lysis, binding and / or washing reagents for isolating and / or purifying nucleic acids

Abstract

The present invention provides a lysis, binding and / or washing reagent for isolating and / or purifying nucleic acids and a method of isolating and / or purifying nucleic acids.

Description

Technical Field

[0001] This invention relates to lysis, binding, and / or washing reagents and methods for isolating and / or purifying nucleic acids. The lysis, binding, and / or washing reagents and methods are particularly suitable for molecular diagnostic purposes. Background of the Invention

[0002] Existing technologies have revealed a variety of methods for isolating and / or purifying nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) from cells, cell cultures or viral cultures.

[0003] In this respect, many "classic" manual methods for isolating nucleic acids are one-step processes, involving extraction after the addition of an aqueous buffer and an organic extractant. The nucleic acids remain in the aqueous phase and can be isolated after the removal of the organic phase containing undesirable concomitant substances.

[0004] These methods first use normal, harmless organic extractants such as chloroform or phenol, then water-soluble contaminants remain in the aqueous phase containing nucleic acids and require further purification steps to remove them.

[0005] Therefore, alternative methods in this field have gained attention, which are based on the selective adsorption of nucleic acids onto solid, primarily mineral supports such as silica. The binding principle is based on the reversible binding of nucleic acids to the silica surface under the influence of "liquid salts" and / or alcohols. In multi-step methods, various solutions or mixtures, typically lysis, binding, washing, and / or elution solutions or mixtures, are added to the nucleic acid-containing sample, and in the final method step, the purified nucleic acids are eluted from the support just added in the binding step.

[0006] The basic principle of both methods is based on the lysis of cells in the first step, especially plant, animal, human, bacterial, and viral cells. For this purpose, the cells are first incubated with a cell-destroying lysis buffer.

[0007] Existing technologies reveal buffers and methods for lysing biological sample cellular materials. Known lysis buffers often contain the surfactant polyoxyethylene dehydrated sorbitan monolaurate (POD). 20). The surfactant is used to convert contaminants generated during cell lysis into a soluble or stable state, thereby removing them from nucleic acids.

[0008] Unfortunately, it contains polyoxyethylene dehydrated sorbitan monolaurate ( The lysis buffers used in 20) are unstable during storage. For example, the pH may decrease. A specific drawback is that the yield of isolated nucleic acids is reduced after storage if these lysis buffers are used. Another drawback is that the eluent containing nucleic acids is opaque, indicating the presence of contamination that may interfere with further applications of the isolated nucleic acids.

[0009] Therefore, the object of the present invention is to provide a method that overcomes at least one of the disadvantages of the prior art described above, wherein the method has the best or better cleavage, binding and / or washing properties possible.

[0010] The objective of the present invention is achieved by the cleavage, binding, and / or washing agent as described in claim 1. Therefore, a cleavage, binding, and / or washing agent is provided, comprising:

[0011] -At least one liquid-free compound,

[0012] - At least one buffer compound, preferably selected from: tris(hydroxymethyl)aminomethane (TRIS), N-(tris(hydroxymethyl)methyl)glycine (TRICINE), N,N-bis(2-hydroxyethyl)glycine (BICINE), N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS) and / or phosphate buffers, and

[0013] - At least one nonionic surfactant based on polyoxyethylene, selected from polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers and / or polyoxyethylene-polyoxypropylene block copolymers, in an amount ranging from ≥8% by weight / volume to ≤50% by weight / volume based on the total volume of the reagent.

[0014] For the purposes of this invention, the term "lysis, binding, and / or washing reagent" refers to a reagent that is a lysis reagent, a binding reagent, or a washing reagent, or a reagent that can be used as a lysis reagent, a binding reagent, or a washing reagent. More specifically, for the purposes of this invention, the term "lysis, binding, and / or washing reagent" may also refer to a mixture of the lysis reagent, binding reagent, and / or washing reagent of this invention.

[0015] For the purposes of this invention, the term "reagent" refers to a cleavage, binding, and / or washing agent.

