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KR-20260065870-A - Method for isolating nucleic acids from inhibitor-rich biological samples

KR20260065870AKR 20260065870 AKR20260065870 AKR 20260065870AKR-20260065870-A

Abstract

The present invention provides a method for isolating nucleic acids from a sample, preferably a stool or intestinal sample, comprising the following: (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved sample is a sample (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one type of protein precipitating agent, and (iv) at least one inhibitor removal agent A step including bringing into contact with, (b) a step of purifying the dissolved material; (c) Step of isolating nucleic acid from the liquid fraction of the purified lysis. The method is particularly useful for isolating total nucleic acids, DNA, or RNA from inhibitor-rich samples, such as stool samples. In contrast to the prior art method, this method advantageously omits a second inhibitor removal step after the lysate purification step (b) and before performing the nucleic acid isolation step (b).

Inventors

  • 오닐, 도미닉
  • 슈뢰어, 슈테파니

Assignees

  • 키아겐 게엠베하

Dates

Publication Date
20260511
Application Date
20240902
Priority Date
20230901

Claims (20)

  1. A method for isolating nucleic acids from a sample, preferably a stool or intestinal sample, comprising the following: (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved sample is a sample (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one type of protein precipitating agent, and (iv) at least one inhibitor removal agent A step including bringing into contact with, (b) a step of purifying the dissolved material; (c) Step of isolating nucleic acid from the liquid fraction of the purified lysis.
  2. A method according to 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) a chaotropic salt comprising an SCN- anion or a ClO4- anion paired with a cation weaker than Mg2 + for solubilizing proteins; (iii) a chaotropic salt comprising a CO32- anion paired with a cation stronger than NH4 + for solubilizing proteins; (iv) a chaotropic salt selected from NaSCN, NaCO3 , KSCN, NH4SCN , LiSCN, LiClO4 , guanidine sulfate, and combinations thereof, wherein the chaotropic agent is preferably selected from NaSCN and NaCO3 ; (v) The chaotropic salt is sodium thiocyanate; (vi) The inclusion of a chaotropic agent in the dissolution solution added in step (a).
  3. A method according to claim 1 or 2, wherein the RNase inhibitor is characterized by one or more of the following features: (i) an organic extraction solvent; (ii) an organic extraction solvent comprising phenol, benzyl alcohol, benzaldehyde, chloroform, isoamyl alcohol, dichloromethane, or a combination of two or more of the above; (iii) an organic extraction solvent selected from phenol, phenol-chloroform-isoamyl alcohol, phenol-chloroform, benzyl alcohol-benzaldehyde, benzyl alcohol-chloroform, and phenol-dichloromethane, wherein, preferably, the organic extraction solvent is phenol-chloroform-isoamyl alcohol; (iv) a reducing agent, optionally DTT or beta-mercaptoethanol, a detergent, optionally an anionic detergent such as SDS, and diethyl pyrocarbonate; (v) What is included in the solution added in step (a).
  4. In any one of paragraphs 1 through 3, (aa) 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; (bb) A method in which the inhibitor removal agent used in step (a) is characterized by one or more of the following features: (i) metal salts; (ii) a 3- or 4-valent salt containing a cation having a valence of 3 or 4, wherein, preferably, the inhibitor scavenger is a 3- or 4-valent metal salt; (iii) an inhibitor removal agent selected from aluminum chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof; (iv) The inhibitor removal agent is a trivalent aluminum salt, more preferably aluminum chloride.
  5. A method according to any one of claims 1 to 4, wherein the method comprises adding at least one type of phosphate in step (a), wherein the phosphate has one or more of the subsequent features: (i) a dibasic phosphate; (ii) The cationic moiety in the phosphate is selected from ammonium, sodium, potassium, or lithium; (iii) a dibasic sodium phosphate; (iv) included in the solution added in step (a), wherein the phosphate is included in the solution containing the chaotropic agent.
