of bacterial nucleic acids. As nucleic acids to be isolated, either total nucleic acids (RNA and DNA) or specific types of nucleic acids, such as RNA or DNA, are of interest. There is a need for efficient and flexible methods that can isolate the nucleic acids of interest from inhibitor-rich samples with good yield and purity thus allowing to use the isolated nucleic acids in downstream methods. E.g. the isolated nucleic acids can be subsequently analyzed using sensitive amplification-based methods, such as polymerase chain reaction (PCR), reverse transcription PCR, qPCR, digital PCR (dPCR) and next-generation sequencing.

[0006] If contaminating substances are present in the isolated nucleic acids, they can interfere with and inhibit the downstream analysis of the isolated nucleic acids. The removal of inhibiting components during nucleic acid isolation is important and challenging. The above-mentioned samples, such as in particular fecal samples, are very complex and contain large amounts of in some cases quite diverse interfering components. Moreover, if the processing of a higher sample amount is desired, e.g. to recover enough nucleic acids, e.g. RNA, for downstream analysis, concomitant high levels of inhibitors enter the isolation process which makes the isolation of pure, inhibitor-depleted nucleic acids from higher sample amounts particularly challenging. To allow efficient down-stream analysis of the isolated nucleic acids, often involving enzymatic reactions such as PCR amplification, thorough removal of these inhibitors is, however, crucial.

[0007] Methods for isolating nucleic acids from inhibitor-rich samples are described in the art, see e.g. WO 2006 / 073472 and WO2019 / 209597. A multitude of commercially available kits for purification of nucleic acid from inhibitor-rich samples, such as fecal samples are also available.

[0008] However, the existing methods have drawbacks, such as the processing of limited amounts of sample, insufficient inhibitor removal, low purity, moderate to low nucleic acid yields and / or the requirement of multiple handling steps. Existing technologies for isolating nucleic acids from inhibitor rich biological samples, such as fecal samples, usually suffer from low yield and / or low purity or require multiple steps for inhibitor removal and are thus laborious and time consuming. Furthermore, the existing methods are not suitable for automated nucleic acid isolation, which is, however, desirable to increase the number of samples that can be processed in parallel for nucleic acid isolation.

[0009] Improvements and standardization of nucleic acid extraction methods are needed to meet the needs of microbiome research while providing the most unbiased representation of the sample.

[0010] It is the object of the present invention to provide a method for isolating nucleic acids, such as RNA and / or DNA, from inhibitor rich biological samples, in particular stool or gut samples, which overcomes at least one of the above drawbacks of the prior art methods. In particular, it is the object of the present invention to provide an improved method for isolating nucleic acids, such as RNA and / or DNA, from inhibitor-rich samples, in particular stool or gut samples, which achieves an efficient removal of inhibitory contaminants and is suitable for automated nucleic acid isolation. It is also an object of the invention to provide a method for isolating nucleic acids, such as RNA and / or DNA, from inhibitor-rich biological samples, that provides high yield, inhibitor-depleted nucleic acids suitable for downstream analysis in the field of molecular biology.

[0011] It is a particular object to provide an efficient and flexible method that is suitable for isolating microbial nucleic acids (RNA and / or DNA) from microbiome containing samples, such as fecal or gut samples. It is also an object of the present invention to provide an improved method for isolating bacterial and other microbial nucleic acids from stool samples and other fecal samples and gut samples. SUMMARY OF THE INVENTION

[0012] According to a first aspect, a method for isolating nucleic acids from a sample, preferably a stool or gut sample, is provided, the method comprising

[0013] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with

[0014] (i) at least one chaotropic agent and preferably a phosphate,

[0015] (ii) at least one RNase inhibiting agent,

[0016] (iii) at least one protein precipitating agent, and

[0017] (iv) at least one inhibitor removing agent,

[0018] (b) clearing the lysate;

[0019] (c) isolating nucleic acids from the liquid fraction of the cleared lysate.

[0020] As is demonstrated by the examples, the method of the invention allows to efficiently recover nucleic acids, such as total nucleic acids, RNA or DNA, of high quality and purity from inhibitor rich samples, in particular microbiome containing samples. Inhibitor rich samples for which the method is particularly suitable include but are not limited to other fecal samples, such as stool, gut samples, sludge samples and wastewater samples. These sample types are rich in microbes (e.g. bacteria, fungi, and viruses) and are often processed for microbiome analysis and other analytic methods that require the detection of microbial nucleic acids.

[0021] The modified sample lysis conditions used in step (a) greatly reduce the amount of inhibitory substances in the isolated nucleic acids.

[0022] Advantageously, this modified step (a) is so efficient that a second inhibitor removal step after lysate clearing (as it is commonly performed in prior art methods) can be omitted. A second inhibitor removal step after lysate clearance, as it is performed in prior art methods, is not necessary and is not performed with the method of the invention. This is highly advantageous, as it inter alia saves time and reduces the number of handling steps. As every handling step (e.g. centrifugation, supernatant removal etc.) will be accompanied with some loss of yield e.g. due to dead volume or pipetting steps, it is highly advantageous that the invention allows to omit the second inhibitor removal step while achieving a high degree of inhibitor depletion and purity. This allows to achieve an increase in yield, as is also demonstrated by the examples.

[0023] Furthermore, nucleic acids can be isolated from the cleared lysate in an automated manner. The method of the invention is also scalable, thereby allowing the processing of large sample volumes. The method is highly efficient in isolating DNA and RNA and provides high and specific yields of both DNA and / or RNA. The method of the invention in particular allows isolating inhibitor-free total DNA and RNA simultaneously from microbiome samples, such as human microbiome samples (e.g. fecal or gut samples). The method allows to isolate bacterial, archael, fungal, and viral nucleic acids. The method can also be modified to allow isolation of RNA (only) or DNA (only) and is thus very flexible. The present invention, therefore, provides significant improvements to the current state of the art.

[0024] According to a second aspect, the present invention pertains to the use of a kit for isolating nucleic acids from a sample, preferably a stool or gut sample, for performing the method according to the first aspect, the kit comprising

[0025] (a) a first solution comprising a chaotropic agent and preferably a phosphate;

[0026] (b) a second solution comprising at least one protein precipitating agent and at least one inhibitor removing agent;

[0027] (c) a solid phase for nucleic acid binding, preferably magnetic particles;

[0028] (d) a binding solution for binding nucleic acids to the solid phase.

[0029] Also provided are advantageous liquid lysis compositions, lysis mixtures and lysed sample preparations. Other aspects, objects, features, and advantages of the present application will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, while indicating preferred embodiments of the application, are given by way of illustration only. The headings provided herein are not limitations of the various aspects or embodiments of this invention which can be read by reference to the specification as a whole.

[0030] In the following description, any ranges provided herein include all the values in the ranges.

[0031] It should also be noted that the term “or” is generally employed in its sense including “and / or” ( / .e., to mean either one, both, or any combination thereof of the alternatives) unless the content dictates otherwise.

[0032] Also, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content dictates otherwise.

[0033] The term “a combination thereof’ as used herein refers to one of all possible combinations of the listed items preceding the term. For example, “A, B, C, or a combination thereof” is intended to refer to any one of: A, B, C, AB, AC, BC, or ABC. Similarly, the term “combinations thereof” as used herein refers to all possible combinations of the listed items preceding the term. For instance, “A, B, C, and combinations thereof” is intended to refer to all of: A, B, C, AB, AC, BC, and ABC.

[0034] The terms “include,” “have,” “comprise” and their variants are used synonymously and to be construed as non-limiting. Throughout the specification, where compositions or solutions are described as comprising components or materials, it is additionally contemplated that the compositions or solutions can in embodiments also consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise.

[0035] It is preferred to select and combine preferred embodiments described herein and the specific subject-matter arising from a respective combination of preferred embodiments also belongs to the present disclosure.

[0036] DETAILED DESCRIPTON OF THE INVENTION

[0037] The present invention provides an improved method for isolating nucleic acids from biological samples, e.g. stool samples, and in particular provides means for efficiently reducing inhibitory substances in the lysed sample, thereby enabling the isolation of high purity nucleic acids with a reduced number of handling steps. It is highly advantageous for isolating nucleic acids (RNA and / or DNA) for microbiome analysis. The method provides a highly useful microbiome extraction technology which is suitable for a wide spectrum of human microbiome samples. The method of the invention will therefore aid the standardization and comparability across microbiome samples. The high-quality nucleic acids isolated using the method of the invention is suitable for numerous downstream applications in the field of molecular biology, including amplification-based methods such as RT-PCR, PCR, qPCR, dPCR and next generation sequencing. As is demonstrated in the examples, the method provides significant advantages over prior art methods.

[0038] METHOD FOR ISOLATING NUCLEIC ACIDS FROM AN INHIBITOR-RICH SAMPLE

[0039] According to a first aspect, a method for isolating nucleic acids from a sample, preferably a fecal sample, is provided, the method comprising

[0040] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with

[0041] (i) at least one chaotropic agent and preferably a phosphate,

[0042] (ii) at least one RNase inhibiting agent,

[0043] (iii) at least one protein precipitating agent, and

[0044] (iv) at least one inhibitor removing agent,

[0045] (b) clearing the lysate;

[0046] (c) isolating nucleic acids from the liquid fraction of the cleared lysate.

[0047] As noted, the sample is preferably an inhibitor-rich sample, such as microbiome containing sample. The method advantageously allows to process fecal samples, such as stool samples, gut samples, sludge sample and wastewater samples, among others. The individual steps and preferred embodiments of the method are described in the following. Step (a) - preparing a lysed sample

[0048] Step (a) comprises preparing a lysed sample, wherein lysate preparation comprises contacting the sample with (i) at least one chaotropic agent, (ii) at least one RNase inhibiting agent, (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removing agent. Preferably, a phosphate is also added in step (a).

[0049] It is a special feature of the method of the invention that a protein precipitating agent and at least one inhibitor removing agent and an RNase inhibiting agent, such as an organic solvent, are already included during the lysis step. This allows to precipitate and / or complex interferents, in particular inhibitors and proteins, during step (a) that can then be removed during lysate clearing step (b). This provides an interferent-depleted cleared lysate.

[0050] The liquid fraction of the cleared lysate can be collected as supernatant and can be directly used in nucleic acid isolation in step (c). A second inhibitor removal step after lysate clearance step (b), as it is e.g. performed in prior art methods, is not necessary and a second inhibitor removal step is not performed in the method of the invention after lysate clearance step (b) and prior to performing step (c). The supernatant obtained after lysate clearance step (b) can be directly subjected to nucleic acid step (c) and contacted with the binding agents and the nucleic acid binding solid phase for nucleic acid isolation.

[0051] Chaotropic agent and phosphate

[0052] A chaotropic salt is preferably used as chaotropic agent. It is preferred to use a relatively mild chaotropic agent. Such chaotropic agent denatures proteins less than the strong chaotropic agents GuSCN or GuHCL. Suitable chaotropic salts include salts having the strong anion, SON paired with a cation weaker than Mg2+in solubilizing proteins; salts having the strong anion, CIOT, paired with a cation weaker than Mg2+in solubilizing proteins; and salts having the weak anion, CO32; paired with a cation stronger than NH4+in solubilizing proteins. The order can be determined according to the Hofmeister series which is a classification of ions in order of their ability to salt out or salt in proteins.

[0053] The chaotropic salt may be selected from NaSCN, NaaCOa, KSCN, NH4SCN, LiSCN, LiCIO4, guanidine sulfate and combinations thereof. These relatively mild chaotropic agents can be used to generate a lysate. While strong chaotropic agents can achieve complete cell lysis, this sometimes is at the expense of degraded biomolecules (e.g., degraded RNA), in particular when lysis is assisted by mechanical disruption. The less aggressive chaotropic agents that are preferably used in conjunction with the present method are unique in their capacity to solubilize biomolecules, including RNA, while minimizing their degradation. This also allows to assist the lysis process by mechanical disruption. According to one embodiment, the chaotropic agent is selected from sodium thiocyanate, potassium thiocyanate, ammonium thiocyanate, lithium thiocyanate and combinations thereof. Such chaotropic agents are particularly suitable to generate a lysate.

[0054] According to one embodiment, the chaotropic agent is NaSCN or Na2COs, preferably NaSCN. According to one embodiment, only one chaotropic agent is used for lysis which is NaSCN.

[0055] The chaotropic agent may be comprised in a lysis solution that is contacted with the sample. As disclosed elsewhere herein, also two or more different solutions comprising the core components of the invention may be added to the sample to establish the lysis conditions.

[0056] The concentration of the at least one chaotropic agent in the lysis solution that is contacted with the sample may be 2.5M or less, e.g. 2M or less, 1.75M or less, 1.5M or less, 1.3M or less, 1.2M or less or 1.125M or less. Suitable concentrations of the at least one chaotropic agent in the lysis solution may be in a range from 0.5M to 2.5M, e.g. selected from 0.6M to 2M, 0.7M to 1.75M, 0.75M to 1.5M and preferably 0.75M to 1.25M or 0.8M to 1.25M. If multiple chaotropic agents are present in the lysis solution, the total concentration of chaotropic agents in the lysis solution may be and preferably lies in the above-described range. The chaotropic agent is preferably a thiocyanate salt as described above, more preferably NaSCN. The above concentrations were found particularly suitable for such mild thiocyanate salts, such as NaSCN. Particularly preferred is a concentration of NaSCN in the lysis solution in the range of 0.7M to 1.75M, 0.75M to 1.5M and preferably 0.75M to 1.25M.

[0057] According to a preferred embodiment, the method comprises adding at least one phosphate in step (a). Without wishing to be bound by theory, it is believed that the free phosphate group (PO43") prevents or reduces complex formation between the additionally used inhibitor removing agent (e.g., AICI3) and the phosphodiester groups of nucleic acids by competitively interacting with the inhibitor removing agent. Therefore, the at least one phosphate is preferably added in step (a) and is thus included during lysis. The at least one phosphate may be included in the lysis solution that comprises the chaotropic agent. Therefore, for lysis in step (a), the sample may be contacted with a solution that comprises the at least one chaotropic agent and the at least one phosphate. According to one embodiment, the lysis solution comprises sodium thiocyanate and a phosphate.

[0058] Exemplary phosphates include phosphate monobasics, phosphate dibasics, and phosphate tribasics, and other compounds that contain one or more free phosphate groups, such as sodium phosphate monobasic, sodium phosphate dibasic, sodium phosphate, potassium phosphate monobasic, potassium phosphate dibasic, potassium phosphate, ammonium phosphate monobasic, ammonium phosphate dibasic, ammonium phosphate, lithium phosphate monobasic, lithium phosphate dibasic, lithium phosphate, trisodium phosphate, sodium poly(vinylphosphonate), sodium hexametaphosphate, pyrophosphate, sodium triphosphate, sodium polyphosphate, other phosphorus-containing oxyanions, and combinations thereof. The cationic moieties in the phosphates include but are not limited to ammonium, sodium, potassium, and lithium. In one embodiment, the cationic moiety is provided by an alkali metal ion, preferably selected from sodium, potassium and lithium, more preferably sodium. Preferably, the phosphate is a phosphate dibasic and more preferably is sodium phosphate dibasic.

[0059] The concentration of the at least one phosphate in the lysis solution that is contacted with the sample may be selected from 0.05M to 0.75M, e.g. 0.075M to 0.5M, 0.1M to 0.3M and 0.1M to 0.25M or may be 0.125M to 0.2M. The concentration of the at least one phosphate in the lysis solution is preferably in the range of 0.1 M to 0.3M, 0.1M to 0.25M or 0.125M to 0.2M. It is particularly preferred to use sodium phosphate dibasic in this concentration.

[0060] According to one embodiment, the lysis solution comprises sodium thiocyanate and at least one phosphate, preferably sodium phosphate dibasic.

[0061] According to one embodiment, the lysis solution that is contacted with the sample in step (a) comprises sodium thiocyanate in a concentration selected from 0.7M to 1.75M, 0.75M to 1.5M and preferably 0.75M to 1.25M and the at least one phosphate, preferably sodium phosphate dibasic, in a concentration selected from 0.075M to 0.3M, 0.1M to 0.25M and 0.1 M to 0.2M. Preferably, the concentration of the at least one phosphate, preferably sodium phosphate dibasic, is in the range of 0.1 M to 0.3M or 0.1 M to 0.25M.

[0062] According to one embodiment, step (a) comprises contacting the sample with a lysis solution that comprises sodium thiocyanate in a concentration of 0.75M to 1.5M and at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.1 M to 0.3M. The lysis solution may comprise sodium thiocyanate in a concentration of 0.8M to 1.25M and at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.1M to 0.25M.

[0063] The lysis solution, may comprise, consist essentially of, or consist of one or more chaotropic agents and one or more phosphates, both as described above. It may be an aqueous solution. Preferably, the one or more relatively mild chaotropic agents comprise or is NaSCN. The one or more phosphates preferably comprise or are sodium phosphate dibasic. An exemplary preferred lysis solution comprises, consists essentially of, or consists of 0.5M to 1.5M NaSCN and 0.1M to 0.25M Na2HPC>4. The pH of the lysis solution may be in the range of pH 4 to pH 10, e.g. pH 5 to pH 9 and pH 6 to 8.0.

[0064] As is also described in further detail below, the lysis solution comprising the chaotropic agent and preferably the phosphate is combined with the RNase inhibiting agent and a further solution that comprises the protein precipitating agent and the inhibitor removing agent to prepare a liquid lysis composition, in which the sample is lysed. This lysis composition is in contact with the sample and comprises (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibiting agent, (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removing agent. The contacting / combining may be performed in any order.

[0065] RNase inhibiting agent

[0066] During step (a), the sample is also contacted with a RNase inhibiting agent. Including an RNase inhibiting agent is advantageous to protect the RNA that is released during the lysis process. This allows to isolate RNA with high quality, either in form of total nucleic acids, or RNA only.