[0016] For the purposes of this invention, the term "liquid-free compound" refers to a compound that acts on proteins in a denaturing manner, specifically by disrupting the conventional structure of liquid water based on hydrogen bonding.

[0017] For the purposes of this invention, the term "buffer compound" refers to a compound that can provide buffering or pH stabilization for aqueous solutions.

[0018] For the purposes of this invention, the term "phosphate buffer" refers to phosphates, such as dihydrogen phosphates like potassium dihydrogen phosphate (KH₂PO₄) or sodium dihydrogen phosphate (NaH₂PO₄), and hydrogen phosphates like disodium hydrogen phosphate (Na₂HPO₄·2H₂O) or dipotassium hydrogen phosphate. Similarly, mixtures of phosphates may be used. Another commonly used phosphate buffer is PBS (phosphate-buffered saline), which contains sodium chloride, Na₂HPO₄, potassium chloride, and KH₂PO₄.

[0019] For the purposes of this invention, the term "nucleic acid" means, but is not limited to: natural, preferably isolated, linear, branched, or circular nucleic acids such as RNA, specifically mRNA, siRNA, miRNA, snRNA, tRNA, hnRNA, or ribozymes; DNA, plasmid DNA, etc.; synthetic or modified nucleic acids; in vitro transcripts, such as oligonucleotides; more specifically primers, probes, or standards usable for PCR; digoxigenin, biotin, or fluorescent dye-labeled nucleic acids; methylated nucleic acids; or "PNA" ("peptide nucleic acid").

[0020] For the purposes of this invention, the term "surfactant" refers to interfacial and / or surface-active substances.

[0021] For the purposes of this invention, the term "fatty alcohol" refers to an alcohol with a chain length of 6-22 carbon atoms, preferably 8-20 carbon atoms, more preferably 10-18 carbon atoms, and even more preferably 12-18 carbon atoms. Alcohols having 12, 14, 16, or 18 carbon atoms are particularly preferred. Although fatty alcohols can be monounsaturated or polyunsaturated, they are preferably saturated fatty alcohols.

[0022] For the purposes of this invention, "polyoxyethylene" represents HO-(CH2CH2O). nThe unit, n, is preferably an integer from 2 to 150, more preferably from 4 to 120, even more preferably from 8 to 80, and most preferably an integer selected from the following group: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33. 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 7 8, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 1 17, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, and 150.

[0023] For the purposes of this invention, "polyoxypropylene" represents HO-(CH2CH2CH2O). n The unit n is preferably an integer from 10 to 90, more preferably from 20 to 80, even more preferably from 30 to 70, and most preferably an integer selected from the following group: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 4 2, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90.

[0024] For the purposes of this invention, the information “% weight / volume”, “% (weight / volume)” or “% (w / v)” indicates, for example, the number of grams of surfactant in 100 ml of reagent or composition.

[0025] Unexpectedly, the cleavage, binding, and / or washing reagents of the present invention were found to have improved stability during storage. Thus, for example, the cleavage, binding, and / or washing reagents of the present invention maintain a stable pH after being stored at room temperature for 3 months, preferably 6 months, and more preferably at least 8 months. More specifically, the cleavage, binding, and / or washing reagents of the present invention also maintain a stable pH after being stored at elevated temperatures, such as at 50°C, for several weeks, preferably several months.

[0026] This is beneficial for lysis, binding, and / or washing reagents, as pH instability is suspected to be related to contamination in the eluent containing nucleic acids obtained after separation.

[0027] According to the present invention, the preferred nonionic surfactant based on polyoxyethylene is polyoxyethylene fatty alcohol ether.

[0028] Suitable examples of polyoxyethylene fatty alcohol ethers are polyethoxylated lauryl, cetyl, oleyl, or stearyl alcohols, which can be used alone or as a mixture.

[0029] According to a preferred embodiment of the present invention, the polyoxyethylene fatty alcohol ether comprises a fatty alcohol moiety having 6-22 carbon atoms and a polyoxyethylene moiety having 2-150 (CH2CH2O) units.

[0030] According to a particularly preferred embodiment of the present invention, the polyoxyethylene fatty alcohol ether is selected from: polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, and / or polyoxyethylene oil-based ether.