  6. In any one of claims 1 to 5, step (a) comprises preparing a liquid dissolution composition by combining two or more solutions, wherein (aa) The first solution comprises a chaotropic agent and preferably a phosphate, wherein the first solution has one or more of the following characteristics: (i) comprising at least one chaotropic agent at a concentration of 0.5M to 2.5M, optionally selected from 0.6M to 2M, 0.7M to 1.75M, 0.75M to 1.5M, and 0.75M to 1.25M; (ii) comprising a thiocyanate salt at a concentration of 0.7M to 1.75M, 0.75M to 1.5M, or 0.75M to 1.25M, preferably NaSCN; (iii) comprising at least one type of phosphate, preferably a dibasic sodium phosphate, at a concentration of 0.05 M to 0.75 M, optionally selected from 0.075 M to 0.5 M, 0.1 M to 0.3 M, and 0.1 M to 0.2 M; (iv) comprising sodium thiocyanate and dibasic sodium phosphate; (v) comprising sodium thiocyanate at a concentration selected from 0.7M to 1.75M, 0.75M to 1.5M, and 0.75M to 1.25M, and at least one phosphate at a concentration selected from 0.075M to 0.3M, 0.1M to 0.25M, and 0.1M to 0.2M, preferably a dibasic sodium phosphate; (bb) A method wherein the second solution comprises a protein precipitating agent and an inhibitor removing agent, wherein the second solution has one or more of the following features: (i) comprising at least one precipitating agent, preferably ammonium acetate, having a concentration of 0.5 M to 10 M, optionally selected from 1.0 M to 8 M, 1.5 M to 6 M, 2 M to 5 M, 2.5 M to 4.5 M, and 3 M to 4 M; (ii) an inhibitor scavenger at a concentration of 10 mM to 500 mM, optionally selected from 25 mM to 300 mM, 50 mM to 250 mM, 50 mM to 200 mM, 50 mM to 175 mM, and 75 mM to 150 mM, preferably comprising a trivalent aluminum salt, more preferably an aluminum chloride; (iii) comprising ammonium acetate at a concentration of 2.5 M to 5 M or 3 M to 4 M and a trivalent aluminum salt, preferably aluminum chloride, at a concentration of 50 mM to 200 mM or 75 mM to 150 mM.
  7. In claim 6, preparing the liquid dissolution 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 an RNase inhibitor, and wherein, preferably, the volume ratio of the third solution to the second solution is 1:1, Optionally, the organic solvent here is as defined in paragraph 3, preferably phenol-chloroform-isoamyl alcohol, phenol-chloroform or benzyl alcohol-chloroform, and the second solution is as defined in paragraph 6.
  8. A method according to any one of claims 1 to 7, wherein the dissolution step (a) comprises mechanical crushing, optionally wherein the mechanical crushing in the dissolution step (a) is assisted by crushed particles added to the sample.
  9. A method according to any one of claims 1 to 8, wherein step (a) comprises forming a dissolution mixture by contacting a sample with at least one chaotropic agent, a phosphate, at least one RNase inhibitor, at least one protein precipitating agent, at least one inhibitor removal agent, and optionally a crushed particle, wherein the dissolution mixture comprises these agents at the following concentrations, wherein, to determine the concentration, the sample and, if added, the crushed particle are excluded: (i) The dissolved mixture comprises at least one chaotropic agent, preferably NaSCN, at a concentration of 2.5 M or less, optionally selected from 0.5 M to 2 M, 0.5 M to 1.75 M, 0.5 M to 1.5 M, and 0.5 M to 1.25 M; (ii) The dissolved mixture comprises at least one type of phosphate, preferably a dibasic sodium phosphate, at a concentration of 0.05 M to 0.75 M, optionally selected from 0.075 M to 0.5 M, 0.1 M to 0.3 M, 0.1 M to 0.25 M, and 0.1 M to 0.2 M; (iii) the dissolution mixture comprises an organic extraction solvent as an RNase inhibitor, preferably phenol-chloroform-isoamyl alcohol, phenol-chloroform or benzyl alcohol-chloroform, wherein the liquid dissolution composition comprises an organic extraction solvent at 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 dissolution mixture is selected from 2% to 25%, 3% to 20%, and 5% to 15%; (iv) The dissolution mixture comprises a precipitating agent, preferably ammonium acetate, at a concentration selected from 0.1 M to 5 M, optionally 0.1 M to 2.5 M, 0.15 M to 2 M, 0.15 M to 1.5 M, 0.2 M to 1 M, and 0.2 M to 0.8 M; (v) The dissolved mixture comprises an inhibitor removal agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, at a concentration selected from 5 mM to 250 mM, optionally 5 mM to 200 mM, 5 mM to 150 mM, 7.5 mM to 100 mM, 7.5 mM to 75 mM, 7.5 mM to 50 mM, or 7.5 mM to 30 mM.
  10. A method according to any one of claims 1 to 9, wherein the purification of the lysate in step (b) provides a nucleic acid-containing supernatant as a liquid fraction applied to the nucleic acid isolation step (c).