[0067] According to a preferred embodiment, the RNase inhibiting agent that is included for lysis is an organic extraction solvent. It may be a segregating chemical. It may comprise phenol. In preferred embodiments, the RNase inhibiting agent is an organic extraction solvent comprising phenol, benzyl alcohol, benzaldehyde, chloroform, isoamylalcohol, dichloromethane or a combination of two or more of the foregoing. The organic extraction solvent that is used in step (a) may be selected from phenol, phenol-chloroform-isoamylalcohol, phenol-chloroform, benzyl alcohol-benzaldehyde and phenol-dichloromethane. Preferably, the organic extraction solvent is phenol-chloroform-isoamylalcohol. In the examples, phenol-chloroform- isoamylalcohol (25:24:1 , pH 6.5-8.0) was used. Alternatively, a combination of benzyl alcohol and chloroform may be used (e.g. in a ration of 9:1). Using an according organic extraction solvent as RNase inhibiting agent is associated with important advantages. It protects the RNA against degradation but also assists in the lysis process. An organic solvent such as phenolchloroform or phenol-chloroform-isoamylalcohol maximizes the lysing efficiency and thus the RNA yield. Lysed cell components are trapped in the solvent and proteins are denatured leaving the nucleic acid in solution. This is particularly preferred in combination with the protein precipitating agent and the inhibitor removal agent that are also used in lysis step (a) to prepare the lysed sample.

[0068] The organic extraction solvent acting as RNase inhibiting agent may be added over a wide concentration range. In the lysis mixture that comprises the sample and optionally disrupting particles (if added), the organic solvent may be comprised in a concentration v / v (calculation excludes the sample and disrupting particles, if additionally used for lysis) of 30% or less, 25% or less or 20% or less, such as 15% or less. Particularly suitable concentrations in v / v (excluding for the calculation the sample and disrupting particles (if used)) for the organic extraction solvent may lie in the range of 2% to 25%, 3% to 20% and 5% to 15%. As noted, phenol-chloroform-isoamyl alcohol or phenol-chloroform are particularly preferred.

[0069] Other known RNase inhibiting agents that can be used during step (a) include, but are not limited to a reducing agent, optionally DTT or beta-mercaptoethanol, a detergent, optionally an anionic detergent such as SDS, and diethyl pyrocarbonate. Such RNase inhibiting agents may also be comprised in the lysis solution that comprises the chaotropic agent.

[0070] Protein precipitating agent and inhibitor removing agent

[0071] According to one embodiment, the at least one protein precipitating agent that is contacted with the sample during lysis step (a) is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride and cesium acetate. Some precipitating agents (e.g., ammonium acetate) may function as a protein precipitating agent at a relatively high concentration but as a molecular screen at a relatively low concentration (e.g., at a concentration 5 to 15 times less than the concentration when functioning as a protein precipitation agent). The use of ammonium acetate as protein precipitating agent is preferred. It may assist in the precipitation of proteins, that may act as interferents, during the lysis step. This assists in providing an interferent-depleted cleared lysate.

[0072] The inhibitor removing agent may form a complex with an inhibitor compound that is comprised in the sample. The formed complex may be precipitated and can be removed during lysate clearing step (b). This assists in providing an interferent-depleted cleared lysate, which is preferably collected as supernatant. The inhibitor removing agent is preferably a metal salt. The metal salt may be selected from aluminium chloride, aluminium sulfate, erbium (III) acetate, erbium (III) chloride, holmium chloride, zirconium (IV) chloride, hafnium (IV) chloride, aluminium ammonium sulfate, aluminium ammonium sulfate dodecahydrate, aluminium potassium sulfate, aluminium chlorohydrate, calcium oxide, iron (III) chloride, iron (II) sulfate, sodium aluminate, sodium silicate, magnesium chloride, and combinations thereof.

[0073] In embodiments, the inhibitor removing agent is a tri- or tetravalent salt that contains a cation having a valence of three or four. Preferably, the inhibitor removing agent is a tri- or tetravalent metal salt. The inhibitor removing agent may be selected from aluminium chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof. In preferred embodiments, the inhibitor removing agent is a trivalent aluminium salt, more preferably aluminium chloride. As is demonstrated in the examples, aluminium chloride is particularly suitable for the purpose of the present invention.

[0074] According to one embodiment, the precipitating agent is ammonium acetate, and the inhibitor removing agent is a trivalent aluminium salt, more preferably aluminium chloride.

[0075] According to one embodiment, which is preferred, the sample is contacted in step (a) with a solution that comprises the at least one precipitating agent and the at least one inhibitor removing agent. For simplicity, this solution comprising both agents is also referred to as inhibitor removal technology solution or inhibitor removal solution (IRT) herein. According to one embodiment, the IRT solution comprises, consists essentially of, or consists of (i) one or more precipitating agents selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, cesium acetate, and combinations thereof,

[0076] (ii) one or more inhibitor removing agents selected from aluminium chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof, and

[0077] (iii) optionally water.

[0078] In embodiments, the total concentration of the one or more precipitating agents in the IRT solution that is added in step (a) for lysis is in the range of 0.5M to 10M, e.g. 1.0M to 8M, or 1.5M to 7.5M, preferably 1.5M to 6M, 2M to 5M, 2.5 to 4.5M and 3M to 4M. The described concentrations are particularly suitable when using ammonium acetate.

[0079] In embodiments, the total concentration of the one or more inhibitor removing agents in the inhibitor removal solution is in the range of 10mM to 500mM, e.g. 25mM to 400mM, 50mM to 350mM, 75mM to 300mM, 90mM to 250mM, preferably 50mM or 100mM to 200mM, such as 50mM to 175mM, 75mM to 150mM or 100mM to 150mM. The use of a trivalent aluminium salt such as aluminium chloride is particularly preferred as inhibitor removing agent and it is in one embodiment comprised in such concentration in the solution. According to one embodiment, the IRT solution that is added in step (a) for lysis comprises aluminium chloride in a concentration of 50mM to 250mM. Particularly preferred concentrations of aluminium chloride include 50mM to 200mM, 50mM to 175mM and 75mM to 150mM.

[0080] Exemplary preferred solutions that comprise a precipitating agent and an inhibitor removal agent that can be added during step (a) include:

[0081] (1) a solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20mM to 200mM aluminium chloride;

[0082] (2) a solution containing 1 M to 10M (preferably 1M to 8M) sodium acetate and 20 mM to 200mM aluminium chloride;

[0083] (3) a solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20 mM to 200mM aluminium chloride;

[0084] (4) a solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20 mM to 200mM erbium (III) acetate;

[0085] (5) a solution containing 1 M to 10M (preferably 1M to 8M) sodium acetate and 20 mM to 200mM erbium (III) acetate;

[0086] (6) a solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20 mM to 200mM erbium (III) acetate;

[0087] (7) a solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20 mM to 200mM erbium (III) chloride;

[0088] (8) a solution containing 1 M to 10M (preferably 1M to 8M) sodium acetate and 20 mM to 200mM erbium (III) chloride; (9) a solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20 mM to 200mM erbium (III) chloride;

[0089] (10) a solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20 mM to 200mM holmium chloride;

[0090] (11) a solution containing 1 M to 10M (preferably 1M to 8M) sodium acetate and 20 mM to 200mM holmium chloride; and

[0091] (12) a solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20 mM to 200mM holmium chloride.

[0092] According to one embodiment, the precipitating agent that is contacted with the sample in step (a) is selected from ammonium acetate, sodium acetate, cesium acetate, or a combination thereof, preferably ammonium acetate, and the inhibitor removing agent is aluminium chloride. Suitable concentrations are described above.

[0093] As disclosed herein, it is preferred to add the precipitating agent and the inhibitor removal agent at the same time in lysis step (a), e.g. by adding a solution that comprises the at least one precipitating agent and the at least one inhibitor removing agent.

[0094] As used herein, the term “inhibitor” in particular refers to any substance that interferes with a reaction involving DNA and / or RNA isolated from a sample and has a detrimental effect on DNA and / or RNA manipulation. An inhibitor may inhibit PCR amplification of isolated nucleic acids and is also referred to herein as “a PCR inhibitor.” “PCR amplification” as used herein includes various types of PCR reactions, such as qPCR and RT-PCR. Inhibitors include, for example, inhibitors of an enzymatic reaction that uses DNA or RNA as a substrate and a contaminant that disrupts hybridization of DNA or RNA. Depending on the types of samples, inhibitors may vary. For example, inhibitors in stool samples often include polysaccharides and bile salts. Inhibitors comprised in stool may also comprise lipids, urates and heme compounds. Common inhibitors are haemoglobin and the metabolites thereof, bilirubin, bile acids and bile acid derivatives, undigested or partially digested fiber, or undigested or partially digested food, and polysaccharides. Inhibitors may also include humic acids and fulvic acids (e.g. comprised in wastewater). Inhibitors may furthermore comprise polycyclic aromatics, saccharides, peptides and phenols.

[0095] Use of solutions for preparing a lysis composition for sample lysis

[0096] In step (a), the sample may be contacted with a liquid lysis composition that comprises the at least one chaotropic agent, the at least one RNase inhibiting agent, the at least one protein precipitating agent, the at least one inhibitor removing agent, and preferably the at least one phosphate.

[0097] Step (a) may comprise preparing the liquid lysis composition by combining two or more solutions, wherein the first solution comprises the chaotropic agent and preferably a phosphate, and wherein the second solution comprises the protein precipitating agent and the inhibitor removing agent. Preparing the liquid lysis composition in step (a) may comprise combining a third solution with the first solution and the second solution, wherein the third solution comprises the at least one RNase inhibiting agent, preferably an organic extraction solvent.

[0098] The first solution, the second solution and the third solution can be combined in any order with each other to prepare the liquid lysis composition that is used to lyse the sample. The sample may also be added at any stage. According to one embodiment, the sample is added to a container (e.g. a tube), followed by the addition of a first solution that comprises the chaotropic agent and optionally the phosphate, followed by the addition of the second solution that comprises the protein precipitating agent and the inhibitor removing agent, followed by the addition of the third solution which comprises the at least one RNase inhibiting agent, which preferably is an organic extraction solvent such as phenol-chloroform-isoamyl alcohol.

[0099] According to one embodiment, the third solution comprises at least one organic extraction solvent as RNase inhibiting agent. Preferably, the volumetric ratio of the third solution comprising the organic extraction solvent to the second solution comprising the protein precipitating agent and the inhibitor removing agent is 1 :1 , wherein the organic solvent is preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform. As is demonstrated by the examples, this embodiment provides good results and is convenient for the workflow.

[0100] The liquid lysis composition that is contacted with the sample, and optionally solid particles to assist disruption of the sample, may comprise these agents in the following concentrations (calculation of the concentrations exclude the sample and disrupting particles, if used for disruption of the sample during lysis):

[0101] - The liquid lysis composition may comprise the at least one chaotropic agent in in a concentration of 2.5M or less, e.g. 2M or less, 1.75M or less, 1.5M or less, 1.3M or less, 1.2M or less or 1.125M or less. In embodiments, the concentration is 1.0M or less. Suitable concentrations of the at least one chaotropic agent in the liquid lysis composition may be in a range from 0.5M to 2.5M, e.g. selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and preferably 0.5M to 1.25M or 0.6M to 1.25M. If multiple chaotropic agents are present in the lysis composition, the total concentration of chaotropic agents in the lysis composition may be and preferably lies in the abovedescribed range. The chaotropic agent is preferably a thiocyanate salt as described above, more preferably NaSCN. The above concentrations were found particularly suitable for such mild thiocyanate salts, such as NaSCN. Particularly preferred is a concentration of NaSCN in the liquid lysis composition in the range of 0.5M to 1.5M. - The liquid lysis composition may comprise the at least one phosphate, which is preferably included during lysis as disclosed herein, in a concentration of 0.05M to 0.75M, e.g. 0.075M to 0.5M, 0.1M to 0.3M and 0.1 M to 0.25M or may be 0.1 M to 0.2M. The concentration of the at least one phosphate in the liquid lysis composition is preferably in the range of 0.1 M to 0.3M or 0.1 M to 0.2M. It is particularly preferred to use sodium phosphate dibasic in this concentration.

[0102] - The liquid lysis composition may comprise an organic extraction solvent as RNase inhibiting agent. This organic extraction solvent may be comprised in a concentration (v / v) of e.g. 30% or less, 25% or less, 20% or less or 15% or less. Particularly suitable concentrations in v / v for the organic extraction solvent in the liquid lysis composition that is contacted with the sample are in the range of 2% to 25%, 3% to 20% and 5% to 15%, such as e.g. 10%. As noted, phenol-chloroform-isoamyl alcohol or phenolchloroform are particularly preferred as the organic extraction solvent used as RNase inhibiting agent.

[0103] The liquid lysis composition may comprise the precipitating agent in a concentration in the range of 0.1 M to 5M, e.g. 0.1M to 2.5M or 0.15M to 2M, preferably 0.15M to 1.5M, 0.2M to 1 M or 0.2M to 0.8M. The described concentrations are particularly suitable when using ammonium acetate.

[0104] The liquid lysis composition may comprise the inhibitor removing agent in a concentration in the range of 5mM to 250mM, e.g. 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM. Particularly suitable is a concentration of 7.5mM to 25mM. The use of a trivalent aluminium salt such as aluminium chloride is particularly preferred as inhibitor removing agent and it is in one embodiment comprised in such concentration in the liquid lysis composition.

[0105] Suitable concentrations in the liquid lysis composition for the individual agents can also be determined by the skilled person based on routine experiments in view of the detailed disclosure presented herein.

[0106] Assisting lysis

[0107] The lysis procedure may be assisted by other sample lysis methods, such as physical disruption and enzymatic lysis.

[0108] Physical disruption of sample includes sonication, temperature change, mechanical disruption using a mechanical force, shear force, mechanical vibration, or a vortexer, or a combination of such methods. Mechanical lysis is the most efficient method for extracting microbiomes. Preferably, lysis step (a) comprises mechanical disruption. Mechanical disruption assists the sample lysis and may include the use of bead beating and / or homogenizing methods. Preferably, disrupting particles, such as beads, are used for mechanical disruption in the method of the invention. The beads useful for mechanical disruptions may be made of or comprise glass, ceramic, metal, mineral, or a combination of two or more of such materials. The size of the beads may range from 0.05 mm to 3 mm. Exemplary beads include 0.7 mm garnet beads, 0.15 mm garnet beads, 0.1 mm glass beads, 0.5 mm glass beads, 0.1 mm ceramic beads, 0.5 mm ceramic beads, 1.4 mm ceramic beads, 0.1 mm yttrium-stabilized zirconium beads, 0.5 mm yttrium-stabilized zirconium beads, or a combination of such beads (e.g., 0.1 mm glass beads and 0.5 mm glass beads in the same amount). In certain preferred embodiments, the beads are high density beads with density (g / cc) at least 6.0, such as yttrium-stabilized zirconium beads, cerium stabilized beads, and stainless steel beads. Bead beating may be performed using a vortex mixer with bead tube adapter or bead beater, such as TissueLyzer II (QIAGEN), AMBION™ Vortex Adapter (Thermo Fisher Scientific, Waltham, MA) and the Omini Bead Rupter Homogenizer, OMNI Int’l, Kennesaw, GA), and various homogenizers by OPS Diagnostics. The speed and duration of bead beating may vary depending on the type and size of the sample (see e.g., Gibbons etal., Bead Beating: A Primer, OPS Diagnostics, LLC). For example, bead beating may be performed at the maximum speed of a bead beater for 1 to 20 minutes, such as 5 to 10 minutes, 10 to 20 minutes, or 5 to 15 minutes.

[0109] Lysis may also be assisted by enzymatic lysis, which includes the use of proteases or the like. However, in embodiments, lysis step (a) is not assisted by enzymatic lysis but only mechanical lysis. This may save costs and enzymatic lysis may change community composition.

[0110] Preferably, the sample is homogenized in the liquid lysis composition that comprises the chaotropic agent, the RNase inhibiting agent, the protein precipitating agent, the inhibitor removal agent and optionally but preferably, the phosphate and which is combined with disrupting particles to support the lysis by mechanical disruption. Lysis of the sample and e.g. microbial cells comprised therein is facilitated by both mechanical collisions between the disrupting particles / beads and chemical disruption of cell membranes. Lysis is preferably combined with mechanical disruption (e.g., bead beating) when isolating nucleic acids from a complex sample, such as a stool sample. The nucleic acid of interest for downstream applications may be of a microbial origin.

[0111] During step (a) also further components may be added to assist the lysis process, such as detergents, EDTA or PVP. In embodiments, the lysis procedure used in step (a) does not include SDS. In embodiments, the lysis procedure used in step (a) does not include any detergent. As disclosed herein, a lysis mixture may be formed when contacting the sample with (i) at least one chaotropic agent and, preferably, a phosphate, (ii) at least one RNase inhibiting agent, (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removing agent. As disclosed herein, the sample may be contacted with a liquid lysis composition that comprises (i) at least one chaotropic agent and, preferably, a phosphate, (ii) at least one RNase inhibiting agent (preferably and organic extraction agent), (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removing agent. The liquid lysis composition may be prepared by combining different solutions, as disclosed herein. The solutions and the sample may be contacted in any order to prepare the lysis mixture. Forming of a mixture may be assisted, e.g. by vortexing. Additionally, the lysis mixture preferably comprises disrupting particles to assist during lysis step (a) the disruption of the sample and e.g. any bacteria or other micoorganisms comprised therein. Excluding the sample and disrupting particles (if added) for the calculation, this lysis mixture may comprise these agents in the following concentrations:

[0112] - The lysis mixture may comprise the at least one chaotropic agent in in a concentration of 2.5M or less, e.g. 2M or less, 1 .75M or less, 1 ,5M or less, 1 ,3M or less, 1 ,2M or less or 1.125M or less. In embodiments, the concentration is 1.0M or less. Suitable concentrations of the at least one chaotropic agent in the lysis mixture may be in a range from 0.5M to 2.5M, e.g. selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and preferably 0.5M to 1.25M. If multiple chaotropic agents are present in the lysis mixture, the total concentration of chaotropic agents in the lysis mixture may be and preferably lies in the above-described range. The chaotropic agent is preferably a thiocyanate salt as described above, more preferably NaSCN. The above concentrations were found particularly suitable for such mild thiocyanate salts, such as NaSCN. Particularly preferred is a concentration of NaSCN in the lysis mixture in the range of 0.5M to 1.5M.