[0031] Nonionic surfactants based on polyoxyethylene, particularly polyoxyethylene fatty alcohol ethers, are beneficial for the wide application within this invention. Specifically, improved storage stability is observed in cleavage, binding, and / or washing agents comprising buffer compounds preferably selected from the group consisting of: tris(hydroxymethyl)aminomethane (TRIS), N-(tris(hydroxymethyl)methyl)glycine (TRICINE), N,N-bis(2-hydroxyethyl)glycine (BICINE), N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), and / or phosphate buffers.

[0032] It has been found that the pyrolysis, binding, and / or washing agents of the present invention are particularly effective when the content of polyoxyethylene-based nonionic surfactants is in the range of ≥8% (weight / volume) to ≤50% (weight / volume) based on the total volume of the pyrolysis, binding, and / or washing agents, wherein the polyoxyethylene-based nonionic surfactants are selected from: polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, and / or polyoxyethylene-polyoxypropylene block copolymers.

[0033] If a mixture of surfactants is used, the concentration information, based on the total volume of the reagent, is preferably the total surfactant content, for example, from ≥8% (weight / volume) to ≤50% (weight / volume).

[0034] This is particularly beneficial for the lysis reagent of the present invention.

[0035] Preferred polyoxyethylene fatty alcohol ethers are ethoxylated lauryl, cetyl, oleyl, or stearyl alcohols, selected from: polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, and / or polyoxyethylene oleyl ether.

[0036] Preferred polyoxyethylene fatty alcohol ethers are selected from the group consisting of: polyoxyethylene (4) lauryl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (2) cetyl ether, polyoxyethylene (10) cetyl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (2) stearyl ether, polyoxyethylene (10) stearyl ether, polyoxyethylene (20) stearyl ether, polyoxyethylene (2) oil-based ether, polyoxyethylene (10) oil-based ether, polyoxyethylene (20) oil-based ether, and / or polyoxyethylene (100) stearyl ether. The numbers represent the average number of ethylene oxide units.

[0037] Particularly suitable for the present invention are, for example, products manufactured by ICI Surfactants under the trade name... Polyoxyethylene fatty alcohol ethers for sale.

[0038] Suitable examples of polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oil-based ether, or polyoxyethylene stearyl ether are preferably selected from: polyoxyethylene (4) lauryl ether Polyoxyethylene (23) lauryl ether ( 35) Polyoxyethylene (2) cetyl ether Polyoxyethylene (10) cetyl ether Polyoxyethylene (20) cetyl ether Polyoxyethylene (2) stearyl ether Polyoxyethylene (10) stearyl ether Polyoxyethylene (20) stearyl ether Polyoxyethylene (2) oil-based ether Polyoxyethylene (10) oil-based ether Polyoxyethylene (20) oil-based ether and / or polyoxyethylene (100) stearyl ether

[0039] Suitable polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oil-based ether, or polyoxyethylene stearyl ether can also be used in powder form, such as polyoxyethylene (21) stearyl ether powder. 721P).

[0040] Another advantage of the lysis, binding, and / or washing reagents of this invention is that, when used for the isolation and / or purification of nucleic acids, the lysis, binding, and / or washing reagents consistently exhibit excellent yields of isolated nucleic acids, even if the lysis, binding, and / or washing reagents have been stored at room temperature or at elevated temperatures (e.g., up to 50°C) for weeks or months, unlike prior art buffers, especially those containing... The 20% buffer showed lower nucleic acid yield after storage.

[0041] A specific advantage of using the lysis, binding, and / or washing reagents of this invention is that the eluent containing nucleic acids remains turbid or only slightly turbid even after storage for weeks or months. Therefore, the advantage of the eluent being free of contaminants or at least significantly less contaminant makes the further application of the nucleic acid-containing eluent significantly more beneficial, as it eliminates the need for time-consuming further purification steps that reduce nucleic acid yield.