  11. A method according to any one of claims 1 to 10, wherein isolating the nucleic acid in step (c) has one or more of the following features: (aa) In step (c), isolating the nucleic acid involves binding the nucleic acid to a solid support, preferably a silica-containing solid support; (bb) The isolation of the nucleic acid in step (c) involves binding the nucleic acid to magnetic particles, preferably magnetic silica or glass particles; (cc) In step (c), the isolation of nucleic acids is performed in an automated manner; (dd) The isolation of nucleic acid in step (c) comprises binding the nucleic acid to a solid phase, washing the bound nucleic acid, and eluting the bound nucleic acid from the solid phase; (ee) isolating the nucleic acid in step (c) optionally comprises binding the nucleic acid onto a silica solid in the presence of a chaotropic salt, preferably a guanidium salt, and/or a C1-C5 alcohol, preferably isopropanol; (ff) The isolation of the nucleic acid in step (c) comprises binding the nucleic acid to magnetic silica or glass particles, wherein the 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) In step (c), isolating the nucleic acid comprises preparing a binding mixture containing an agent to induce binding to the sample and the solid phase, wherein the binding mixture (i) The concentration of the chaotropic salt is in the range of 1M to 3M, wherein, preferably, the chaotropic salt is selected from guanidium thiocyanate and guanidinium chloride, and (ii) The concentration of the C1-C5 aliphatic alcohol is in the range of 10-25% (v/v), wherein the alcohol is preferably isopropanol; (iii) The concentration of the non-ionic detergent is in the range of 1%-10% (w/v), preferably 2%-10%; (hh) Performing DNase digestion to isolate RNA depleted of DNA.
  12. A method according to any one 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) is (i) bringing the liquid fraction of the purified lysate into contact with the binding solution and the nucleic acid-binding solid support under conditions allowing the binding of the nucleic acid to the solid support; (ii) washing the bound nucleic acids; and (iii) Eluting nucleic acids from a solid phase Including; (bb) method (d) a step of processing, preferably analyzing, the isolated nucleic acid Additionally, including, optionally, that the analysis in step (d) satisfies one or more of the following features: - Step (d) includes performing 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 by the following: (i) The sample is selected from the group consisting of fecal samples, intestinal samples, sludge samples, wastewater samples, swab samples, e.g., 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, intestinal samples, sludge samples, and wastewater samples; or (ii) The sample is a stool or intestinal sample.
  13. In any one of paragraphs 1 through 12, (a) A step for preparing a dissolved sample, wherein the preparation of the dissolved sample is A sample is brought into contact with a liquid dissolution composition comprising at least one chaotropic agent, a phosphate, at least one organic extraction solvent as an RNase inhibitor, at least one protein precipitating agent, and at least one inhibitor removal agent, wherein the liquid dissolution composition is prepared by combining at least three solutions that may 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 preferably comprises an organic extraction solvent as defined in claim 3, and an agent of a concentration as defined in claim 9, thereby providing a dissolution mixture. Here, the dissolution is a step assisted by mechanical crushing in the presence of crushed particles, (b) a step of purifying the dissolved material; (c) a liquid fraction of the purified lysate, preferably a step of isolating nucleic acid from the nucleic acid-containing supernatant obtained in the lysate purification step (b). A method including
  14. In any one of paragraphs 1 through 13, (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved sample involves contacting the sample with crushed particles and a liquid dissolution composition, wherein the liquid dissolution composition is (i) at least one chaotropic agent at a concentration of 0.5 M to 2 M, wherein the chaotropic agent is selected from the group consisting of NaSCN, NaCO₃ , KSCN, NH₄SCN , LiSCN, LiClO₄ , and guanidine sulfate, preferably NaSCN; (ii) at least one organic extraction solvent as an RNase inhibitor at a concentration (v/v) of 3% to 20%; (iii) at least one protein precipitating agent at a concentration of 0.1M to 2.5M, wherein the protein precipitating agent is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, and cesium acetate, and wherein, preferably, ammonium acetate; (iv) at least one inhibitor scavenger at a concentration of 5 mM to 250 mM, wherein the inhibitor scavenger is a trivalent or tetravalent metal salt Including; and a step comprising mechanically crushing a sample in a provided melting mixture, (b) a step of purifying the dissolved material; (c) Step of isolating nucleic acids from the liquid fraction of the purified lysis. A method including