[0113] - The lysis mixture may comprise the at least one phosphate, which is preferably included in the lysis mixture as disclosed herein, in a concentration of 0.05M to 0.75M, e.g. 0.075M to 0.5M, 0.1M to 0.3M and 0.1 M to 0.25M or may be 0.1M to 0.2M. The concentration of the at least one phosphate in the lysis mixture is preferably in the range of 0.1M to 0.3M or 0.1M to 0.2M. It is particularly preferred to use sodium phosphate dibasic in this concentration.

[0114] - The lysis mixture may comprise an organic extraction solvent as RNase inhibiting agent. This organic extraction solvent may be comprised in a concentration (v / v) of e.g. 30% or less, 25% or less, 20% or less or 15% or less. Particularly suitable concentrations in v / v (excluding for the calculation the sample and particles (if used)) for the organic extraction solvent in the lysis mixture are in the range of 2% to 25%, 3% to 20% and 5% to 15%. As noted, phenol-chloroform-isoamylalcohol, phenolchloroform or a combination of benzyl alcohol and chloroform, are particularly preferred as the organic extraction solvent used as RNase inhibiting agent.

[0115] The lysis mixture may comprise the precipitating agent in a concentration in the range of 0.1 M to 5M, e.g. 0.1M to 2.5M or 0.15M to 2M, preferably 0.15M to 1.5M, 0.2M to 1M or 0.2M to 0.8M. The described concentrations are particularly suitable when using ammonium acetate.

[0116] The lysis mixture may comprise the inhibitor removing agent in a concentration in the range of 5 mM to 250mM, e.g. 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM. Particularly suitable is a concentration of 7.5mM to 25mM. The use of a trivalent aluminium salt such as aluminium chloride is particularly preferred as inhibitor removing agent and it is in one embodiment comprised in such concentration in the lysis mixture.

[0117] Suitable concentrations in the lysis mixture for the individual agents can also be determined by the skilled person based on routine experiments in view of the detailed disclosure presented herein.

[0118] Step (b) - lysate clearing

[0119] Step (b) comprises clearing the lysate that was provided in step (a). Lysis of the sample in step (a), which is preferably assisted by mechanical disruption with solid disrupting particles, provides a mixture which comprises solid components from the sample, including precipitates and inhibitor complexes formed during step (a), and a liquid fraction which comprises the released nucleic acids. To remove the solids and precipitated and / or complexed inhibitors, proteins and contaminants from the nucleic acid containing liquid fraction, the lysate is cleared in step (b).

[0120] This clearing step may comprise separating the lysed mixture into a solid fraction and a liquid fraction. The separated solid components may be discarded, as they predominantly comprise sample remainders, debris, contaminants, precipitated proteins and inhibitors. Separation of the liquid fraction may be assisted by sedimentation, centrifugation, or filtration, preferably by centrifugation. Also combinations of such methods can be used. Preferably, lysate clearing is assisted by centrifugation.

[0121] The liquid fraction of the cleared lysate (e.g. supernatant) is depleted from interfering components and comprises the nucleic acids.

[0122] This cleared lysate is further processed in step (c). As disclosed herein, the liquid fraction of the cleared lysate may be collected as supernatant and is directly further processed in step (c) for nucleic acid isolation. A second inhibitor removal step after lysate clearance step (b), as it is commonly performed in prior art methods, is not necessary and is not performed with the method of the invention. This is associated with numerous advantages as was already described above. Step (c) - recovering nucleic acids from the cleared lysate

[0123] In step (c), nucleic acids are isolated from the liquid fraction of the cleared lysate, which is preferably recovered as supernatant. Isolation step (c) allows to obtain the nucleic acids of interest in purified form. As is demonstrated by the Examples, the method of the invention allows to isolate highly pure nucleic acids without requiring a second inhibitor removal step after lysate clearing. This is advantageous, as it saves handling steps and renders the method suitable for performing the nucleic acid isolation step in an automated manner after lysate clearing.

[0124] The improved lysis and inhibitor removal technology provided by the present invention provides a liquid phase that comprises large amounts of nucleic acids and which is advantageously depleted from inhibitors (due to the use of the precipitating agent, the inhibitor removing agent and the RNase inhibiting agent during step (a)). Therefore, nucleic acids can be isolated with high yield and purity from the provided liquid phase.

[0125] The liquid fraction of the cleared lysate comprises RNA and DNA, as both types of nucleic acids are released during the lysis process. The method thus allows to recover total nucleic acids. Alternatively, only DNA (i.e. DNA depleted from RNA) or only RNA (i.e. RNA depleted from DNA) may be isolated as is demonstrated in the examples. The flexibility of the method of the invention is an important advantage.

[0126] The isolated nucleic acid of interest may be further used in downstream applications, e.g. in amplification-based methods such as PCR, qPCR, dPCR and next-generation-sequencing.

[0127] Suitable nucleic acid isolation methods are known in the art. In embodiments where both DNA and RNA are isolated from a sample, total nucleic acids may be isolated. Alternatively, DNA isolation and RNA isolation may be performed in parallel. The liquid phase obtained in step (d) may be divided into at least two portions: one for RNA isolation, and one for DNA isolation. DNA and RNA may also be isolated sequentially (see e.g., U.S. Patent No. 8,889,393, WO 2004 / 108925).

[0128] Essentially any nucleic acid isolation method can be used in order to isolate the target nucleic acid from the liquid fraction of the cleared lysate. RNA isolation methods are well-known in the art and therefore, do not need to be described in detail herein.

[0129] Preferably, a solid support is used for RNA isolation. Exemplary solid support suitable for binding RNA and DNA includes silica matrices, glass particles, diatomaceous earth, magnetic beads, nitrocellulose, nylon, and anion-exchange materials. The solid support may be in the form of loose particles, filters, membranes, fibers or fabrics, or lattices, and contained in a vessel, including tubes, columns, and preferably a spin column. To facilitate or strengthen the binding to a solid support, a binding solution is used. The binding solution may be added to the liquid fraction of the cleared lysate.

[0130] An exemplary binding solution may comprise a chaotropic agent (e.g., GuSCN or GuHCI, preferably GuSCN) and at least one C1-C5 alcohol (e.g., ethanol or isopropanol, preferably isopropanol). It may further comprise a buffer substance, such as Tris HCI.

[0131] In embodiments, the binding mixture additionally comprises a non-ionic detergent. As is demonstrated by the Examples, including a non-ionic detergent into the binding mixture improves the recovery of nucleic acids, in particular RNA, in a concentration dependent manner. Suitable non-ionic detergents include, but are not limited to polysorbate 20, polysorbate 80, Triton X-100, Triton X-114, Brij 58, Brij 35, Tween 20, Tween 80, NP-40 (nonyl phenoxypolyethoxylethanol), CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate), and CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1- propanesulfonate).

[0132] After binding to a solid phase, the nucleic acids bound to the solid phase may be washed, and subsequently eluted from the solid phase. The wash solution may comprise a chaotropic agent (e.g., GuHCI), an alcohol (e.g., ethanol, isopropanol), or both. It may further comprise a buffer substance (e.g., Tris HCI), a chelating agent (e.g., EDTA (ethylenediaminetetraacetic acid)), and / or a salt (e.g., NaCI). In embodiments, at least two different wash solutions are used. Preferably, at least one wash solution comprises a chaotropic salt (e.g. guanidium hydrochloride) and a C1-C5 aliphatic alcohol (e.g. ethanol), and optionally a non-ionic detergent, and another wash solution comprises a C1-C5 aliphatic alcohol (e.g. ethanol), but no chaotropic salt.

[0133] For elution, an elution solution comprising a buffer (e.g., a Tris buffer) or water may be used. RNA may be eluted from a solid support using DEPC-treated or other RNase-free water.

[0134] Preferably, nucleic acid isolation occurs by binding the nucleic acids to magnetic particles, such as magnetic silica or glass particles. This can be performed using a manual protocol as well as an automated protocol that runs on a robotic instrument. It is a significant advantage that the liquid fraction of the cleared lysate that is obtained after step (b) can be directly processed for isolating nucleic acids therefrom in an automated manner in step (c). Suitable embodiments are disclosed in the examples and are also disclosed elsewhere herein.

[0135] As is demonstrated by the examples, as the lysis conditions used preserve the RNA, RNA and DNA may also be isolated in form of total nucleic acids.

[0136] Where the isolation of DNA is not of interest, the DNA may also be destroyed to provide the recovered RNA in a form where it is depleted from DNA. To destroy the comprised DNA, step (c) may comprise performing a DNase digestion step. The DNase digestion may be performed on the liquid fraction of the cleared lysate, or, preferably, the DNase digestion step may be performed after binding the nucleic acids to the solid phase. Such an embodiment is also illustrated in the examples.

[0137] As is demonstrated in the examples, nucleic acids recovered by the method of the invention (RNA and / or DNA) are of high purity and quality. As shown by the examples, inhibitors are very effectively removed, and the inhibitor removal is significantly improved compared to other methods.

[0138] The method provided herein is capable of substantially removing one or more inhibitors from a sample. Specifically, the method of the present invention is very efficient in removing PCR inhibitors. The removal of such inhibitors by a particular inhibitor removal process may be evaluated by comparing certain features (e.g., Ct values) of PCR reactions using the nucleic acids isolated with the inhibitor removal process with PCR reactions using nucleic acids isolated without the inhibitor removal process or compared. The degree of reduction in Ct values between the PCR reactions may indicate the effectiveness of the inhibitor removal process in depleting PCR inhibitor(s). The high effectiveness in removing inhibitors and in particular in removing PCR inhibitors is also evidenced by the low delta Ct values that are achieved with RNA that has been isolated using the method of the present invention. The efficient inhibitor removal technology of the invention thereby allows to use higher eluate volumes in inhibitory sensitive downstream applications, which can increase the sensitivity of the downstream methods.

[0139] Step (d) - processing, preferably analyzing, the recovered nucleic acids

[0140] The nucleic acids isolated in step (c) are pure and of high quality as is demonstrated by the examples. The recovered nucleic acids, e.g. total nucleic acids, RNA or DNA, may thus be used immediately in downstream applications. As shown in the examples, the method is particularly suitable for isolating microbial nucleic acids, e.g. derived from bacteria, fungi or virus.

[0141] In preferred embodiments, the method of the invention thus comprises a step (d) of processing, preferably analyzing, the recovered nucleic acids. RNA may be reverse transcribed in step (d).

[0142] In embodiments, analyzing in step (d) comprises performing a PCR, qPCR, RT-PCR and / or nucleic acid sequencing. Other downstream applications include, but are not limited to cDNA synthesis, Northern, dot, and slot blot analyses and microarray analysis.

[0143] Analyzing in step (d) may comprise detecting a nucleic acid of interest derived from a microorganism or virus. In preferred embodiments, the nucleic acid of interest is derived from bacteria or fungi. The microbial nucleic acid of interest may be derived from bacteria and fungi, such as gram-positive bacteria, gram-negative bacteria, fungus, mold and spores, or a combination of the foregoing. Of particular interest are nucleic acids derived from bacteria. The method of the invention allows to isolate total microbial nucleic acids as well as microbial genomic DNA or microbial RNA from the samples, such as in particular stool samples and gut samples.

[0144] As discussed elsewhere herein, where microbial nucleic acids are of interest, lysis in step (a) is preferably assisted by mechanical disruption, more preferably bead beating. This ensures that the bacteria comprised in the sample, such as a stool sample, gut sample, sludge sample or wastewater sample, are efficiently lysed to release their microbial nucleic acids.

[0145] Samples

[0146] The method of the invention may be used to recover nucleic acids from various sample types, and in particular from inhibitor-rich samples that are difficult to process with common methods. Advantageously, the sample can be a microbiome sample. It can be a biological or environmental sample.

[0147] The sample may be selected from the group consisting of fecal samples, gut samples, sludge samples, wastewater samples, swab samples, such as skin swabs, genital swabs, rectal swabs, oral swabs, plaque swabs, buccal swabs, cadaver swabs, urine samples and saliva samples. These are common sample types for doing microbiome analysis on and therefore suitable samples for the method of the invention. In embodiments, the sample is a human sample.

[0148] The sample may be selected from a fecal sample, a stool sample, a gut sample, a wastewater sample and a sludge sample. Preferably, the sample is a stool sample. As is demonstrated by the examples, the method of the invention is particularly efficient in isolating nucleic acids (total nucleic acids or RNA or DNA) with good yield from stool samples, while substantially depleting comprised inhibitors thereby providing the nucleic acids in pure form. In embodiments, the sample is not a soil sample.

[0149] It is a particular advantage that the method of the invention allows the processing of a variety of sample amounts. In embodiments, the amount of sample lysed in step (a) is in the range selected from 1mg to 500mg, 25mg to 300mg and 50mg to 250mg. The amount of sample lysed in step (a) may be in the range of 50mg to 150mg. The foregoing amounts are particularly suitable when processing a stool sample or other fecal sample. Particular embodiments

[0150] Suitable and preferred embodiments of the method of the present invention, the individual steps (a) to (c) and optionally step (d), as well as the used components were described in detail above. As will be appreciated by the skilled person, the disclosure with respect to the individual steps and components and reagents used in said method can be combined with each other. The subject-matter resulting from a respective combination of individual features also belongs to the present disclosure. Non-limiting, particularly preferred embodiments of the present invention are again disclosed in the following.

[0151] According to one embodiment, the method comprises

[0152] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with at least one chaotropic agent, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent and disrupting particles and providing a lysis mixture, and mechanically disrupting the sample in the provided lysis mixture,

[0153] (b) clearing the lysate;

[0154] (c) isolating nucleic acids from the liquid fraction of the cleared lysate.

[0155] Suitable concentrations of the individual agents in the lysis mixture were disclosed above and it is referred thereto for the sake of conciseness. Particularly preferred is the use of NaSCN as chaotropic salt, sodium phosphate dibasic as phosphate, ammonium acetate as protein precipitating agent and a tri-or tetravalent metal salt, preferably a trivalent aluminium salt such as aluminium chloride, as inhibitor removing agent. The method is particularly suitable for processing stool samples, also on larger scale. As disclosed herein, the method may comprise a step (d) of processing, preferably analyzing the purified nucleic acids of interest. Suitable embodiments of step (d) are also described elsewhere herein, and it is referred to the respective disclosure. E.g. step (d) may comprise analyzing the purified nucleic acids of interest (DNA and / or RNA) e.g. by detecting RNA or DNA derived from bacteria or fungi using amplification based methods.

[0156] According to one embodiment, the method comprises

[0157] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with a liquid lysis composition that comprises at least one chaotropic agent, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent and at least one inhibitor removing agent, wherein the liquid lysis composition is prepared by combining at least three solutions that can be added in any order, and providing a lysis mixture, wherein lysis is assisted by mechanical disruption in the presence of disrupting particles,

[0158] (b) clearing the lysate;

[0159] (c) isolating nucleic acids from the liquid fraction of the cleared lysate. As disclosed herein, the first solution preferably comprises the chaotropic agent and the phosphate, the second solution comprises the protein precipitating agent and the inhibitor removing agent and the third solution comprises the organic extraction solvent as RNase inhibiting agent. Suitable embodiments for the first, second and third solution are described in detail herein and it is referred to the respective disclosure which also applies here. As disclosed herein, the method may comprise a step (d) of processing, preferably analyzing, the purified nucleic acids of interest. Step (d) may e.g. comprise detecting DNA and / or RNA derived from bacteria or fungi using amplification-based methods. Suitable embodiments of step (d) are also described elsewhere herein, and it is referred to the respective disclosure.

[0160] In embodiments of the method of the invention, step (a) comprises combining with the sample

[0161] - a first solution that comprises the chaotropic agent and the phosphate,

[0162] - a second solution that comprises the protein precipitating agent and the inhibitor removal agent, and

[0163] - a third solution that comprises and preferably consists of the organic extraction solvent, which in preferred embodiments is selected from phenol-chloroform-isoamyl alcohol, phenol-chloroform and benzyl alcohol-chloroform.

[0164] As disclosed herein, it is advantageous if the volumetric ratio of the third solution to the second solution used in step (a) is 1 :1.

[0165] In embodiments, the protein precipitating agent and the inhibitor removing agent are added in form of a solution in step (a). This solution is preferably characterized by one or more of the following features:

[0166] (i) it comprises the at least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5M to 10M, optionally in a concentration selected from 1.0M to 8M, 1 ,5M to 6M, 2M to 5M, 2.5M to 4.5M and 3M to 4M;

[0167] (ii) it comprises the inhibitor removing agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, in a concentration of 10mM to 500mM, optionally in a concentration selected from 25mM to 300mM, 50mM to 250mM, 50mM to 200mM, 50mM to 175mM and 75mM to 150mM;

[0168] (iii) it comprises ammonium acetate in a concentration of 2.5M to 5M or 3M to 4M and a trivalent aluminum salt, preferably aluminum chloride, in a concentration of 50mM to 200mM or 75mM to 150mM.

[0169] According to one embodiment, the method of the invention comprises:

[0170] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with a liquid lysis composition that comprises at least one chaotropic salt, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent and at least one inhibitor removing agent, wherein the liquid lysis composition is prepared by combining at least three solutions that can be added in any order, wherein the first solution comprises sodium thiocyanate in a concentration of 0.75M to 1.5M and sodium phosphate dibasic in a concentration of 0.1 M to 0.3M, the second solution comprises ammonium acetate in a concentration of 2M to 5M and aluminium chloride in a concentration of 75mM to 150mM, and the third solution comprises the organic extraction solvent, wherein preferably, the organic extraction solvent is phenol-chloroform-isoamyl alcohol, and providing a lysis mixture, wherein lysis is assisted by mechanical disruption in the presence of disrupting particles,

[0171] (b) clearing the lysate;

[0172] (c) isolating nucleic acids from the liquid fraction of the cleared lysate which is obtained as supernatant.

[0173] Suitable embodiments for the used solutions and the mixture provided in step (a) are also disclosed elsewhere herein and the respective disclosure also applies here. As disclosed herein, the volumetric ratio of the third solution to the second solution used in step (a) is preferably 1 :1. The sample is preferably selected from stool or other fecal sample, gut samples, sludge or wastewater and more preferably is a stool sample. As evidenced by the examples, the method of the invention allows to process various amounts of stool and allows to provide high quality, pure nucleic acids (DNA and RNA) with high yield. It is a particular advantage that the method allows to process sample amounts over a broad range. As disclosed herein, the method may comprise a step (d) of processing, preferably analyzing the purified nucleic acids of interest, e.g. by detecting DNA or RNA derived from bacteria, fungi or virus using amplification-based methods. Suitable embodiments of step (d) are also described elsewhere herein and it is referred to the respective disclosure.