[0042] The next preferred option includes polyoxyethylene lauryl ether, such as polyoxyethylene (4) lauryl ether. Or polyoxyethylene (23) lauryl ether The cleavage, binding, and / or washing agents are not polyoxyethylene fatty alcohol ethers. Therefore, in a preferred embodiment, the cleavage, binding, and / or washing agents do not contain any of these substances. In a particularly preferred embodiment, the polyoxyethylene fatty alcohol ether of the cleavage, binding, and / or washing agents is not a polyoxyethylene lauryl ether.

[0043] In a preferred embodiment of the present invention, the polyoxyethylene fatty alcohol ether is selected from: polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and / or polyoxyethylene oil ether.

[0044] Preferred polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, or polyoxyethylene stearyl ether, selected from: polyoxyethylene (10) cetyl ether Polyoxyethylene (20) cetyl ether Polyoxyethylene (20) stearyl ether and / or polyoxyethylene (20) oil-based ether

[0045] Especially preferred are polyoxyethylene cetyl alcohol ether or polyoxyethylene oleyl alcohol ether, preferably selected from: polyoxyethylene (10) cetyl alcohol ether Polyoxyethylene (20) cetyl ether and / or polyoxyethylene (20) oil-based ether

[0046] More specifically, by comparison, the present invention comprises polyoxyethylene fatty alcohol ethers, specifically polyoxyethylene cetyl ethers or polyoxyethylene oleyl alcohol ethers, whose cleavage, binding, and / or washing agents have particularly excellent yields of nucleic acids, especially viral DNA. More specifically, compared with those containing... Compared to a lysis buffer of 20%, the use of freshly prepared lysis and / or binding reagents containing polyoxyethylene cetyl ether, as well as those stored at 50°C for several weeks or even months, to isolate hepatitis B virus (HBV) unexpectedly yielded significantly increased viral DNA yields. This specifically provides a particular advantage of the lysis and / or binding reagents of the present invention, as hepatitis B virus (HBV) is known to be difficult to lyse. The lysis reagents of the present invention are particularly suitable for isolating viral DNA.

[0047] Furthermore, suitable options include polyethoxylated lauryl alcohol, cetyl alcohol, stearyl alcohol, or oleyl alcohol, which are obtained under the INCI names laureth, cetyl alcohol, stearyl alcohol, or oleyl alcohol, respectively.

[0048] Particularly suitable examples of ethoxylated dodecyl alcohol, lauryl alcohol, cetyl alcohol, stearyl alcohol, or oleyl alcohol can be obtained from the following group of names: lauryl alcohol polyether-9, lauryl alcohol polyether-4, lauryl alcohol polyether-23, cetyl alcohol polyether-2, cetyl alcohol polyether-20, stearyl alcohol polyether-2, stearyl alcohol polyether-10, stearyl alcohol polyether-20, oleoyl alcohol polyether-2, oleoyl alcohol polyether-10, and / or oleoyl alcohol polyether-20.

[0049] A further preferred nonionic surfactant based on polyoxyethylene is a polyoxyethylene alkylphenyl ether. Preferred polyoxyethylene alkylphenyl ethers have an alkyl group containing 5-15 carbon atoms, preferably 6-10 carbon atoms. More preferably, they are branched or unbranched C7- to C6-20-20-30-4 ... 10 -alkyl, more preferably branched or unbranched C8- and C9-alkyl, more preferably isooctyl and nonyl.

[0050] In a preferred embodiment of the invention, the polyoxyethylene alkylphenyl ether is selected from: polyoxyethylene nonylphenyl ether and / or polyoxyethylene isooctylphenyl ether. Suitable polyoxyethylene nonylphenyl ether and polyoxyethylene isooctylphenyl ether are, for example, marketed under trade names. Obtained from BASF.