  15. In claim 14, the liquid dissolution composition prepared in step (a) (i) a chaotropic agent at a concentration of 0.5 M to 1.25 M, wherein, preferably, the chaotropic agent is NaSCN, (ii) an organic extraction solvent at a concentration of 5% to 15%, wherein the organic extraction solvent is preferably selected from phenol-chloroform-isoamyl alcohol, phenol-chloroform and benzyl alcohol-chloroform; (iii) a precipitating agent at a concentration of 0.2M to 1M, preferably an ammonium acetate; (iv) a trivalent or tetravalent metal salt as an inhibitor scavenger at a concentration of 7.5 mM to 75 mM, preferably selected from aluminum chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, and zirconium (IV) chloride, optionally a trivalent aluminum salt, more preferably aluminum chloride; and (v) A phosphate at a concentration of 0.1M to 0.3M, preferably a dibasic sodium phosphate. A method that includes
  16. In any one of paragraphs 13 to 15, isolating nucleic acid from the liquid fraction of the purified lysis according to step (c) (i) under conditions allowing the binding of nucleic acid to a solid support, the liquid fraction of the purified solution is brought into contact with the binding solution and the nucleic acid binding solid support, preferably magnetic silica or glass particles, and Here, the bonding 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, and Here, in the prepared combination mixture containing the sample - The concentration of the chaotropic salt is in the range of 1M to 3M, wherein, preferably, the chaotropic salt is selected from guanidium thiocyanate and guanidinium chloride, and - The concentration of the C1-C5 aliphatic alcohol is in the range of 10-25% (v/v), wherein the alcohol is preferably isopropanol; - The concentration of the non-ionic detergent is in the range of 1%-10% (w/v); (ii) washing the bound nucleic acid, wherein preferably, at least two washing steps using different washing solutions are performed; (iii) Eluting nucleic acids from a solid phase A method that includes
  17. In any one of paragraphs 1 through 16, (a) A step for preparing a dissolved sample, wherein the preparation of the dissolved sample is A sample is brought into contact with a liquid dissolution composition comprising at least one chaotropic salt, a phosphate, at least one organic extraction solvent as an RNase inhibitor, at least one protein precipitating agent, and at least one inhibitor removal agent; The liquid dissolution composition is prepared by combining at least three solutions that may be added in any order, wherein the first solution comprises sodium thiocyanate at a concentration of 0.75 M to 1.5 M and dibasic sodium phosphate at a concentration of 0.1 M to 0.3 M, the second solution comprises ammonium acetate at a concentration of 2 M to 5 M and aluminum chloride at a concentration of 75 mM to 150 mM, and the third solution comprises an organic extraction solvent, preferably phenol-chloroform-isoamyl alcohol, and provides a dissolution mixture. Here, the dissolution is a step assisted by mechanical crushing in the presence of crushed particles, (b) a step of purifying the dissolved material; (c) Step of isolating nucleic acids from the liquid fraction of the purified lysis. A method including
  18. A method according to any one of claims 13 to 17, wherein step (c) comprises isolating RNA from which RNA has been depleted, wherein step (c) comprises the following: - Binding RNA and DNA to magnetic silica or glass particles; - Preferably, washing the bound nucleic acid using two different washing solutions; - Adding DNase and performing DNase digestion; - Rebinding RNA to magnetic particles by adding an additional binding buffer; - Preferably, washing the bound RNA using two different washing solutions; and - The process of eluting RNA from magnetic particles.
  19. A method according to any one of paragraphs 13 to 18, wherein the sample is a fecal sample or an intestinal sample, preferably a stool sample.
  20. The kit is intended for isolating nucleic acids from a sample for performing a method according to any one of claims 1 to 19, and the kit is (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 a magnetic particle; (h) Binding solution for binding nucleic acids to a solid phase Includes; Optionally, the kit here has one or more of the following features: (i) comprising a third solution comprising an RNase inhibitor, wherein preferably, the third solution comprises an organic solvent as the RNase inhibitor, such as phenol-chloroform-isoamyl alcohol, phenol-chloroform, or benzyl alcohol-chloroform; (ii) comprising one or more washing solutions and eluent solutions; (iii) The chaotropic agent contained in the first solution is the same as defined in paragraph 2; (iv) The phosphate contained in the first solution is as defined in paragraph 5; The first solution is as defined in paragraph 6; (v) The protein precipitant contained in the second solution is as defined in paragraph 4; (vi) inhibitor removal agents as defined in paragraph 5; (vii) The second solution is as defined in paragraph 6; (viii) The RNase inhibitor contained in the third solution is as defined in paragraph 3, The kit here is intended for use in automated nucleic acid isolation.