[0174] USE OF A KIT

[0175] According to a second aspect, the present invention relates to the use of a kit for recovering RNA from a sample for performing the method according to the first aspect, the kit comprising

[0176] (a) a first solution comprising a chaotropic agent and preferably a phosphate;

[0177] (b) a second solution comprising at least one protein precipitating agent and at least one inhibitor removing agent;

[0178] (c) a solid phase for nucleic acid binding;

[0179] (d) a binding solution for binding nucleic acids to the solid phase.

[0180] As discussed above and demonstrated in the examples, high and specific yields of pure DNA and / or RNA can be isolated using the same kit from various samples. Microbiome samples, such as fecal or gut samples, are of particular interest. The kit allows isolating microbial nucleic acids, such as bacterial, archael, fungal, and viral nucleic acids from inhibitor rich sample types. Details of the kit components were already described in conjunction with the method of the invention and it is referred to the above disclosure which also applied to the components of the kit that is used for performing the method.

[0181] Details of the first solution and the included chaotropic agent were described above and it is referred to the respective disclosure. The same applies to the comprised phosphate. Multiple embodiments for the lysis solution comprising the chaotropic agent were also disclosed above in conjunction with the method and the disclosed lysis solutions may be comprised as first solution in the provided kit.

[0182] Details of the second solution and comprised at least one protein precipitating agent and at least one inhibitor removing agent were described above and it is referred to the respective disclosure. Multiple embodiments for the inhibitor removal solution comprising the comprising the at least one protein precipitating agent and at least one inhibitor removing agent, also referred to herein as IRT solution, were also disclosed above in conjunction with the method and the disclosed inhibitor removal solutions may be comprised as second solution in the provided kit.

[0183] In embodiments, the kit also comprises a third solution comprising an RNase inhibiting agent. Suitable embodiments were described above in conjunction with the method, and it is referred to the respective disclosure. As discussed in detail in this context, the third solution may comprise an organic solvent as described, such as phenol-chloroform-isoamylalcohol, phenolchloroform or benzyl alcohol-chloroform.

[0184] Suitable solid phases for binding nucleic acids were disclosed in conjunction with the claimed method. In embodiments, the solid phase is provided by a column. The column may comprise a silica solid phase, e.g. in form of particles or a membrane.

[0185] Furthermore, as the method of the invention is suitable for automation, the kit may comprise magnetic particles that are capable of binding nucleic acids, such as magnetic silica or glass particles.

[0186] The kit may also comprise a binding solution for binding nucleic acids of interest to the solid phase. The binding solution may comprise a chaotropic salt for supporting binding to the solid phase.

[0187] The kit may furthermore comprise at least one wash solution. Preferably, the kit comprises at least two wash solutions. Preferably, at least one wash solution comprises a chaotropic salt (e.g. guanidium hydrochloride) and a C1-C5 aliphatic alcohol (e.g. ethanol), and optionally a non-ionic detergent, and another wash solution comprises a C1-C5 aliphatic alcohol (e.g. ethanol), but no chaotropic salt. The kit may also comprise disrupting particles to assist the lysis of the sample. Suitable embodiments were described above in conjunction with the method, and it is referred to the respective disclosure.

[0188] FURTHER ITEMS OF THE INVENTION

[0189] Also disclosed in the context of the present invention are the following items as embodiments of the present invention:

[0190] 1. A method for isolating nucleic acids from a sample, preferably a stool or gut sample, the method comprising

[0191] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with

[0192] (i) at least one chaotropic agent and preferably a phosphate,

[0193] (ii) at least one RNase inhibiting agent,

[0194] (iii) at least one protein precipitating agent, and

[0195] (iv) at least one inhibitor removing agent,

[0196] (b) clearing the lysate;

[0197] (c) isolating nucleic acids from the liquid fraction of the cleared lysate.

[0198] 2. The method of item 1 , wherein the chaotropic agent used in step (a) is characterized by one or more of the following features:

[0199] (i) it is a chaotropic salt;

[0200] (ii) it is a chaotropic salt comprising the SCN' anion or the CIC>4' anion paired with a cation that is weaker than Mg2+in solubilizing proteins;

[0201] (iii) it is a chaotropic salt comprising the COa2' anion paired with a cation stronger than NH4+in solubilizing proteins;

[0202] (iv) it is a chaotropic salt selected from NaSCN, NaCOa, KSCN, NH4SCN, LiSCN, UCIO4, guanidine sulfate, and combinations thereof, wherein preferably the chaotropic agent is selected from NaSCN and NaCOa;

[0203] (v) the chaotropic salt is sodium thiocyanate;

[0204] (vi) the chaotropic agent is comprised in a lysis solution that is added in step (a).

[0205] 3. The method according to item 1 or 2, wherein the RNase inhibiting agent is characterized by one or more of the following features:

[0206] (i) it is an organic extraction solvent;

[0207] (ii) it is an organic extraction solvent comprising phenol, benzyl alcohol, benzaldehyde, chloroform, isoamylalcohol, dichloromethane or a combination of two or more of the foregoing;

[0208] (iii) it is an organic extraction solvent selected from phenol, phenol-chlorofrom- isoamylalcohol, phenol-chloroform, benzyl alcohol-benzaldehyde, benzylalcohol- chlororofm and phenol-dichloromethane, wherein preferably, the organic extraction solvent is phenol-chlorofrom-isoamylalcohol; (iv) it is selected from a reducing agent, optionally DTT or beta-mercaptoethanol, a detergent, optionally an anionic detergent such as SDS, and diethyl pyrocarbonate;

[0209] (v) it is comprised in a solution that is added in step (a).

[0210] 4. The method according to one or more of items 1 to 3, wherein the protein precipitating agent used in step (a) is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride and cesium acetate, wherein preferably, the protein precipitating agent is ammonium acetate, and wherein preferably, the protein precipitating agent is comprised in a solution that is added in step (a).

[0211] 5. The method according to one or more of items 1 to 4, wherein the inhibitor removing agent used in step (a) is characterized by one or more of the following features:

[0212] (i) it is a metal salt;

[0213] (ii) it is a tri- or tetravalent salt that contains a cation having a valence of three or four, wherein preferably, the inhibitor removing agent is a tri- or tetravalent metal salt;

[0214] (iii) the inhibitor removing agent is selected from aluminium chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof;

[0215] (iv) the inhibitor removing agent is a trivalent aluminium salt, more preferably aluminium chloride;

[0216] (v) it is comprised in a solution that is added in step (a), wherein preferably, the inhibitor removing agent and the precipitating agent are comprised in the same solution.

[0217] 6. The method according to one or more of items 1 to 5, wherein the method comprises adding at least one phosphate in step (a), wherein preferably, the phosphate is added together with the chaotropic agent, optionally wherein the phosphate is comprised in a solution that comprises the chaotropic agent.

[0218] 7. The method of item 6, wherein the phosphate has one or more of the subsequent characteristics:

[0219] (i) it is a phosphate dibasic;

[0220] (ii) the cationic moiety in the phosphate is selected from ammonium, sodium, potassium, or lithium;

[0221] (iii) it is sodium phosphate dibasic;

[0222] (iv) it is comprised in a solution that is added in step (a).

[0223] 8. The method of item 7, wherein in step (a) the sample is contacted with sodium thiocyanate as chaotropic agent and sodium phosphate dibasic.

[0224] 9. The method according to one or more of items 1 to 8, wherein in step (a) the sample is contacted with ammonium acetate as protein precipitating agent and aluminium chloride as inhibitor removing agent, wherein preferably, ammonium acetate and aluminium chloride are comprised in a solution that is added in step (a).

[0225] 10. The method according to one or more of items 1 to 9, wherein in step (a), the sample is contacted with a liquid lysis composition that comprises the at least one chaotropic agent, the at least one RNase inhibiting agent, the at least one protein precipitating agent, the at least one inhibitor removing agent, and at least one phosphate, optionally wherein the liquid lysis composition has one or more, preferably two or more, or all of the following characteristics:

[0226] (i) it comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;

[0227] (ii) it comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;

[0228] (iii) the RNase inhibiting agent is an organic extraction solvent, preferably phenol- chloroform-isoamyl alcohol, phenol-chloroform or benzyl alcohol-chloroform, and the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the liquid lysis composition is selected from 2% to 25%, 3% to 20% and 5% to 15%;

[0229] (iv) it comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1 M to 5M, optionally in a concentration selected from 0.1M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1M and 0.2M to 0.8M;

[0230] (v) it comprises the inhibitor removing agent, preferably a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

[0231] 11. The method according to one or more of items 1 to 10, wherein step (a) comprises preparing a liquid lysis composition by combining two or more solutions, wherein the first solution comprises the chaotropic agent and preferably the phosphate, and wherein the second solution comprises the protein precipitating agent and the inhibitor removing agent.

[0232] 12. The method according to one or more of items 1 to 11 , wherein step (a) comprises adding a solution that has one or more of the following characteristics:

[0233] (i) it comprises the at least one chaotropic agent in a concentration of 0.5M to 2.5M, optionally in a concentration selected from 0.6M to 2M, 0.7M to 1.75M, 0.75M to 1.5M and 0.75M to 1.25M;

[0234] (ii) it comprises a thiocyanate salt, preferably NaSCN, in a concentration of 0.7M to 1.75M, 0.75M to 1.5M or 0.75M to 1.25M;

[0235] (iii) it comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M and 0.1 M to 0.2M;

[0236] (iv) it comprises sodium thiocyanate and sodium phosphate dibasic;

[0237] (v) it comprises sodium thiocyanate in a concentration selected from 0.7M to 1.75M, 0.75M to 1.5M and 0.75M to 1.25M and the at least one phosphate, preferably sodium phosphate dibasic, in a concentration selected from 0.075M to 0.3M, 0.1 to 0.25M and 0.1 M to 0.2M;

[0238] (vi) the solution provides the first solution according to item 11 , wherein preferably, the first solution comprises a chaotropic salt and a phosphate, more preferably sodium thiocyanate and sodium phosphate dibasic.

[0239] 13. The method according to one or more of items 1 to 12, wherein step (a) comprises adding a solution that comprises the protein precipitating agent and the inhibitor removing agent, wherein said solution has one or more of the following characteristics:

[0240] (i) it comprises the at least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5 M to 10M, optionally in a concentration selected from 1.0M to 8M, 1.5M to 6M, 2M to 5M, 2.5M to 4.5M and 3M to 4M;

[0241] (ii) it comprises the inhibitor removing agent, preferably a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 10mM to 500mM, optionally in a concentration selected from 25mM to 300mM, 50mM to 250mM, 50mM to 200mM, 50mM to 175mM and 75mM to 150mM;

[0242] (iii) it comprises ammonium acetate in a concentration of 2.5M to 5M or 3M to 4M and a trivalent aluminium salt, preferably aluminium chloride, in a concentration of 50mM to 200mM or 75mM to 150mM;

[0243] (iv) the solution provides the second solution according to item 11 , wherein preferably, the second solution comprises ammonium acetate and a trivalent aluminium salt, preferably aluminium chloride.

[0244] 14. The method according to any one of items 11 to 13, wherein preparing the liquid lysis composition in step (a) comprises adding a third solution in addition to the first solution and the second solution, wherein the third solution comprises the at least one RNase inhibiting agent, wherein preferably, the RNase inhibiting agent is an organic extraction solvent as defined in item 3.

[0245] 15. The method according to item 14, wherein the first, second and third solution may be contacted in any order with the sample to provide the liquid lysis composition in which the sample is lysed or the first, second and third solution may be combined in advance to prepare the liquid lysis composition comprising all three solutions that is then contacted with the sample for lysis.

[0246] 16. The method according to item 14 or 15, wherein the third solution comprises at least one organic extraction solvent as RNase inhibiting agent, and wherein preferably, the volumetric ratio of the third solution to the second solution is 1 :1 , optionally wherein the organic solvent is as defined in item 3, preferably phenol-chloroform- isoamyl alcohol, phenol-chloroform or benzyl alcohol-chloroform and the second solution is as defined in item 13.

[0247] 17. The method according to one of more of items 1 to 16, wherein step (a) comprises adding 500pl to 1000pl, preferably 600pl to 800pl, of a first solution that comprises the chaotropic agent and a phosphate, 50pl to 150pl, preferably 75pl to 125pl, of a second solution that comprises the protein precipitating agent and the inhibitor removal agent, and 50pl to 150pl, preferably 75pl to 125pl , of a third solution that comprises and preferably consists of an organic extraction solvent serving as RNase inhibiting agent, which in preferred embodiments is selected from phenol-chloroform-isoamyl alcohol, phenol-chloroform and benzyl alcoholchloroform. 18. The method according to one or more of items 1 to 17, wherein lysis step (a) comprises mechanical disruption, optionally wherein mechanical disruption in lysis step (a) is assisted by disrupting particles that are added to the sample.

[0248] 19. The method according to item 18, wherein disrupting particles are used for mechanical disruption and wherein said particles are characterized by one or more of the following features:

[0249] (i) the particles are crystalline particles;

[0250] (ii) the particles comprise or consist of zirconium, zircon (zirconium silicate), zirconia (zirconium dioxide), yttrium-stabilized zirconium, quartz, aluminium oxide, silicon carbide, ceramic, glasses (e.g. silicon dioxide glass or silica) or a combination of the foregoing;

[0251] (iii) the particles are substantially spherical;

[0252] (iv) the particles have a size that lies in the range selected from 0.05mm to 0.9mm, 0.07mm to 0.8mm, 0.08mm to 0.75mm and 0.09mm to 0.7mm;

[0253] (v) the particles are substantially spherical and comprise or consist of zirconium, zircon (zirconium silicate), zirconia (zirconium dioxide) or yttrium-stabilized zirconium having on average a size that lies in the range of 0.08mm to 0.7mm, preferably 0.09mm to 0.6mm, wherein preferably, zirconium beads are used;

[0254] (vi) the particles have a density of at least 2.0 g / cc, at least 2.5 g / cc, at least 3.0 g / cc, at least 3.5 g / cc, at least 4.0 g / cc, at least 4.5 g / cc, at least 5.0 g / cc or at least 5.5 g / cc;

[0255] (vii) the particles have a density that lies in a range selected from 2.0g / cc to 15g / cc, 2.5g / cc to 12g / cc, 3.0g / cc to 10g / cc, 3.5g / cc to 9g / cc, 4.0g / cc to 8g / cc, 4.5g / cc to 7.5g / cc and 5g / cc to 7g / cc;

[0256] (viii) the particles have at least two different sizes, wherein (i) the first particle size lies on average in a range selected from 0.05mm to 0.25mm, 0.07mm to 0.2mm, 0.08mm to 0.175mm and 0.9mm to 0.15mm and (ii) the second particle size lies on average in a range selected from 0.3mm to 0.9mm, 0.35mm to 0.8mm, 0.4mm to 0.7mm and 0.45mm to 0.6mm.

[0257] 20. The method according to any one of items 1 to 19, wherein step (a) comprises forming a lysis mixture by contacting the sample with at least one chaotropic agent, a phosphate, at least one RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent, and optionally disrupting particles, wherein the lysis mixture comprises these agents in the following concentrations, wherein for determining the concentration, the sample and disrupting particles, if added, are excluded

[0258] (i) the lysis mixture comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;

[0259] (ii) the lysis mixture comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1 M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;

[0260] (iii) the lysis mixture comprises an organic extraction solvent as RNase inhibiting agent, preferably phenol-chloroform-isoamylalcohol, phenol-chloroform or benzyl alcoholchloroform, and wherein the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the lysis mixture is selected from 2% to 25%, 3% to 20% and 5% to 15%;

[0261] (iv) the lysis mixture comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1M to 5M, optionally in a concentration selected from 0.1 M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1 M and 0.2M to 0.8M;

[0262] (v) the lysis mixture comprises the inhibitor removing agent, which preferably is a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5 mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

[0263] 21. The method according to one or more of items 1 to 20, wherein lysate clearing in step (b) comprises removing solids.

[0264] 22. The method according to one or more of items 1 to 21 , wherein lysate clearing is assisted by centrifugation, sedimentation and / or filtration.

[0265] 23. The method according to one or more of items 1 to 22, wherein lysate clearing provides a nucleic acid containing supernatant as liquid fraction which is subjected to nucleic acid isolation step (c).

[0266] 24. The method according to one or more of items 1 to 23, wherein no inhibitor removal step is performed between lysate clearing step (b) and nucleic acid isolation step (c).

[0267] 24. The method according to one or more of items 1 to 23, wherein after step (b), the cleared lysate is not contacted with at least one protein precipitating agent and at least one inhibitor removing agent prior to nucleic acid isolation step (c).

[0268] 25. The method according to one or more of items 1 to 24, wherein isolating nucleic acids in step (c) is performed in an automated manner.

[0269] 26. The method according to one or more of items 1 to 25, wherein isolating nucleic acids in step (c) involves binding nucleic acids to a solid support, preferably a silica containing solid support.

[0270] 27. The method according to one or more of items 1 to 26, wherein isolating nucleic acids in step (c) involves binding nucleic acids to magnetic particles, preferably to magnetic silica or glass particles.

[0271] 28. The method according to one or more of items 1 to 27, wherein step (c) comprises isolating nucleic acids from a nucleic acid containing supernatant obtained as liquid fraction in lysate clearing step (b).

[0272] 29. The method according to one or more of items 1 to 28, wherein isolating nucleic acids in step (c) comprises binding nucleic acids to a solid phase, washing bound nucleic acids, and eluting bound nucleic acids from the solid phase.

[0273] 30. The method according to one or more of items 1 to 29, wherein isolating nucleic acids in step (c) comprises

[0274] (i) contacting the liquid fraction of the cleared lysate with a binding solution and a nucleic acid binding solid support under conditions that allow binding of the nucleic acids to the solid support; (ii) washing the bound nucleic acids; and

[0275] (iii) eluting the nucleic acids from the solid phase.

[0276] 31. The method according to one or more of items 1 to 30, wherein isolating nucleic acids comprises binding nucleic acids to a silica solid phase, optionally in the presence of a chaotropic salt, preferably a guanidium salt, and / or a C1-C5 alcohol, preferably isopropanol.