[0051] Suitable examples of polyoxyethylene nonylphenyl ether and polyoxyethylene isooctylphenyl ether are preferably selected from: polyoxyethylene (2) nonylphenyl ether Polyoxyethylene (2) isooctylphenyl ether Polyoxyethylene (5) Nonylphenyl ether Polyoxyethylene (5) isooctylphenyl ether Polyoxyethylene (9) Nonylphenyl ether Polyoxyethylene (9) isooctylphenyl ether Polyoxyethylene (12) Nonylphenyl ether Polyoxyethylene (12) isooctylphenyl ether and / or polyoxyethylene (100) nonylphenyl ether

[0052] A more preferred nonionic surfactant based on polyoxyethylene is a polyoxyethylene-polyoxypropylene block copolymer. Polyoxyethylene-polyoxypropylene block copolymers are also known as "poloxamers." The preferred empirical formula is HO(C₂H₄O). a (C3H6O) b (C2H4O) a H is a polyoxyethylene-polyoxypropylene block copolymer, wherein "a" represents the number of polyoxyethylene units and "b" represents the number of polyoxypropylene units, and the a / b weight ratio is preferably in the range of 0.1 to 3.

[0053] "a" is more preferably 2-150, more preferably 4-120, more preferably 8-80, and even more preferably "a" is an integer selected from the following group: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36. 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 12 4, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 and 150, the optimal choice "a" is an integer selected from 2, 4, 10, 20, 23, 40, 55, 70 and 100.

[0054] "b" is more preferably 10-90, more preferably 20-80, more preferably 30-70, and even more preferably "b" is an integer selected from the following group: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47. 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90, the optimal choice "b" is an integer selected from 15, 18, 23, 40, 55, 67, and 75.

[0055] More preferably, a polyoxyethylene-polyoxypropylene block copolymer having polyoxyethylene and polyoxypropylene blocks of different lengths, wherein a polyoxypropylene block having 15-67 polypropylene units is surrounded by two polyoxyethylene blocks each having 2-130 polyethylene units.

[0056] Suitable polyoxyethylene-polyoxypropylene block copolymers can be traded under the name or For example, it can be obtained from BASF.

[0057] Suitable examples of polyoxyethylene-polyoxypropylene block copolymers are preferably selected from: PE6200 PE6400 PE6800 PE10300 PE10500 F127 F108 F108 F127 and / or F68.

[0058] According to a preferred embodiment of the present invention, the cleavage, binding and / or washing agent comprises a nonionic surfactant, the amount of which is ≥9% (weight / volume) to ≤40% (weight / volume) based on the total volume of the agent, preferably ≥10% (weight / volume) to ≤30% (weight / volume), and preferably ≥15% (weight / volume) to ≤20% (weight / volume).

[0059] According to a preferred embodiment of the present invention, the ionizing compound is a sodium salt or a guanidine salt, preferably selected from: sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, and / or a mixture of two or more salts. The ionizing compound is preferably a guanidine salt, preferably selected from: guanidine hydrochloride, guanidine thiocyanate, and / or guanidine isothiocyanate.

[0060] Specifically, the combination of the aforementioned liquid-dissolving compound and the polyoxyethylene-based nonionic surfactant is beneficial for lysing viral cells and isolating nucleic acids from viral cells.

[0061] The appropriate concentration and amount of the ionizing compound can vary depending on the sample type or lysis parameters. Generally, the concentration of the ionizing compound, based on the total volume of the reagent, is preferably ≥0.1M to ≤10M. The concentration of the ionizing compound in the lysis, binding, and / or washing reagent is preferably ≥0.5M to ≤8M, and more preferably ≥0.9M to ≤6M.

[0062] The concentration of the ionized compound in the lysis reagent is preferably ≥3M to ≤7M, more preferably ≥4M to ≤6M. The concentration of the ionized compound in the binding reagent is preferably ≥0.5M to ≤7M, more preferably ≥1M to ≤6M. The concentration of the ionized compound in the washing reagent is preferably ≥0.5M to ≤3.5M, more preferably ≥0.9M to ≤3M.

[0063] According to another preferred embodiment, the cleavage, binding, and / or washing agent comprises at least one buffer compound selected from the group consisting of tris(hydroxymethyl)aminomethane (TRIS), N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), and / or a phosphate buffer.

[0064] According to a particularly preferred embodiment, the cleavage, binding and / or washing agent comprises at least one buffer compound selected from the group consisting of tris(hydroxymethyl)aminomethane (TRIS) and / or N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES).