Description

Method for isolating nucleic acids from inhibitor-rich biological samples The present invention provides a method and kit for isolating nucleic acids, such as bacterial and other microbial nucleic acids, from inhibitor-rich samples, particularly fecal samples, such as stool samples. The technology of the present invention advantageously reduces the steps required for inhibitor depletion and is suitable for automation. This provides pure DNA and/or RNA in high yield. Biological samples, such as gut-derived samples and particularly stool samples, represent a rich source of information regarding the microbiome, and many microbiome studies investigate these sample types. Microorganisms include bacteria, fungi, and viruses. There is growing interest in isolating genetic information from these sample types, which demands accurate and easy-to-use methods to recover nucleic acids from them, for example, to support microbiome research or for diagnostic and/or medical purposes. The primary focus is usually on the isolation of bacterial nucleic acids. As for the nucleic acids to be isolated, 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 capable of isolating nucleic acids of interest from inhibitor-rich samples with excellent yield and purity, thereby allowing the isolated nucleic acids to be used in downstream methods. For example, 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. If contaminants are present in the isolated nucleic acids, they can interfere with and inhibit downstream analysis of the isolated nucleic acids. The removal of inhibitory components during nucleic acid isolation is critical and challenging. The aforementioned samples, such as fecal samples in particular, are highly complex and, in some cases, contain a wide variety of inhibitory components. Furthermore, when processing larger sample volumes is intended to recover sufficient nucleic acids—e.g., RNA—for downstream analysis, high levels of inhibitors simultaneously enter the isolation process, making the isolation of pure, inhibitor-depleted nucleic acids from such large sample volumes particularly challenging. However, thorough removal of these inhibitors is crucial to allow for efficient downstream analysis of the isolated nucleic acids, which typically involves enzymatic reactions such as PCR amplification. Methods for isolating nucleic acids from inhibitor-rich samples are described in the art, see, for example, WO 2006/073472 and WO 2019/209597. A number of commercially available kits for purifying nucleic acids from inhibitor-rich samples, such as fecal samples, are also available. However, existing methods have drawbacks, such as the processing of limited sample volumes, insufficient inhibitor removal, low purity, moderate to low nucleic acid yields, and/or the requirement of multiple processing steps. Existing techniques for isolating nucleic acids from inhibitor-rich biological samples, such as fecal samples, typically suffer from low yields and/or low purity, or require multiple steps for inhibitor removal, making the process difficult and time-consuming. Furthermore, existing methods are not suitable for automated nucleic acid isolation (although this is desirable for increasing the number of samples that can be processed in parallel for nucleic acid isolation). Improvement and standardization of nucleic acid extraction methods are necessary to satisfy the demands of microbiome research while providing the most unbiased representation of samples. It is an object of the present invention to provide a method for isolating nucleic acids, such as RNA and/or DNA, from inhibitor-rich biological samples, particularly stool or intestinal samples, which overcomes at least one of the aforementioned problems of the prior art method. In particular, it is an object of the present invention to provide an improved method for isolating nucleic acids, such as RNA and/or DNA, from inhibitor-rich samples, particularly stool or intestinal samples, which achieves efficient removal of inhibitory contaminants and is suitable for automated nucleic acid isolation. It is also an object of the present invention to provide a method for isolating nucleic acids, such as RNA and/or DNA, from inhibitor-rich biological samples, which provides high-yield, inhibitor-depleted nucleic acids suitable for downstream analysis in the field of molecular biology. The objective is to provide an efficient and flexible method particularly suitable for isolating microbial nucleic acids (RNA and/or DNA) from microbiome-containing samples, such as fecal or intestinal samples. Additionally, the objective of the present invention is to provide an improved method for isolating bacterial and other mic