[0277] 32. The method according to one or more of items 1 to 31 , wherein the nucleic acids of interest are RNA and wherein step (c) comprises performing a DNase digestion step to remove undesired DNA, optionally wherein the DNase digestion step is performed while the RNA is bound to a solid phase.

[0278] 33. The method according to one or more of items 1 to 32, wherein the nucleic acids of interest are DNA and wherein the method comprises performing a RNase digestion step to remove undesired RNA, optionally wherein the RNase digestion step is performed on the liquid fraction of the cleared lysate or while the DNA is bound to a solid phase.

[0279] 34. The method according to one or more of items 1 to 33, wherein the method comprises sequential isolation of DNA and RNA from the cleared lysate.

[0280] 35. The method according to one or more of items 1 to 34, wherein the method comprises isolating total nucleic acids from the cleared lysate.

[0281] 36. The method according to one or more of items 1 to 35, wherein in nucleic acid isolation step c) the binding mixture comprises a non-ionic detergent.

[0282] 37. The method according to one or more of items 1 to 36, wherein isolating nucleic acids in step (c) comprises binding nucleic acids to magnetic silica or glass particles, wherein binding occurs in the presence of (i) a chaotropic salt, preferably a guanidium salt, (ii) a C1-C5 aliphatic alcohol, preferably isopropanol; and (iii) a non-ionic detergent.

[0283] 38. The method according to item 37, wherein in the binding mixture comprising the sample

[0284] (i) the concentration of the chaotropic salt is in the range of 1 M-3M, wherein preferably, the chaotropic salt is selected from guanidium thiocyanate and guanidinium chloride,

[0285] (ii) the concentration of the C1-C5 aliphatic alcohol is in the range of 10-25% (v / v), wherein preferably the alcohol isopropanol; and

[0286] (iii) the concentration of the non-ionic detergent is in the range of 1 %-10% (w / v), preferably 2%-10%.

[0287] 39. The method according to item 37 or 38, wherein in the binding mixture comprising the sample

[0288] (i) the concentration of the chaotropic salt is in the range of 1 M-2M, and wherein the chaotropic salt is guanidium thiocyanate,

[0289] (ii) the concentration of the C1-C5 aliphatic alcohol is in the range of 12-20% (v / v) or 15-20% (v / v), wherein the alcohol isopropanol; and

[0290] (iii) the concentration of the non-ionic detergent is in the range of 2%-10% (w / v) or preferably 4%-10%.

[0291] 40. The method according to one or more of items 1 to 39, comprising

[0292] (d) processing, preferably analyzing, the isolated nucleic acids.

[0293] 41. The method according to item 40, wherein analyzing in step (d) comprises performing a PCR, qPCR, RT-PCR and / or nucleic acid sequencing.

[0294] 42. The method according to item 40 or 41 , wherein analyzing in step (d) comprises detecting RNA and / or DNA derived from bacteria, fungi and / or viruses.

[0295] 43. The method according to one or more of items 1 to 42, wherein the sample is characterized as follows:

[0296] (a) the sample is selected from the group consisting of fecal samples, gut samples, sludge samples, wastewater samples, swab samples, such as skin swabs, genital swabs, rectal swabs, oral swabs, plaque swabs, buccal swabs, cadaver swabs, urine samples and saliva samples;

[0297] (b) the sample is selected from the group consisting of fecal samples, preferably stool, gut samples, sludge samples and wastewater samples; or

[0298] (c) the sample is a stool or gut sample.

[0299] 44. The method according to one or more of items 1 to 43, said method comprising

[0300] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with at least one chaotropic agent, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent and disrupting particles and providing a lysis mixture, wherein preferably, the lysis mixture comprises the agents in the concentrations as defined in item 20, and mechanically disrupting the sample in the provided lysis mixture,

[0301] (b) clearing the lysate;

[0302] (c) isolating nucleic acids from the liquid fraction of the cleared lysate, preferably from a nucleic acid containing supernatant obtained in lysate clearance step (b).

[0303] 45. The method according to item 44, wherein the protein precipitating agent used in step (a) is ammonium acetate and wherein the inhibitor removing agent used in step (a) is a trivalent aluminium salt, preferably aluminium chloride.

[0304] 46. The method according to one or more of items 1 to 45, said method comprising

[0305] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with a liquid lysis composition that comprises at least one chaotropic agent, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent and at least one inhibitor removing agent, wherein the liquid lysis composition is prepared by combining at least three solutions that can be added in any order, wherein the first solution is as defined in item 12, the second solution is as defined in item 13 and the third solution comprises the organic extraction solvent, wherein preferably, the organic extraction solvent is as defined in item 3, and providing a lysis mixture, wherein the lysis mixture comprises the agents in the concentrations as defined in item 20, and wherein lysis is assisted by mechanical disruption in the presence of disrupting particles,

[0306] (b) clearing the lysate;

[0307] (c) isolating nucleic acids from the liquid fraction of the cleared lysate, preferably from a nucleic acid containing supernatant obtained in lysate clearance step (b).

[0308] 47. The method according to item 46, wherein the volumetric ratio of the third solution to the second solution used in step (a) is 1 :1. 48. The method according to one or more of items 44 to 47, wherein the chaotropic agent used is step (a) is as defined in item 2, the organic extraction solvent used as RNase inhibiting agent is as defined in item 3 (ii), (iii) or (iv), the phosphate is as defined in item 7, the precipitating agent used in step (a) is as defined in item 4 and the inhibitor removing agent used in step (a) is as defined in item 5, wherein preferably, the chaotropic agent is NaSCN, the phosphate is sodium phosphate dibasic, the protein precipitating agent used in step (a) is ammonium acetate and the inhibitor removing agent used in step (a) is a trivalent aluminium salt, preferably aluminium chloride.

[0309] 49. The method according to one or more of items 44 to 49, wherein the sample is selected from the group consisting of a fecal sample, a gut sample, a sludge sample and a wastewater sample, and wherein preferably, the sample is a stool sample.

[0310] 50. The method according to one or more of items 1 to 49, said method comprising

[0311] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with disrupting particles and a liquid lysis composition, wherein the liquid lysis composition comprises

[0312] (i) at least one chaotropic agent in a concentration of 0.5M to 2M, wherein the chaotropic agent is selected from the group consisting of NaSCN, NaCOs, KSCN, NH4SCN, LiSCN, LiCICU, and guanidine sulfate, wherein preferably the chaotropic agent is NaSCN;

[0313] (ii) at least one organic extraction solvent as RNase inhibiting agent in a concentration (v / v) 3% to 20%;

[0314] (iii) at least one protein precipitating agent in a concentration of 0.1 M to 2.5M, wherein the protein precipitating agent is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride and cesium acetate, wherein preferably, the protein precipitating agent is ammonium acetate;

[0315] (iv) at least one inhibitor removing agent in a concentration of 5mM to 250mM, wherein the inhibitor removing agent is a tri- or tetravalent metal salt; mechanically disrupting the sample in the provided lysis mixture,

[0316] (b) clearing the lysate;

[0317] (c) isolating nucleic acids from the liquid fraction of the cleared lysate.

[0318] 51. The method according to item 50, wherein the liquid lysis composition prepared in step (a) additionally comprises a phosphate, preferably sodium phosphate dibasic, in a concentration of 0.1 M to 0.3M, and wherein the liquid lysis composition comprises

[0319] (i) the chaotropic agent in a concentration of 0.5M to 1.25M, wherein preferably, the chaotropic agent is NaSCN,

[0320] (ii) the organic extraction solvent in a concentration of 5% to 15%, wherein preferably the organic extraction solvent is selected from phenol-chloroform-isoamyl alcohol, phenolchloroform and benzyl alcohol-chloroform;

[0321] (iii) the precipitating agent in a concentration of 0.2M to 1M, wherein preferably, the precipitating agent is ammonium acetate;

[0322] (iv) the tri- or tetravalent metal salt as inhibitor removing agent in a concentration of 7.5mM to 75mM, wherein preferably, the inhibitor removing agent is selected from aluminium chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride and zirconium (IV) chloride, optionally wherein the inhibitor removing agent is a trivalent aluminium salt, more preferably aluminium chloride.

[0323] 52. The method according to item 50 and 51 , wherein isolating nucleic acids from the liquid fraction of the cleared lysate according to step (c) comprises

[0324] (i) contacting the liquid fraction of the cleared lysate with a binding solution and a nucleic acid binding solid support, preferably magnetic silica or glass particles, under conditions that allow binding of the nucleic acids to the solid support, wherein binding occurs in the presence of (i) a chaotropic salt, preferably a guanidium salt, (ii) a C1-C5 aliphatic alcohol, preferably isopropanol; and (iii) a non-ionic detergent;

[0325] (ii) washing the bound nucleic acids, wherein preferably, at least two wash steps using different wash solutions are performed;

[0326] (iii) eluting the nucleic acids from the solid phase.

[0327] 53. The method according to any one of items 50 to 52, wherein in the binding mixture comprising the sample

[0328] (i) the concentration of the chaotropic salt is in the range of 1 M-3M, wherein preferably, the chaotropic salt is selected from guanidium thiocyanate and guanidinium chloride,

[0329] (ii) the concentration of the C1-C5 aliphatic alcohol is in the range of 10-25% (v / v), wherein preferably the alcohol is isopropanol; and

[0330] (iii) the concentration of the non-ionic detergent is in the range of 1 %-10% (w / v).

[0331] 54. The method according to item 53, wherein in the binding mixture comprising the sample

[0332] (i) the concentration of the chaotropic salt is in the range of 1 M-2M, and wherein the chaotropic salt is guanidium thiocyanate,

[0333] (ii) the concentration of the C1-C5 aliphatic alcohol is in the range of 12-20% (v / v) or 15-20% (v / v), wherein the alcohol is isopropanol; and

[0334] (iii) the concentration of the non-ionic detergent is in the range of 2%-10% (w / v) or preferably 4%-10%.

[0335] 55. The method according to one or more of items 1 to 54, said method comprising

[0336] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with a liquid lysis composition that comprises at least one chaotropic salt, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent and at least one inhibitor removing agent, wherein the liquid lysis composition is prepared by combining at least three solutions that can be added in any order, wherein the first solution comprises sodium thiocyanate in a concentration of 0.75M to 1.5M and sodium phosphate dibasic in a concentration of 0.1 M to 0.3M, the second solution comprises ammonium acetate in a concentration of 2M to 5M and aluminium chloride in a concentration of 75mM to 150mM, and the third solution comprises the organic extraction solvent, wherein preferably, the organic extraction solvent is phenol- chloroform-isoamyl alcohol, and providing a lysis mixture, wherein lysis is assisted by mechanical disruption in the presence of disrupting particles,

[0337] (b) clearing the lysate; (c) isolating nucleic acids from the liquid fraction of the cleared lysate.

[0338] 56. The method according to one or more of items 50 to 55, wherein step (c) isolates RNA depleted from RNA, and wherein step (c) comprises:

[0339] - binding RNA and DNA to magnetic silica or glass particles;

[0340] - washing the bound nucleic acids, preferably using two different wash solutions;

[0341] - adding DNase and performing a DNase digest;

[0342] - adding additional binding buffer to rebind RNA to the magnetic particles;

[0343] - washing the bound RNA, preferably using at least two different wash solutions; and

[0344] - eluting the RNA from the magnetic particles.

[0345] 57. The method according to one or more of items 50 to 56, wherein the sample is a fecal sample or gut sample, preferably a stool sample.

[0346] 58. Use of a kit for isolating nucleic acids from a sample for performing the method according to any one of items 1 to 57, the kit comprising

[0347] (a) a first solution comprising a chaotropic agent and preferably a phosphate;

[0348] (b) a second solution comprising at least one protein precipitating agent and at least one inhibitor removing agent;

[0349] (c) a solid phase for nucleic acid binding, preferably magnetic particles;

[0350] (d) a binding solution for binding nucleic acids to the solid phase.

[0351] 59. Use according to item 58, wherein the kit comprises a third solution comprising an RNase inhibiting agent, wherein preferably the third solution comprises an organic solvent as RNase inhibiting agent, such as phenol-chloroform-isoamylalcohol or phenol-chloroform.

[0352] 60. Use according to item 58 or 59, wherein the kit comprises a wash solution and an elution solution.

[0353] 61. Use according to any one of items 58 to 60, wherein the kit has one or more of the following characteristics:

[0354] (i) the chaotropic agent comprised in the first solution is as defined in item 2;

[0355] (ii) the phosphate comprised in the first solution is as defined in item 7;

[0356] (iii) the first solution is as defined in item 12;

[0357] (iv) the protein precipitating agent comprised in the second solution is as defined in item 4;

[0358] (v) the inhibitor removing agent is as defined in item 5;

[0359] (vi) the second solution is as defined in item 13;

[0360] (vii) the RNase inhibiting agent comprised in the third solution is as defined in item 3.

[0361] 62. Use according to any one of items 58 to 61 , wherein the kit is for use for automated nucleic acid isolation.

[0362] 63. Use according to items 58 to 62, wherein the kit comprises magnetic silica or glass particles for nucleic acid binding.

[0363] 64. A liquid lysis composition suitable for lysing a sample, the composition comprising

[0364] (i) at least one chaotropic agent and preferably a phosphate,

[0365] (ii) at least one RNase inhibiting agent,

[0366] (iii) at least one protein precipitating agent, and

[0367] (iv) at least one inhibitor removing agent. 65. The liquid lysis composition according to item 64, wherein

[0368] (i) the chaotropic agent is as defined in item 2, optionally wherein the liquid lysis composition comprises a phosphate as defined in item 7;

[0369] (ii) the RNase inhibiting agent is as defined in item 3, preferably as defined in item 3 (iii);

[0370] (iii) the protein precipitating agent is as defined in item 4; and

[0371] (iv) the inhibitor removing agent is as defined in item 5.

[0372] 66. The liquid lysis composition according to item 64 or 65, wherein the liquid lysis composition has one or more, preferably two or more, or all of the following characteristics:

[0373] (i) it comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;

[0374] (ii) it comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;

[0375] (iii) the RNase inhibiting agent is an organic extraction solvent, preferably phenol-chloroform- isoamyl alcohol or phenol-chloroform, and the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the liquid lysis composition is selected from 2% to 25%, 3% to 20% and 5% to 15%;

[0376] (iv) it comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1 M to 5M, optionally in a concentration selected from 0.1 M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1M and 0.2M to 0.8M;

[0377] (v) it comprises the inhibitor removing agent, preferably a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

[0378] 67. The liquid lysis composition according to any one of items 64 to 66, wherein the liquid lysis composition is in contact with the sample and disrupting particles, optionally wherein the disrupting particles are as defined in item 19.

[0379] 68. The liquid lysis composition according to any one of items 64 to 67, prepared by combining three solutions, wherein the first solution comprises the chaotropic agent and preferably the phosphate, the second solution comprises the protein precipitating agent and the inhibitor removing agent and the third solution comprises the at least one RNase inhibiting agent, wherein preferably, the first solution is as defined in item 12, the second solution is as defined in item 13 and the third solution comprises an organic extraction solvent as defined in item 3, preferably an organic solvent selected from phenol, phenol-chloroform-isoamylalcohol, phenol-chloroform, benzyl alcohol-benzaldehyde and phenol-dichloromethane, wherein optionally, the organic extraction solvent is phenol-chloroform-isoamylalcohol.

[0380] 69. The liquid lysis composition according to item 68, wherein (i) the first solution comprises sodium thiocyanate and sodium phosphate dibasic, (ii) the second solution comprises ammonium acetate and a trivalent aluminium salt, preferably aluminium chloride, and (iii) the third solution comprises an organic solvent as defined in item 3, preferably phenol-chloroform- isoamylalcohol or phenol-chloroform.

[0381] 70. The liquid lysis composition according to item 68 or 69, wherein the first solution comprises sodium thiocyanate in a concentration of 0.8M to 1.25M and sodium phosphate dibasic in a concentration of 0.1 M to 0.25M, and the second solution comprises ammonium acetate in a concentration of 3M to 4M and AICI3 in a concentration of 100mM to 150mM.

[0382] 71. The liquid lysis composition according to any one of items 68 to 70, wherein the first, second and third solution may be contacted in any order with the sample to provide the liquid lysis composition in which the sample is lysed or the first, second and third solution may be combined in advance to prepare the liquid lysis composition comprising all three solutions that is then contacted with the sample for lysis.

[0383] 72. Use of a liquid lysis composition according to any one of items 64 to 71 for lysing a sample.

[0384] 73. A lysis mixture comprising a sample and

[0385] (i) at least one chaotropic agent and preferably a phosphate,

[0386] (ii) at least one RNase inhibiting agent,

[0387] (iii) at least one protein precipitating agent, and

[0388] (iv) at least one inhibitor removing agent.

[0389] 74. A lysed sample preparation comprising a lysed sample and

[0390] (i) at least one chaotropic agent and preferably a phosphate,

[0391] (ii) at least one RNase inhibiting agent,

[0392] (iii) at least one protein precipitating agent, and

[0393] (iv) at least one inhibitor removing agent.

[0394] 75. The lysis mixture or lysed sample preparation according to item 73 or 74, wherein

[0395] (i) the chaotropic agent is as defined in item 2, optionally wherein the lysis mixture or lysed sample preparation comprises a phosphate as defined in item 7;

[0396] (ii) the RNase inhibiting agent is as defined in item 3, preferably as defined in item 3(iii);

[0397] (iii) the protein precipitating agent is as defined in item 4; and

[0398] (iv) the inhibitor removing agent is as defined in item 5.

[0399] 76. The lysis mixture or lysed sample preparation according to any one of items 73 to 75, comprising disrupting particles, optionally wherein the disrupting particles are as defined in item 19.