[0065] The cleavage, binding, and / or washing reagents are preferably aqueous solutions.

[0066] According to a further preferred embodiment, the pH of the cleavage, binding and / or washing agent is from ≥4 to ≤12, more preferably ≥6 to ≤11, more preferably ≥7 to ≤10, and more preferably ≥8 to ≤9.

[0067] In a preferred embodiment, the lysis, binding, and / or washing reagents, especially the lysis reagents, may also contain enzymes, such as lysins, specifically, for example, proteinase K, proteases (e.g., QIAGEN protease), digestive enzymes, lysozymes, colorless peptidases, lysozymes, lysozymes, and, depending on the application, nucleases, such as DNases and / or RNases.

[0068] The cleavage, binding, and / or washing reagents of the present invention may be cleavage reagents, binding reagents, or washing reagents, or mixtures of the cleavage reagents, binding reagents, and / or washing reagents of the present invention.

[0069] Nucleic acids are immobilized onto a matrix based on one or more silica compounds in the presence of a liquid-bound compound, preferably in the presence of branched or unbranched alkanols. Therefore, a binding agent comprising at least one branched or branched alkanol is preferred.

[0070] Preferred and useful are short-chain branched or unbranched alkanols having 1-5 carbon atoms. According to a preferred embodiment of the invention, the branched or unbranched alkanol is an alcohol having 1-5 carbon atoms, preferably selected from: methanol, ethanol, isopropanol, n-propanol, branched or unbranched butanol or pentanol, and / or mixtures thereof.

[0071] Unless otherwise stated, the definition of "branched or unbranched alkanols" specifically includes propanol, butanol, and pentanol, encompassing any isomer of the specific consumable group. Thus, for example, branched or unbranched propanol includes n-propanol and isopropanol, branched or unbranched butanol includes isobutanol, sec-butanol, and tert-butanol, and branched or unbranched pentanol includes, for example, n-pentanol and isopentanol. Alcohols selected from the group consisting of methanol, ethanol, isopropanol, and / or mixtures thereof are preferred, and mixtures thereof are particularly preferred.

[0072] According to a preferred embodiment of the present invention, the binding reagent comprises, by volume, branched or unbranched alkanols with a content ranging from ≥20% to ≤80%, preferably ≥40% to ≤70%, and more preferably ≥50% to ≤60% based on the total volume of the binding reagent.

[0073] When it comes to volume and / or weight content, those skilled in the art will readily understand that the volume and / or weight content of each component is selected such that the total volume or total weight of the components does not exceed 100% by volume or 100% by weight.

[0074] This invention also relates to the use of the lysis, binding and / or washing reagents of this invention in the isolation and / or purification of nucleic acids.

[0075] The present invention also relates to a method for isolating and / or purifying nucleic acids from biological samples containing nucleic acids, the method comprising the following steps:

[0076] a) Lysing biological samples,

[0077] b) Immobilizing released nucleic acids onto a matrix based on one or more silica compounds in the presence of a liquid-dissolving compound and / or branched or unbranched alkanols.

[0078] c) Optionally wash the nucleic acids immobilized on the matrix.

[0079] d) Optional removal of bound nucleic acids,

[0080] The cleavage and / or fixation are carried out in the presence of a cleavage and / or binding composition comprising the following components:

[0081] - at least one liquid-free compound, and

[0082] - At least one nonionic surfactant based on polyoxyethylene, selected from polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers and / or polyoxyethylene-polyoxypropylene block copolymers, is used in an amount from ≥0.1% by weight / volume to ≤50% by weight / volume, based on the total volume of the composition.

[0083] For the purposes of this invention, the term "composition" refers to a cleavage and / or binding composition.

[0084] In a preferred embodiment of the method of the present invention, the sample is lysed using the lysis reagent of the present invention. The lysis reagent is brought into contact with the biological sample to be lysed. Depending on the application, one or more enzymes are added independently at various time points. The sample may be liquid, for example, in the case of liquid clinical samples. Typically, clinical samples containing solid components, such as fecal samples or swab samples, are suspended in a suitable aqueous solution and then further analyzed. Cell cultures are usually obtained from a culture medium before lysis, but in most cases, completely dried samples are avoided. In the case of completely dried samples, such as lyophilized samples, the sample is reconstituted in an aqueous solution before further processing, such as lyophilized viral standards. Therefore, samples requiring lysis typically contain some liquid. The liquid present in the sample is brought into contact with the lysis reagent. In this respect, methods for isolating and / or purifying nucleic acids from a sample typically involve a lysis composition comprising the lysis reagent and other liquids of the sample or a solution to which the sample has been added.

[0085] For the purposes of this invention, the term "lysis and / or binding composition" refers to a lysis and / or binding reagent used in methods for isolating and / or purifying nucleic acids from a sample, which may also contain liquids in addition to lysis, binding, and / or washing reagents. The lysis and / or binding composition preferably contains the lysis and / or binding reagents of this invention.

[0086] According to another preferred embodiment of the method, in the presence of the inventive combination composition, the released nucleic acids are immobilized onto a matrix based on one or more silica compounds.

[0087] Preferably, the lysis and / or binding reagent of the present invention is brought into contact with the lysed sample. The lysis composition or another solution used for lysis may be removed before the sample is brought into contact with the binding reagent. Preferably, the lysis composition is not removed. Preferably, the binding reagent is brought into contact with the sample containing the lysis composition.

[0088] According to a particularly preferred embodiment of the method, pyrolysis is carried out in the presence of the pyrolysis composition, and fixation is carried out in the presence of the binding composition. Therefore, fixation is preferably carried out in the presence of a mixture of the pyrolysis composition and the binding composition.

[0089] Optionally, the lysis reagent can also be used as a binding reagent. Optionally, the binding reagent can also be used as a lysis reagent. Optionally, the binding reagent can also be used as a washing reagent.

[0090] The cleavage and / or binding composition comprises at least one liquid-ionizing compound and at least one polyoxyethylene-based nonionic surfactant selected from polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, and / or polyoxyethylene-polyoxypropylene block copolymers, wherein the polyoxyethylene-based nonionic surfactant comprises from ≥0.1% (weight / volume) to ≤50% (weight / volume) by total volume of the composition. If a mixture of surfactants is used, the total surfactant content is preferably from, for example, from ≥0.1% (weight / volume) to ≤50% (weight / volume) by total volume of the composition.

[0091] The advantage of such methods for isolating and / or purifying nucleic acids from nucleic acid-containing biological samples is that, if a lysis and / or binding composition comprising at least one ionizing compound and at least one polyoxyethylene-based nonionic surfactant is used, the nucleic acid-containing eluent will not become turbid or will only become slightly turbid, even after storage at room temperature or elevated temperatures (e.g., 50°C) for several weeks or months. The at least one polyoxyethylene-based nonionic surfactant is selected from polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, and / or polyoxyethylene-polyoxypropylene block copolymers, and is used in amounts ranging from ≥0.1% (weight / volume) to ≤50% (weight / volume) based on the total volume of the composition. Therefore, advantageously, the eluent is free of contaminants or contains at least significantly fewer contaminants. This makes the further use of the nucleic acid-containing eluent significantly more beneficial, as it eliminates the need for time-consuming further purification steps that reduce nucleic acid yield.

[0092] Another advantage of such methods for isolating and / or purifying nucleic acids from biological samples containing nucleic acids is that, for example, particularly excellent yields of isolated nucleic acids, especially viral DNA, such as hepatitis B virus (HBV) DNA, are possible.

[0093] "Biological sample" can be understood as referring to particulate or molecular-based materials, specifically viruses, bacteriophages, and cells such as bacterial cells, yeast or mold cells, or human, animal, or plant cells. The method is particularly suitable for isolating nucleic acids such as DNA or RNA from sample materials of human or animal origin, such as clinical samples like blood, plasma, serum, oral, larynx, nasal lavage fluid, bronchoalveolar lavage fluid, urine, cerebrospinal fluid, sputum, saliva, feces, aspirates, smears / swabs, such as nasal smears / swabs, buccal smears / swabs, neck smears / swabs, vaginal smears / swabs, urethral smears / swabs, pharyngeal smears / swabs, perineal smears / swabs, and rectal smears / swabs, feces, aspirates, epithelial smears / swabs, biopsies and other tissue or bone marrow samples, and cultures of these sample materials in suitable nutrient media.