[0400] 77. The lysis mixture or lysed sample preparation according to any one of items 73 to 76, comprising at least one chaotropic agent, a phosphate, at least one RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent, and preferably disrupting particles, wherein the lysis mixture or lysed sample preparation comprises these agents in the following concentrations, wherein for determining the concentration, the sample and furthermore disrupting particles, if added, are excluded:

[0401] (i) the lysis mixture or lysed sample preparation comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;

[0402] (ii) the lysis mixture or lysed sample preparation comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;

[0403] (iii) the lysis mixture or lysed sample preparation comprises an organic extraction solvent as RNase inhibiting agent, preferably phenol-chloroform-isoamylalcohol or phenol-chloroform, and wherein the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the lysis mixture is selected from 2% to 25%, 3% to 20% and 5% to 15%;

[0404] (iv) the lysis mixture or lysed sample preparation comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1 M to 5M, optionally in a concentration selected from 0.1 M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1 M and 0.2M to 0.8M;

[0405] (v) the lysis mixture or lysed sample preparation comprises the inhibitor removing agent, which preferably is a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5 mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

[0406] 78. The lysis mixture or lysed sample preparation according to any one of items 73 to 77, prepared by combining three solutions with the sample and optionally disrupting particles, wherein the first solution comprises the chaotropic agent and preferably the phosphate, the second solution comprises the protein precipitating agent and the inhibitor removing agent and the third solution comprises the at least one RNase inhibiting agent, wherein preferably, the first solution is as defined in item 12, the second solution is as defined in item 13 and the third solution comprises an organic extraction solvent as defined in item 3, preferably an organic solvent selected from phenol, phenol-chloroform-isoamylalcohol, phenol-chloroform, benzyl alcohol-benzaldehyde and phenol-dichloromethane, wherein optionally, the organic extraction solvent is phenol-chloroform-isoamylalcohol.

[0407] 79. The lysis mixture or lysed sample preparation according to item 78, wherein (i) the first solution comprises sodium thiocyanate and sodium phosphate dibasic, (ii) the second solution comprises ammonium acetate and a trivalent aluminium salt, preferably aluminium chloride, and (iii) the third solution comprises an organic solvent as defined in item 3, preferably phenol- chloroform-isoamylalcohol or phenol-chloroform.

[0408] 80. The lysis mixture or lysed sample preparation according to item 78 or 79, wherein the first solution comprises sodium thiocyanate in a concentration of 0.8M to 1.25M and sodium phosphate dibasic in a concentration of 0.1 M to 0.25M, and the second solution comprises ammonium acetate in a concentration of 3M to 4M and AICI3 in a concentration of 100mM to 150mM.

[0409] 81. The lysis mixture according to any one of items 78 to 80, wherein the first, second and third solution may be contacted in any order with the sample to provide the lysis mixture or the first, second and third solution may be combined in advance to prepare a liquid lysis composition comprising all three solutions that is then contacted with the sample for providing the lysis mixture. 82. The subject-matter of any one of claims 64 to 81 , wherein the sample is selected from soil, waste water and a fecal sample.

[0410] 83. The subject-matter of claim 82, wherein the sample is a soil sample.

[0411] 84. The subject-matter of claim 82, wherein the sample is a fecal sample, preferably selected from stool, gut samples and sludge.

[0412] EXAMPLE SECTION

[0413] I. Materials

[0414] PowerMax Bead Pro Tube (50ml)

[0415] Collection Tubes (50ml)

[0416] MB Maxi Spin Columns (QIAGEN) - comprise silica for nucleic acid binding

[0417] Mag G beads (magnetic silica particles, QIAGEN)

[0418] Mag B beads (magnetic silica particles, QIAGEN)

[0419] Lysis Solution (LS): The lysis solution comprises NaSCN and Na2HPO4. Preferred concentrations are described herein. E.g. NaSCN can be present in the lysis solution in a concentration in a range of 0.8M to 1.25M. Na2HPO4 can be present in a concentration in a range of 0.1 M to 0.25M or 0.15M to 0.2M. Na2HPO4 is preferably comprised in the lysis solution but could also be added separately. An according lysis solution was used in the examples below.

[0420] Inhibitor removal solution (IRT): The inhibitor removal solution (IRT) comprises ammonium acetate as precipitating agent and AICI3 as inhibitor removing agent. Preferred concentrations for both agents are described herein. E.g. ammonium acetate can be comprised in the IRT solution in a concentration in a range of 3M to 4M. Aluminium chloride can be comprised in the IRT solution in a concentration that lies in a range of 100mM to 150mM. An according solution was used in the examples below.

[0421] Phenol-chloroform-isoamyl alcohol (25:24:1 ; pH 6.5-8.0) - PCIA

[0422] TissueLyzer II instrument (TLI I , QIAGEN)

[0423] MVL (QIAGEN, binding buffer comprising a high concentration of a guanidinium salt and a non-ionic detergent)

[0424] MVB (QIAGEN, binding buffer comprising isopropanol, a guanidinium salt and a non-ionic detergent) DNase I, RNase free

[0425] Buffer RDD (QIAGEN; DNase I buffer)

[0426] RNase- free water

[0427] II. Overview reference extraction workflows

[0428] Magnetic bead based reference workflow (manual protocol)

[0429] As reference bead-extraction workflow, a manual bead-based protocol was performed which is based on the QIAsymphony® PowerFecal® Pro chemistry (QIAGEN).

[0430] The method was performed as follows unless indicated otherwise: Spin column-based reference workflows

[0431] The spin-column based methods (commercial kits) were performed using the standard protocol as follows:

[0432] Spin REF-DNA (QIAamp® PowerFecal® Pro DNA Kit, QIAGEN)

[0433] Spin REF-RNA (RNeasy® PowerFecal® Pro, QIAGEN)

[0434]

[0435] III. Analytics

[0436] Determination of yield using UV-Vis based and fluorometric technique

[0437] UV-Vis: Nanodrop (ThermoFisher), blanked against elution buffer

[0438] Fluoromteric: Qubit (ThermoFisher), dsDNA BR Assay Kit Q32853 and RNA BR Assay Kit Q10211

[0439] Detection of the presence of inhibitors - Inhibition Assay

[0440] Removal of inhibitors during extraction was investigated by spiking the same volume of eluate of DNA into the amplification reaction of an IC DNA. Afterwards the delta Cq value is determined and delta Cq >2, >4 considered as being indicative of the presence of inhibitors. Sample material as well as amount of starting material and eluate volume spiked into the assay must be considered when assessing the inhibition. As assay, the QuantiFast Pathogen PCR +IC Kit (100) was used.

[0441] IV. Examples

[0442] 1. Combined Lysis and inhibitor removal with PCIA during sample disruption in step (a) and subsequent inhibitor removal after lysate clearance

[0443] This example demonstrates that the additional use of the RNase inhibiting organic solvent (phenol-chloroform isoamyl alcohol (PCIA)) in the combined lysis and inhibitor removal step (a) results in an improved inhibitor depletion. Adaption of the ratio of the organic solvent and the solution containing the protein precipitation agent during lysis allows to optimize the nucleic acid yield as is shown by the following titration experiments. Total nucleic acid (DNA and RNA) or DNA alone was extracted from 100mg stool material in duplicate determination.

[0444] As reference method, the manual bead-based QIAsymphony® PowerFecal® Pro protocol (see above) was carried out with lysis and inhibitor removal done in separate steps prior to DNA binding using Mag G silica beads with QSB1 binding buffer (QIAGEN) and QSW1 (QIAGEN) and QSW2 (QIAGEN) washes.

[0445] For comparison, manual bead-based purification protocols were performed which utilize a combined lysis and inhibitor removal approach for disruption with conditions as indicated in Fig. 1A and Fig. 1B. After lysate clearing, which was assisted by centrifugation, 600pl lysate supernatant was either treated with RNaseA (to digest RNA) or not. After lysate clearance, the supernatant was subjected to a second inhibitor removal step (using inhibitor removal solution IRT). The so obtained nucleic acid containing liquid phase was then used for isolating either DNA or total nucleic acids (DNA and RNA) from the liquid phase. Nucleic acids were bound to Mag B silica beads (QIAGEN) using 600pl MVL / MVB(1 :1) buffer mix for inducing binding of the nucleic acids to the beads. The binding mixture comprised isopropanol, GTC and a nonionic detergent. The samples were briefly vortexted for 10 seconds. To ensure good nucleic acid binding, the samples were incubated and mixed in a thermoshaker for 10min at 1250rpm at room temperature. The nucleic acids bound to the beads were then washed with 2x Buffer QSW1 (QIAGEN) and 2x Buffer QSW2 (QIAGEN).

[0446] In all protocols the TissueLyzer II instrument was used for mechanical disruption (2x 5min 25HZ) of the sample. A magnetic stand was used for separating the magnetic particles.

[0447] Nucleic acids were finally eluted in 10OpI RNase-free water for all protocols.

[0448] Results

[0449] The results are shown in Fig. 1 .

[0450] The yield was determined by fluorometric measurement (Qubit), see Fig. 1A.

[0451] Eluates were also tested for the presence of remaining inhibitors by spiking 1 l, 2.5pl or 5pl eluate into the amplification reaction of an internal DNA control (QuantiFast Pathogen with IC DNA PCR assay), see Fig. 1 B. In the delta Cq analysis delta Cq values >2 >4 in reference to the IC DNA with H2O as spike are indicative of the presence of inhibitors.

[0452] A decrease in yield was observed when using 200pl inhibitor removal solution (IRT) + 100pl PCIA with 700pl lysis solution (LS) for a combined lysis and inhibitor removal step. However, as can be seen, the yields recovered when reducing the IRT volume to 100pl. In contrast, a reduction of the volume of the organic solvent (here: PCIA) leads to an even greater loss in yield that cannot be recovered by a reduction in IRT.

[0453] Adding 1 OOpI PCIA to a mix of 1 OOpI IRT and 700pl LS for combined lysis and inhibitor removal results in a significant improvement in inhibitor depletion while a loss in DNA and RNA yield is avoided. As the results show, the yields are even improved. Such or a similar ratio therefore represents a preferred embodiment of the combined lysis and inhibitor removal step (a) of the present invention.

[0454] 2. Omitting a second inhibitor removal step in the method of the invention for total nucleic acid and RNA isolation

[0455] It was observed that the cleared lysates (supernatants) that were obtained after sample disruption using a combined lysis with an RNase inhibiting organic extraction solvent (here: PCIA), the lysis solution (LS) and the inhibitor solution (IRT), were no longer turbid but completely clear (unlike those samples prepared without addition of PCIA). Based on these observations and the overall good results for inhibitor depletion when adding an RNase inhibiting organic extraction solvent (here: PCIA) for combined lysis and inhibitor removal, it was assessed whether the second inhibitor removal step after lysate clearing could be omitted to advantageously reduce handling steps and provide a protocol that is after lysate clearing suitable for automated nucleic acid isolation.

[0456] This was further investigated by comparing protocols with and without a second inhibitor removal step after clearing the lysate that was obtained using a combined lysis and inhibitor removal step in the presence of the RNase inhibiting organic solvent (here: PCIA). Fig. 2 shows the results.

[0457] In brief, the protocol according to the invention was performed as follows. Total nucleic acid (DNA and RNA) or RNA alone was extracted from 100mg or 200mg stool material in duplicate determination.

[0458] The protocol used for demonstrating the advantages of the invention, done manually, used the combined lysis, inhibitor removal and organic solvent (here: PCIA) based lysis approach (700pl LS + 10OpI IRT + 1 OOpI PCIA) according to the invention for sample disruption.

[0459] - After sample disruption and lysate clearing, the obtained cleared lysate supernatant (approx. 600pl) was either (1) directly subjected to the nucleic acid isolation step (method according to the invention; no second inhibitor removal step after lysate clearing), or (2) the supernatant obtained after lysate clearing was further processed by performing a second inhibitor removal step with 50pl IRT and means for removing potentially formed precipitates (comparison). For isolating the nucleic acids, 600pl supernatant was used for nucleic acid binding using Mag B silica beads (QIAGEN) with 600pl MVL / MVB(1 :1) binding buffer mix. The nucleic acids bound to the beads were washed with 2x buffer QSW1 (QIAGEN) and 2x buffer QSW2 (QIAGEN).

[0460] As spin reference for RNA, RNA was extracted using the RNeasy® PowerFecal® Pro protocol with 600pl supernatant from disruption in binding and DNasel digestion. As spin reference for DNA the QIAamp® PowerFecal® Pro protocol was carried out.

[0461] In all protocols the TissueLyzer II instrument was used for mechanical disruption (2x 5min 25HZ).

[0462] Nucleic acids were finally eluted in 10OpI RNase-free water for the RNeasy® PowerFecal® Pro and the protocol of the invention (and comparison with 2ndinhibitor removal step) and 1 OOpI Solution C6 for the QIAamp® PowerFecal® Pro protocol.

[0463] Samples from the approach of the invention but also the RNeasy® PowerFecal® Pro reference samples, for reasons of better comparability, were after extraction treated with a DNase I digestion in solution plus clean-up to be able to assess the RNA quality of the mixed samples in the Agilent Bioanalyzer, see Fig. 2C.

[0464] Yield was determined by fluorometric measurement (Qubit), see Fig. 2A.

[0465] Eluates were also tested for the presence of inhibitors by spiking 1 pl, 2,5pl or 5pl eluate into the amplification reaction of an internal DNA control (QuantiFast Pathogen with IC DNA PCR assay), see Fig. 2B. In the delta Cq analysis delta Cq values >2 >4 in reference to the IC DNA with H2O as spike are indicative of the presence of inhibitors.

[0466] Results

[0467] The results demonstrate that the ratio of PCI A and inhibitor removal solution (IRT) in the combined lysis and inhibitor removal step can be optimized to reduce a loss in yield while obtaining good results for inhibitor depletion.

[0468] Lysis with a 1 :1 ratio of the RNase inhibiting organic solvent (here: PCIA) and the inhibitor removal solution (IRT) - here 10OpI each - mixed with a sufficient amount of the lysis solution LS (here: 700pl), provides good results for both DNA and RNA extraction from 100mg stool material. Furthermore, the RNA quality is as good as for the spin RNA REF with 100mg stool material. Importantly, the results furthermore indicated that a second inhibitor removal step after lysate clearing is obsolete if an RNase inhibiting organic extraction solvent (here: PCIA) is included during the lysis and inhibitor removal step (a). Furthermore, by omitting the second inhibitor removal step, the DNA and RNA yield can even be improved as the invention avoids that nucleic acids get lost during such extra handling steps. Inhibitors are efficiently depleted. This is highly advantageous as it allows to reduce handling steps. The method is furthermore suitable for use with magnetic beads for nucleic acid isolation, thus allowing to automate the nucleic acid isolation step that follows lysate clearing step b).

[0469] 3. Additional experiments confirming that a second inhibitor removal step is obsolete when incorporating an RNAse inhibiting organic solvent such as PCIA during step (a)

[0470] To further confirm that a second inhibitor removal step can be omitted if an RNase inhibiting organic extraction solvent such as PCIA is included during combined lysis and inhibitor removal step (a), larger amounts of samples were processed. For this purpose, 5 stool samples (100 mg or 200mg) were processed with the protocol described above with the following variations:

[0471] - with and without addition of PCIA in the combined lysis and inhibitor removal step (a); and with and without performing a second inhibitor removal step after lysate clearing and prior to nucleic acid isolation.

[0472] The protocols were performed as follows:

[0473] DNA was extracted from 100mg and 200mg stool material (5 donors) in duplicate determination.

[0474] The manual bead-based QIAsymphony® PowerFecal® Pro protocol was carried out as bead reference with lysis and inhibitor removal step done in separate steps prior to DNA binding using Mag G silica beads (QIAGEN) plus QSB1 buffer (QIAGEN) and QSW1 (QIAGEN) and QSW2 (QIAGEN) washes.

[0475] The protocol according to the invention, done manually, used for sample disruption the combined lysis and inhibitor removal approach with PCIA (700pl lysis solution (LS) + 100pl inhibitor removal solution (IRT) + 100pl PCIA) and, for comparison, without PCIA (700pl lysis solution (LS) + 200pl inhibitor removal solution (IRT)).

[0476] Lysate clearance was performed by centrifugation for 5min at 18000xg. 600 pl supernatant was obtained as cleared lysate and treated with RNaseA for RNA digestion.

[0477] - Afterwards, the RNase treated supernatant was either (1) directly subjected to the nucleic acid isolation step (method of the invention), or (2) a second inhibitor removal step was performed with the inhibitor removal solution (IRT) and removal of potentially formed precipitates (for comparison). DNA was then bound to Mag B silica beads (QIAGEN) using binding buffer mix MVL / MVB(1 :1) or 360pl CD3 / 240ml isopropanol. The nucleic acids bound to the beads were washed with buffers QSW1 (QIAGEN) and QSW2 (QIAGEN).

[0478] As spin reference DNA was extracted using the QIAamp® PowerFecal® Pro protocol.

[0479] In all protocols the TissueLyzer II instrument was used for mechanical disruption (2x 5min 25HZ).

[0480] DNA was finally eluted in 10OpI Solution C6 for the spin reference and 10OpI RNase-free water for the bead-based protocols.

[0481] Yield was determined by UV-Vis based technology (Nanodrop) and fluorometric measurement (Qubit), see Fig. 3A.

[0482] Eluates were also tested for the presence of inhibitors by spiking 2,5pl or 5pl eluate into the amplification reaction of an internal DNA control (QuantiFast Pathogen with IC DNA PCR assay), see Fig. 3B. In the delta Cq analysis delta Cq values >2 >4 in reference to the IC DNA with H2O as spike are indicative of the presence of inhibitors.

[0483] Results

[0484] The results demonstrate that the presence of the RNase inhibiting organic extraction solvent (here: PCIA) in the combined lysis and inhibitor removal step (a) is sufficient to remove inhibitors from the sample without the need for a further inhibitor removal step after lysate clearance. Even higher amounts of stool material such as 200mg could be efficiently processed with the method of the present invention.

[0485] Overall, for stool, the results for inhibitor and impurity depletion were best when the RNase inhibiting organic extraction solvent (here: PCIA) was included in the sample disruption step (a) that combines lysis and inhibitor removal.

[0486] 4. RNA Extraction

[0487] Previous results showed that the workflow of the present invention which uses an RNase inhibiting organic extraction solvent, such as PCIA, in the combined lysis and inhibitor removal step (a) is very efficient in depleting inhibitors and furthermore allows to isolate high amounts of inhibitor-free DNA and RNA from the cleared lysate supernatant. The results demonstrate that a second inhibitor removal step after lysate clearance is not required for efficient inhibitor removal, which is advantageous as it reduces the required handling steps. In addition, the method of the invention allows to isolate the nucleic acids from the cleared lysate supernatant by using magnetic particles or spin columns. This advantageously allows to process the cleared lysate supernatant in an automated manner for nucleic acid isolation. Suitable automated systems for isolating nucleic acids using magnetic particles or spin columns are known in the art and can be used for the present invention. Furthermore, extracted RNA is of good quality that is comparable to the spin reference (RNeasy® PowerFecal® Pro). Copurification of DNA is advantageously high in the total nucleic acid extraction protocol.

[0488] For isolating RNA only, a DNase digestion step (e.g. using DNasel) can be included in the workflow. Here, different options exist. A DNAse digestion step can be e.g. performed on the cleared lysate supernatant. Alternatively, and as demonstrated in the present examples, the DNA is first co-purified together with RNA by binding to magnetic particles. The DNase digestion is then performed on this purified sample. This has the advantage that the DNase is more effective in such purified environment. The RNA is then further purified by re-binding to the magnetic particles, washing and elution.

[0489] RNA / total nucleic acids were extracted from 100mg stool material in duplicate determination. In brief, the following protocol was performed:

[0490] The protocol according to the invention, done manually, used for sample disruption in step (a) the combined lysis, inhibitor removal and PCIA approach (700pl lysis solution (LS) + 10OpI inhibitor removal solution (IRT) + 1 OOpI PCIA).

[0491] - After lysate clearance using centrifugation, 600pl supernatant was used for RNA / total nucleic acid binding. As solid phase, Mag B silica beads were used and 600pl MVL / MVB (1 :1) buffer mix to adapt the binding conditions. o For total nucleic acid purification, the beads were washed 2x with Buffer QSW1 and 2x with Buffer QSW2. o For isolating only RNA, the beads with the bound nucleic acids were washed first with 700pl Buffer AW1 (QIAGEN) and second with 700pl Buffer QSW2 (QIAGEN). The beads with the bound nucleic acids were incubated and mixed for 10 minutes in 200pl DNasel mix (20pl DNasel + 160pl RDD / 20pl H2O or 140pl RDD / 40pl H2O or 20pl BoosterBuffer / 160pl H2O; all QIAGEN) for digesting the DNA. To ensure tight binding / rebinding of the RNA to the magnetic beads, 200pl binding buffer (MVL / MVB (1 :1)) was added and the samples were incubated and mixed for 10minutes for rebinding. Subsequently, the beads with the bound RNA were washed 1x with Buffer QSW1 and 2x with Buffer QSW2.

[0492] As spin reference RNA was extracted using the RNeasy® PowerFecal® Pro protocol with 600pl supernatant from disruption in binding and DNasel digestion. DNA was extracted using the QIAamp® PowerFecal® Pro Kit. In all protocols the TissueLyzer II instrument was used for mechanical sample disruption (2x 5min 25HZ).

[0493] RNA was finally eluted in 10OpI RNase-free water for all protocols.

[0494] The workflow is illustrated in Fig. 4A.

[0495] RNA quality in the eluates was afterwards assessed by electrophoretic analysis on the Agilent Bioanalyzer, see Fig. 4D.

[0496] Yield was determined by fluorometric measurement (Qubit), see Fig. 4B.

[0497] Eluates were also tested for the presence of inhibitors by spiking 2.5pl or 5pl eluate into the amplification reaction of an internal DNA control (QuantiFast Pathogen with IC DNA PCR assay) or by spiking 500ng RNA into the amplification of an internal RNA control (QuantiNova SG Rt-PCR with IC RNA), see Fig. 4C. In the delta Cq analysis delta Cq values >2 >4 in reference to the IC DNA / IC RNA with H2O as spike are indicative of the presence of inhibitors.

[0498] Results

[0499] DNA depletion is still highly efficient with lower volumes of RDD in the DNasel mix that allow 48 preparations including DNase digestion with the purchase of 2x RNase DNAsel sets. RNA quality and yield is comparable to the spin REF and results for inhibitor removal show similar results to the spin RNA REF in the RT-PCR assay with 500ng RNA spiked in.

[0500] 5. Method of the invention with manual nucleic acid isolation

[0501] In the following, different embodiments of the method of the invention with subsequent manual extraction of the nucleic acids are described.

[0502] In a further embodiment, nucleic acids are isolated from the cleared lysate supernatant (prepared as described above) using the following protocol: Preparation of the magnetic particles binding mixtures. o E.g. the magnetic particles are mixed by vortexing and 350pl [60g / l] Mag B bead suspension (QIAGEN) are pipetted into a 2ml tube. o The magnetic particles are separated on a magnetic rack (e.g. AdnaMag-S) and the storage buffer supernatant is discarded. o The magnetic particles pellet is resuspended in 600pl cleared lysate supernatant and the samples are briefly vortexed. o Afterwards, the binding conditions allowing the nucleic acids to bind to the magnetic particles are established. This can be achieved e.g. by adding 450pl MVL (QIAGEN) to the samples and briefly vortexing. Afterwards 200pl isopropanol is added and the samples are again briefly vortexed.

[0503] To ensure good binding of the nucleic acids to the magnetic particles, the samples are incubated for 10min in a thermoshaker at 1250rpm and room temperature. The magnetic particles with the bound nucleic acids are on a magnetic stand (e.g. AdnaMag -S). The supernatant is removed with a pipette and discarded.

[0504] The nucleic acids bound to the beads are washed. This can be done according to the following protocol: o 700pl QSW1 (QIAGEN) are added to the beads and the samples are incubated for 5min in a thermoshaker at 1400rpm and room temperature. The magnetic particles with the bound nucleic acids are separated on a magnetic stand (e.g. AdnaMag -S). The supernatant is removed and discarded. o 700pl QSW1 (QIAGEN) are added to the beads and the samples are incubated for 5min in a thermoshaker at 1400rpm and room temperature. The magnetic particles with the bound nucleic acids are separated on a magnetic stand (e.g. AdnaMag -S). The supernatant is removed and discarded. o 700pl QSW2 (QIAGEN) are added to the beads and the samples are incubated for 5min in a thermoshaker at 1400rpm and room temperature. The magnetic particles with the bound nucleic acids are separated on a magnetic stand (e.g. AdnaMag -S). The supernatant is removed and discarded. o 700pl QSW2 (QIAGEN) are added to the beads and the samples are incubated for 5min in a thermoshaker at 1400rpm and room temperature. The magnetic particles with the bound nucleic acids are separated on a magnetic stand (e.g. AdnaMag -S). The supernatant is removed and discarded. o Any residual is removed, e.g. using a pipette. o The samples are air dried on the magnetic stand for 10minutes. The nucleic acids are eluted. This can be done according to the following protocol: o 10OpI RNase-free water are added to the beads and the samples are incubated for 5min in a thermoshaker at 1400rpm at room temperature. The magnetic particles are separated on a magnetic stand (e.g. AdnaMag-S). o The supernatant comprising the eluted nucleic acids is transferred into a new tube. o The eluates may be directly processed further or e.g. kept at -20°C.

[0505] 6. Method of the invention with automated nucleic acid isolation

[0506] In the following, different embodiments of the method of the invention are described with subsequent automated nucleic acid extraction.

[0507] Lysis and sample disruption

[0508] For combined lysis and inhibitor removal, 700pl solution LS, 10OpI solution IRT and 10OpI PCIA are added (preferably in this order) to the tube holding the stool material. To assist sample disruption, the tube preferably comprises disrupting particles, such as zirconia beads. Corresponding tubes are commercially available (e.g. PowerBead Pro Tubes, QIAGEN). The samples are briefly vortexed and then mechanically homogenized. Homogenization can be performed e.g. in the TissueLyzer II: 2x 5min 25Hz (in between adapters may be rotated so that the side that was closest to the machine in the first round is then furthest in the second round).

[0509] Lysate clearing

[0510] For lysate clearing, the samples are centrifuged for 5min at 18000xg. In the below example the supernatants were pooled in a 50ml falcon tube for better comparability. The pool was mixed and 600pl aliquots can be used in binding / further processing.

[0511] The cleared lysate supernatant is then further processed on an automated system for nucleic acid isolation. In the examples, the EZ2 instrument (QIAGEN) was used for automated nucleic acid isolation.

[0512] 600pl sample is pipetted into a 2ml EZ2 tube (2ml screw cap tube w / o skirted rim). Afterwards, the tubes are placed into TipRack pos. A. Tip Holder & Tips and elution tubes (1.5ml screw cap tube) are placed into TipRack pos. C and D. The cartridges are loaded with the required components.

[0513] Processing on the EZ2 instrument - isolation of total nucleic acids

[0514] The following protocol can be performed in an automated manner: - Addition of magnetic particles (e.g. 350pl preseparated Mag B silica beads (60g / l)) to the 600pl supernatant, mixing

[0515] - Addition of a binding buffer that comprises a chaotropic salt and preferably a non-ionic detergent (e.g. 470pl MVL, QIAGEN), mixing

[0516] - Addition of an C1-C5 alcohol (e.g. isopropanol) to establish the binding conditions for binding the nucleic acids to the magnetic particles, incubating and mixing;

[0517] Separation of the magnetic particles with the bound nucleic acids;

[0518] Performing two or more wash steps (suitable washing conditions are described above, e.g. 2x QSW1 wash and 2x QSW2 wash) and separating the magnetic particles with the bound nucleic acids;

[0519] - Air drying of the magnetic particles with the bound nucleic acids;

[0520] - Addition of elution solution (e.g. 10OpI RNase free water or elution buffer), incubation and mixing;

[0521] Separation of the magnetic particles and collection of the nucleic acid containing eluate.

[0522] As noted, if it is desired to isolate DNA only, an RNase digest can be performed. If it is desired to isolate RNA only, a DNase digest can be performed. Suitable embodiments are described elsewhere herein and below.

[0523] Processing on the EZ2 instrument - isolation of only RNA

[0524] The following protocol can be performed in an automated manner:

[0525] - Addition of magnetic particles (e.g. 350pl preseparated Mag B silica beads (60g / l)) to the supernatant, mixing

[0526] - Addition of a binding buffer that comprises a chaotropic salt and preferably a non-ionic detergent (e.g. 470pl MVL, QIAGEN), mixing

[0527] - Addition of an C1-C5 alcohol (e.g. isopropanol) to establish the binding conditions for binding the nucleic acids to the magnetic particles, incubating and mixing;

[0528] Separation of the magnetic particles with the bound nucleic acids;

[0529] Performing at least one, preferably two, pre DNase wash steps o E.g. first washing with 800pl QSW1 (QIAGEN) by incubating and mixing followed by magnetic separation and second washing with 800pl QSW2 by incubating and mixing; o separating the magnetic particles with the bound nucleic acids;

[0530] Digesting the DNA by performing a DNAse digestion; o E.g. by adding 200pl DNase mix (comprising 10pl DNase I and 40pl RDD and 150pl RNase free water) and incubating and mixing for 10 minutes;

[0531] Rebinding the RNA to the magnetic silica particles o This can be achieved by adding again a binding buffer comprising a chaotropic salt (e.g. MVL, QIAGEN) and a C1-C5 alcohol (e.g. isopropanol) to reestablish the binding conditions for RNA.

[0532] Performing at least one, preferably two, post DNase wash steps o E.g. first washing with SOO I QSW1 (QIAGEN) by incubating and mixing followed by magnetic separation and second washing with 800pl QSW2 by incubating and mixing; o separating the magnetic particles with the bound RNA;

[0533] - Air drying of the magnetic particles with the bound RNA;

[0534] - Addition of elution solution (e.g. 10OpI RNase free water or elution buffer), incubation and mixing;

[0535] Separation of the magnetic particles and collection of the RNA containing eluate.

[0536] 7. Adjustment of the nucleic acid binding conditions

[0537] When using magnetic silica or glass particles for nucleic acid binding, it is advantageous to include a chaotropic salt, preferably a guanidium salt, more preferably guanidinium isothiocyanate, and an alcohol, preferably a C1-C5 alcohol e.g. selected from methanol, ethanol, propanol, isopropanol, butanol and pentanol, more preferably selected from ethanol and isopropanol, most preferably isopropanol, into the binding mixture to ensure tight binding of the nucleic acids of interest to the solid phase.

[0538] Preferably, the binding mixture including the sample (but excluding the solid phase) comprises GTC in a range of 1M-3M, preferably 1.25M to 2M, and at least 10%, preferably 15-25%, alcohol e.g. isopropanol. It was found that such binding conditions ensure good binding of the nucleic acids to the magnetic particles and thus high yields and low co-purification of inhibitors.

[0539] It was furthermore found that the presence of detergent, preferably a non-ionic detergent, in the binding mixture can further improve the binding efficiency, especially for RNA. It was found that the concentration of the included non-ionic detergent influenced how much RNA is copurified (see Fig. 5). Consequently, binding chemistries without a non-ionic detergent have the potential to make the RNaseA treatment obsolete or at least reduce the amount of RNase required. Furthermore, including a non-ionic detergent into the binding mixture further reduced the co-purification of inhibitors for some stool samples.

[0540] 8. Comparison of established protocols with method of the invention

[0541] Different stool samples were aliquoted and extracted using the method of the invention, targeting either total nucleic acid, DNA only, or RNA only. The performance was benchmarked to well-established commercial nucleic acid extraction kits, as indicated in the figure (Fig. 6A). The extracted eluates were spiked into a qPCR assay for an internal control. Delta Ct was measured to detection of internal control when water was used as a template to measure inhibition (Fig. 6B). The methods of the invention performed very well and rendered high and specific yields of DNA and RNA. The methods of the invention were further benchmarked against established magnetic-beads based microbiome and fecal / stool nucleic acid extraction protocols provided by different suppliers as indicated in the figures (Fig. 7 A-C). The results show that the highest yield for both DNA and RNA was achieved using the methods according to the invention which furthermore efficiently removed inhibitors. Some of the established protocols achieved divergent results between UV-vis and fluorometric methods which is an indicator of contamination. The method of the invention, however, proved very consistent and concordant between measurement methods. Overall, the method of the invention performed better than established protocols.

[0542] Stool samples are often inhibitory, and it is difficult to extract pure nucleic acid from stool samples. This can be seen in the performance of many competing methods. The method according to the invention removed inhibitors efficiently and provided a high purity while several of the established protocols resulted in impurities. The discordant yield values from supplier O are explained by the impurities seen by UV-Vi. Supplier Z and to some degree P and T also have impure eluates. This highlights the importance of using more than one analysis method to measure yield and purity (Fig. 8 A-B).

[0543] The method of the invention furthermore provided highly consistent sequencing results as demonstrated by amplicon sequencing. 16S V4 rRNA was profiled by Illumina amplicon sequencing using the automated method of the invention (run on an EZ2 instrument, EZ2) or the spin-column based QIAamp® PowerFecal® Pro kit (manual) as reference. The results are shown in Fig. 9 A. The high similarity in community compositions indicate the reproducibility of the method of the invention. A principal coordinate analysis based on the Bray-Curtis dissimilarity matrix underlined the similarity of the microbial profiles derived from the two different nucleic acid extraction methods. The data points cluster by stool sample not by extraction method (Fig. 9 B).

[0544] Viral extraction was verified in a dPCR assay against PMMoV (Pepper mild mottle virus, a common indicator target in stool or wastewater). The results show a high sensitivity and comparable results between the established manual protocol RNeasy® PowerFecal Pro (RNy PF Pro) and the automated method of the invention that was run on an EZ2 instrument (Fig. 10).

Claims

CLAIMS1. A method for isolating nucleic acids from a sample, preferably a stool or gut sample, the method comprising(a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with(i) at least one chaotropic agent and preferably a phosphate,(ii) at least one RNase inhibiting agent,(iii) at least one protein precipitating agent, and(iv) at least one inhibitor removing agent,(b) clearing the lysate;(c) isolating nucleic acids from the liquid fraction of the cleared lysate.

2. The method of claim 1 , wherein the chaotropic agent used in step (a) is characterized by one or more of the following features:(i) it is a chaotropic salt;(ii) it is a chaotropic salt comprising the SCN' anion or the CIC>4' anion paired with a cation that is weaker than Mg2+in solubilizing proteins;(iii) it is a chaotropic salt comprising the COa2' anion paired with a cation stronger than NH4+in solubilizing proteins;(iv) it is a chaotropic salt selected from NaSCN, NaCOa, KSCN, NH4SCN, LiSCN, UCIO4, guanidine sulfate, and combinations thereof, wherein preferably the chaotropic agent is selected from NaSCN and NaCOa;(v) the chaotropic salt is sodium thiocyanate;(vi) the chaotropic agent is comprised in a lysis solution that is added in step (a).

3. The method according to claim 1 or 2, wherein the RNase inhibiting agent is characterized by one or more of the following features:(i) it is an organic extraction solvent;(ii) it is an organic extraction solvent comprising phenol, benzyl alcohol, benzaldehyde, chloroform, isoamylalcohol, dichloromethane or a combination of two or more of the foregoing;(iii) it is an organic extraction solvent selected from phenol, phenol-chlorofrom- isoamylalcohol, phenol-chloroform, benzyl alcohol-benzaldehyde, benzylalcohol- chlororofm and phenol-dichloromethane, wherein preferably, the organic extraction solvent is phenol-chlorofrom-isoamylalcohol;(iv) it is selected from a reducing agent, optionally DTT or beta-mercaptoethanol, a detergent, optionally an anionic detergent such as SDS, and diethyl pyrocarbonate;(v) it is comprised in a solution that is added in step (a).

4. The method according to one or more of claims 1 to 3,(aa) wherein the protein precipitating agent used in step (a) is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride and cesium acetate, wherein preferably, the protein precipitating agent is ammonium acetate; and(bb) wherein the inhibitor removing agent used in step (a) is characterized by one or more of the following features:(i) it is a metal salt;(ii) it is a tri- or tetravalent salt that contains a cation having a valence of three or four, wherein preferably, the inhibitor removing agent is a tri- or tetravalent metal salt;(iii) the inhibitor removing agent is selected from aluminium chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof;(iv) the inhibitor removing agent is a trivalent aluminium salt, more preferably aluminium chloride.

5. The method according to one or more of claims 1 to 4, wherein the method comprises adding at least one phosphate in step (a), wherein the phosphate has one or more of the subsequent characteristics:(i) it is a phosphate dibasic;(ii) the cationic moiety in the phosphate is selected from ammonium, sodium, potassium, or lithium;(iii) it is sodium phosphate dibasic;(iv) it is comprised in a solution that is added in step (a), wherein the phosphate is comprised in a solution that comprises the chaotropic agent.

6. The method according to one or more of claims 1 to 5, wherein step (a) comprises preparing a liquid lysis composition by combining two or more solutions, wherein(aa) the first solution comprises the chaotropic agent and preferably the phosphate, wherein said first solution has one or more of the following characteristics:(i) it comprises the at least one chaotropic agent in a concentration of 0.5M to 2.5M, optionally in a concentration selected from 0.6M to 2M, 0.7M to 1.75M, 0.75M to 1.5M and 0.75M to 1.25M;(ii) it comprises a thiocyanate salt, preferably NaSCN, in a concentration of 0.7M to 1.75M, 0.75M to 1.5M or 0.75M to 1.25M;(iii) it comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M and 0.1 M to 0.2M;(iv) it comprises sodium thiocyanate and sodium phosphate dibasic;(v) it comprises sodium thiocyanate in a concentration selected from 0.7M to 1.75M, 0.75M to 1.5M and 0.75M to 1.25M and the at least one phosphate, preferably sodium phosphate dibasic, in a concentration selected from 0.075M to 0.3M, 0.1 to 0.25M and 0.1 M to 0.2M; and(bb) the second solution comprises the protein precipitating agent and the inhibitor removing agent, wherein said second solution has one or more of the following characteristics:(i) it comprises the at least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5 M to 10M, optionally in a concentration selected from 1.0M to 8M, 1.5M to 6M, 2M to 5M, 2.5M to 4.5M and 3M to 4M;(ii) it comprises the inhibitor removing agent, preferably a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 10mM to 500mM, optionally in a concentration selected from 25mM to 300mM, 50mM to 250mM, 50mM to 200mM, 50mM to 175mM and 75mM to 150mM;(iii) it comprises ammonium acetate in a concentration of 2.5M to 5M or 3M to 4M and a trivalent aluminium salt, preferably aluminium chloride, in a concentration of 50mM to 200mM or 75mM to 150mM.

7. The method according to claim 6, wherein preparing the liquid lysis composition in step (a) comprises adding a third solution in addition to the first solution and the second solution, wherein the third solution comprises at least one organic extraction solvent as RNase inhibiting agent, and wherein preferably, the volumetric ratio of the third solution to the second solution is 1 :1 , optionally wherein the organic solvent is as defined in claim 3, preferably phenol-chloroform- isoamyl alcohol, phenol-chloroform or benzyl alcohol-chloroform and the second solution is as defined in claim 6.

8. The method according to one or more of claims 1 to 7, wherein lysis step (a) comprises mechanical disruption, optionally wherein mechanical disruption in lysis step (a) is assisted by disrupting particles that are added to the sample.

9. The method according to any one of claims 1 to 8, wherein step (a) comprises forming a lysis mixture by contacting the sample with at least one chaotropic agent, a phosphate, at least one RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent, and optionally disrupting particles, wherein the lysis mixture comprises these agents in the following concentrations, wherein for determining the concentration, the sample and disrupting particles, if added, are excluded(i) the lysis mixture comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;(ii) the lysis mixture comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1 M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;(iii) the lysis mixture comprises an organic extraction solvent as RNase inhibiting agent, preferably phenol-chloroform-isoamylalcohol, phenol-chloroform or benzyl alcoholchloroform, and wherein the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the lysis mixture is selected from 2% to 25%, 3% to 20% and 5% to 15%;(iv) the lysis mixture comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1M to 5M, optionally in a concentration selected from 0.1 M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1 M and 0.2M to 0.8M;(v) the lysis mixture comprises the inhibitor removing agent, which preferably is a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5 mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

10. The method according to one or more of claims 1 to 9, wherein lysate clearing in step (b) provides a nucleic acid containing supernatant as liquid fraction which is subjected to nucleic acid isolation step (c).

11. The method according to one or more of claims 1 to 10, wherein isolating nucleic acids in step (c) has one or more of the following characteristics:(aa) isolating nucleic acids in step (c) involves binding nucleic acids to a solid support, preferably a silica containing solid support;(bb) isolating nucleic acids in step (c) involves binding nucleic acids to magnetic particles, preferably to magnetic silica or glass particles;(cc) isolating nucleic acids in step (c) is performed in an automated manner;(dd) isolating nucleic acids in step (c) comprises binding nucleic acids to a solid phase, washing bound nucleic acids, and eluting bound nucleic acids from the solid phase;(ee) isolating nucleic acids in step (c) comprises binding nucleic acids to a silica solid phase, optionally in the presence of a chaotropic salt, preferably a guanidium salt, and / or a C1-C5 alcohol, preferably isopropanol;(ff) isolating nucleic acids in step (c) comprises binding nucleic acids to magnetic silica or glass particles, wherein binding occurs in the presence of (i) a chaotropic salt, preferably a guanidium salt, (ii) a C1-C5 aliphatic alcohol, preferably isopropanol; and (iii) a non-ionic detergent;(gg) isolating nucleic acids in step (c) comprises preparing a binding mixture that comprises the sample and agents for inducing binding to the solid phase, wherein in said binding mixture (i) the concentration of the chaotropic salt is in the range of 1M-3M, wherein preferably, the chaotropic salt is selected from guanidium thiocyanate and guanidinium chloride,(ii) the concentration of the C1-C5 aliphatic alcohol is in the range of 10-25% (v / v), wherein preferably the alcohol isopropanol; and(iii) the concentration of the non-ionic detergent is in the range of 1 %-10% (w / v), preferably 2%-10%;(hh) performing a DNase digest for isolating RNA depleted of DNA.

12. The method according to one or more of claims 1 to 11 , wherein the method is characterized by one or more of the following features:(aa) isolating nucleic acids in step (c) comprises(i) contacting the liquid fraction of the cleared lysate with a binding solution and a nucleic acid binding solid support under conditions that allow binding of the nucleic acids to the solid support;(ii) washing the bound nucleic acids; and(iii) eluting the nucleic acids from the solid phase;(bb) the method further comprises(d) processing, preferably analyzing, the isolated nucleic acids, optionally wherein analyzing in step (d) fulfils one or more of the following features:- step (d) comprises performing a PCR, qPCR, RT-PCR and / or nucleic acid sequencing;- step (d) comprises detecting RNA and / or DNA derived from bacteria, fungi and / or viruses;(cc) the sample is characterized as follows:(i) the sample is selected from the group consisting of fecal samples, gut samples, sludge samples, wastewater samples, swab samples, such as skin swabs, genital swabs, rectal swabs, oral swabs, plaque swabs, buccal swabs, cadaver swabs, urine samples and saliva samples;(ii) the sample is selected from the group consisting of fecal samples, preferably stool, gut samples, sludge samples and wastewater samples; or(ii) the sample is a stool or gut sample.

13. The method according to one or more of claims 1 to 12, said method comprising(a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with a liquid lysis composition that comprises at least one chaotropic agent, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent and at least one inhibitor removing agent, wherein the liquid lysis composition is prepared by combining at least three solutions that can be added in any order, wherein the first solution is as defined in claim 6, the second solution is as defined in claim 6 and the third solution comprises the organic extraction solvent, wherein preferably, the organic extraction solvent is as defined in claim 3, and providing a lysis mixture, wherein thelysis mixture comprises the agents in the concentrations as defined in claim 9, and wherein lysis is assisted by mechanical disruption in the presence of disrupting particles,(b) clearing the lysate;(c) isolating nucleic acids from the liquid fraction of the cleared lysate, preferably from a nucleic acid containing supernatant obtained in lysate clearance step (b).

14. The method according to one or more of claims 1 to 13, said method comprising(a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with disrupting particles and a liquid lysis composition, wherein the liquid lysis composition comprises(i) at least one chaotropic agent in a concentration of 0.5M to 2M, wherein the chaotropic agent is selected from the group consisting of NaSCN, NaCOs, KSCN, NH4SCN, LiSCN, UCIO4, and guanidine sulfate, wherein preferably the chaotropic agent is NaSCN;(ii) at least one organic extraction solvent as RNase inhibiting agent in a concentration (v / v) 3% to 20%;(iii) at least one protein precipitating agent in a concentration of 0.1 M to 2.5M, wherein the protein precipitating agent is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride and cesium acetate, wherein preferably, the protein precipitating agent is ammonium acetate;(iv) at least one inhibitor removing agent in a concentration of 5mM to 250mM, wherein the inhibitor removing agent is a tri- or tetravalent metal salt; and mechanically disrupting the sample in the provided lysis mixture,(b) clearing the lysate;(c) isolating nucleic acids from the liquid fraction of the cleared lysate.

15. The method according to claim 14, wherein the liquid lysis composition prepared in step (a) comprises(i) the chaotropic agent in a concentration of 0.5M to 1.25M, wherein preferably, the chaotropic agent is NaSCN,(ii) the organic extraction solvent in a concentration of 5% to 15%, wherein preferably the organic extraction solvent is selected from phenol-chloroform-isoamyl alcohol, phenolchloroform and benzyl alcohol-chloroform;(iii) the precipitating agent in a concentration of 0.2M to 1M, wherein preferably, the precipitating agent is ammonium acetate;(iv) the tri- or tetravalent metal salt as inhibitor removing agent in a concentration of 7.5mM to 75mM, wherein preferably, the inhibitor removing agent is selected from aluminium chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride and zirconium(IV) chloride, optionally wherein the inhibitor removing agent is a trivalent aluminium salt, more preferably aluminium chloride; and(v) a phosphate in a concentration of 0.1 M to 0.3M, wherein preferably, the phosphate is sodium phosphate dibasic.

16. The method according to any one of claims 13 to 15, wherein isolating nucleic acids from the liquid fraction of the cleared lysate according to step (c) comprises(i) contacting the liquid fraction of the cleared lysate with a binding solution and a nucleic acid binding solid support, preferably magnetic silica or glass particles, under conditions that allow binding of the nucleic acids to the solid support, wherein binding occurs in the presence of (i) a chaotropic salt, preferably a guanidium salt, (ii) a C1-C5 aliphatic alcohol, preferably isopropanol; and (iii) a non-ionic detergent, wherein in the prepared binding mixture comprising the sample the concentration of the chaotropic salt is in the range of 1 M-3M, wherein preferably, the chaotropic salt is selected from guanidium thiocyanate and guanidinium chloride,- the concentration of the C1-C5 aliphatic alcohol is in the range of 10-25% (v / v), wherein preferably the alcohol is isopropanol; and- the concentration of the non-ionic detergent is in the range of 1 %- 10% (w / v);(ii) washing the bound nucleic acids, wherein preferably, at least two wash steps using different wash solutions are performed;(iii) eluting the nucleic acids from the solid phase.

17. The method according to one or more of claims 1 to 16, said method comprising(a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with a liquid lysis composition that comprises at least one chaotropic salt, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent and at least one inhibitor removing agent, wherein the liquid lysis composition is prepared by combining at least three solutions that can be added in any order, wherein the first solution comprises sodium thiocyanate in a concentration of 0.75M to 1.5M and sodium phosphate dibasic in a concentration of 0.1 M to 0.3M, the second solution comprises ammonium acetate in a concentration of 2M to 5M and aluminium chloride in a concentration of 75mM to 150mM, and the third solution comprises the organic extraction solvent, wherein preferably, the organic extraction solvent is phenol- chloroform-isoamyl alcohol, and providing a lysis mixture, wherein lysis is assisted by mechanical disruption in the presence of disrupting particles,(b) clearing the lysate;(c) isolating nucleic acids from the liquid fraction of the cleared lysate.

18. The method according to one or more of claims 13 to 17, wherein step (c) comprisesisolating RNA depleted from RNA, and wherein step (c) comprises:- binding RNA and DNA to magnetic silica or glass particles;- washing the bound nucleic acids, preferably using two different wash solutions; adding DNase and performing a DNase digest; adding additional binding buffer to rebind RNA to the magnetic particles;- washing the bound RNA, preferably using two different wash solutions; and eluting the RNA from the magnetic particles.

19. The method according to one or more of claims 13 to 18, wherein the sample is a fecal sample or gut sample, preferably a stool sample.

20. Use of a kit for isolating nucleic acids from a sample for performing the method according to any one of claims 1 to 19, the kit comprising(e) a first solution comprising a chaotropic agent and preferably a phosphate;(f) a second solution comprising at least one protein precipitating agent and at least one inhibitor removing agent;(g) a solid phase for nucleic acid binding, preferably magnetic particles;(h) a binding solution for binding nucleic acids to the solid phase; optionally wherein the kit has one or more of the following characteristics:(i) it comprises a third solution comprising an RNase inhibiting agent, wherein preferably the third solution comprises an organic solvent as RNase inhibiting agent, such as phenol- chloroform-isoamylalcohol, phenol-chloroform or benzyl alcohol-chloroform;(ii) it comprises one or more wash solutions and an elution solution;(iii) the chaotropic agent comprised in the first solution is as defined in claim 2;(iv) the phosphate comprised in the first solution is as defined in claim 5; the first solution is as defined in claim 6;(v) the protein precipitating agent comprised in the second solution is as defined in claim 4;(vi) the inhibitor removing agent is as defined in claim 5;(vii) the second solution is as defined in claim 6;(viii) the RNase inhibiting agent comprised in the third solution is as defined in claim 3, optionally wherein the kit is for use for automated nucleic acid isolation.

21. A liquid lysis composition suitable for lysing a sample, the composition comprising(i) at least one chaotropic agent and preferably a phosphate,(ii) at least one RNase inhibiting agent,(iii) at least one protein precipitating agent, and(iv) at least one inhibitor removing agent.

22. The liquid lysis composition according to claim 21, wherein(i) the chaotropic agent is as defined in claim 2, optionally wherein the liquid lysis composition comprises a phosphate as defined in claim 5;(ii) the RNase inhibiting agent is as defined in claim 3, preferably as defined in claim 3 (iii);(iii) the protein precipitating agent is as defined in claim 4 (aa); and(iv) the inhibitor removing agent is as defined in claim 4 (bb).

23. The liquid lysis composition according to claim 21 or 22, wherein the liquid lysis composition has one or more, preferably two or more, or all of the following characteristics:(i) it comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;(ii) it comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;(iii) the RNase inhibiting agent is an organic extraction solvent, preferably phenol-chloroform- isoamyl alcohol or phenol-chloroform, and the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the liquid lysis composition is selected from 2% to 25%, 3% to 20% and 5% to 15%;(iv) it comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1 M to 5M, optionally in a concentration selected from 0.1 M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1M and 0.2M to 0.8M;(v) it comprises the inhibitor removing agent, preferably a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

24. The liquid lysis composition according to any one of claims 21 to 23, wherein the liquid lysis composition is in contact with the sample and disrupting particles.

25. The liquid lysis composition according to any one of claims 21 to 24, prepared by combining three solutions, wherein (i) the first solution comprises sodium thiocyanate in a concentration of 0.8M to 1.25M and sodium phosphate dibasic in a concentration of 0.1 M to 0.25M, and (ii) the second solution comprises ammonium acetate in a concentration of 3M to 4M and AlCh in a concentration of 100mM to 150mM, and (iii) the third solution comprises an organic solvent as defined in claim3, preferably phenol-chloroform-isoamylalcohol or phenol-chloroform, wherein the first, second and third solution may be contacted in any order with the sample to provide the liquid lysis composition in which the sample is lysed or the first, second and third solution may be combined in advance to prepare the liquid lysis composition comprising all three solutions that is then contacted with the sample for lysis.

26. Use of a liquid lysis composition according to any one of claims 21 to 24 for lysing a sample.

27. A lysis mixture comprising a sample and(i) at least one chaotropic agent and preferably a phosphate,(ii) at least one RNase inhibiting agent,(iii) at least one protein precipitating agent, and(iv) at least one inhibitor removing agent.

28. A lysed sample preparation comprising a lysed sample and(i) at least one chaotropic agent and preferably a phosphate,(ii) at least one RNase inhibiting agent,(iii) at least one protein precipitating agent, and(iv) at least one inhibitor removing agent.

29. The lysis mixture or lysed sample preparation according to claim 27 or 28, wherein(i) the chaotropic agent is as defined in claim 2, optionally wherein the lysis mixture or lysed sample preparation comprises a phosphate as defined in claim 5;(ii) the RNase inhibiting agent is as defined in claim 3, preferably as defined in claim 3(iii);(iii) the protein precipitating agent is as defined in claim 4 (aa); and(iv) the inhibitor removing agent is as defined in claim 4 (bb).

30. The lysis mixture or lysed sample preparation according to any one of claims 27 to 29, comprising disrupting particles.

31. The lysis mixture or lysed sample preparation according to any one of claims 27 to 30, comprising at least one chaotropic agent, a phosphate, at least one RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent, and preferably disrupting particles, wherein the lysis mixture or lysed sample preparation comprises these agents in the following concentrations, wherein for determining the concentration, the sample and furthermore disrupting particles, if added, are excluded:(i) the lysis mixture or lysed sample preparation comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;(ii) the lysis mixture or lysed sample preparation comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;(iii) the lysis mixture or lysed sample preparation comprises an organic extraction solvent as RNase inhibiting agent, preferably phenol-chloroform-isoamylalcohol or phenol-chloroform, and wherein the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the lysis mixture is selected from 2%to 25%, 3% to 20% and 5% to 15%;(iv) the lysis mixture or lysed sample preparation comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1 M to 5M, optionally in a concentration selected from 0.1 M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1 M and 0.2M to 0.8M;(v) the lysis mixture or lysed sample preparation comprises the inhibitor removing agent, which preferably is a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5 mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

32. The lysis mixture or lysed sample preparation according to any one of claims 27 to 31 , prepared by combining three solutions with the sample and optionally disrupting particles in any order, wherein (i) the first solution comprises sodium thiocyanate in a concentration of 0.8M to 1.25M and sodium phosphate dibasic in a concentration of 0.1 M to 0.25M, and (ii) the second solution comprises ammonium acetate in a concentration of 3M to 4M and AlCh in a concentration of 100mM to 150mM, and (iii) the third solution comprises an organic extraction solvent as defined in claim 3, preferably phenol-chloroform-isoamylalcohol or phenolchloroform.

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