[0094] Samples can also come from environmental analysis, food analysis, or molecular biology research fields, such as bacterial cultures, yeast or fungal cultures, viral cultures, bacteriophage lysates, or products of amplification processes, such as polymerase chain reaction (PCR) products.

[0095] The method of this invention is particularly suitable for isolating and / or purifying genomic DNA, mitochondrial DNA, plasmid DNA, viral DNA, and viral RNA from whole blood, for isolating and purifying intracellular RNA from whole blood, for example, for reverse transcription polymerase chain reaction (RT-PCR), and can also be used to isolate and / or purify freely circulating nucleic acids present in cell-free sample materials. The method of this invention is particularly suitable for isolating and / or purifying viral DNA.

[0096] Biological samples are lysed in step a) of the method described below. In principle, the methods listed below are suitable for lysing biological samples, selected from: the use of a chelating salt in the presence of a suitable reagent or buffer, with the aid of ionic and nonionic surfactants such as sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LiDS), or sodium sarcosinate (sarcosyl), mechanical lysis (e.g., by sonication, using a Freund's cell crusher), grinding with particles such as glass beads, ceramic beads, or metal particles, or in liquid nitrogen, by repeated freeze-thaw cycles, or by boiling, enzymatic lysis, lyophilization lysis, osmotic shock lysis, microwave and / or temperature treatment, and / or combinations thereof. Lysis is preferably carried out in the presence of a chelating salt.

[0097] Biological samples are preferably lysed in the presence of a lysis composition comprising at least one ionizing compound and at least one polyoxyethylene-based nonionic surfactant selected from polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers and / or polyoxyethylene-polyoxypropylene block copolymers, wherein the polyoxyethylene-based nonionic surfactant is ≥0.1% (w / v) to ≤50% (w / v) based on the total volume of the lysis composition.

[0098] More specifically, the combination of a liquid release agent and a polyoxyethylene-based nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, and / or polyoxyethylene-polyoxypropylene block copolymers is particularly effective for the lysis of viral cells.

[0099] The cleavage and / or binding composition comprises at least one liquid-ionizing compound and at least one nonionic surfactant based on polyoxyethylene selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, and / or polyoxyethylene-polyoxypropylene block copolymers.

[0100] For information on nonionic surfactants based on polyoxyethylene, please refer to the full contents of the instruction manual.

[0101] Suitable examples of ethoxylated fatty alcohols are ethoxylated dodecyl alcohol, lauryl alcohol, cetyl alcohol, oleyl alcohol, or stearyl alcohol, which can be used alone or in mixtures. Preferred polyoxyethylene fatty alcohol ethers are ethoxylated lauryl alcohol ether, cetyl alcohol ether, oleyl alcohol ether, or stearyl alcohol ether, selected from: polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, and / or polyoxyethylene oleyl ether.

[0102] Preferred polyoxyethylene fatty alcohol ethers are selected from: polyoxyethylene (4) lauryl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (2) cetyl ether, polyoxyethylene (10) cetyl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (2) stearyl ether, polyoxyethylene (10) stearyl ether, polyoxyethylene (20) stearyl ether, polyoxyethylene (2) oil-based ether, polyoxyethylene (10) oil-based ether, polyoxyethylene (20) oil-based ether, and / or polyoxyethylene (100) stearyl ether. The numbers represent the average number of ethylene oxide units.

[0103] Suitable examples of polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, or polyoxyethylene stearyl ether are preferably selected from: polyoxyethylene (4) lauryl ether Polyoxyethylene (23) lauryl ether Polyoxyethylene (2) cetyl ether Polyoxyethylene (10) cetyl ether Polyoxyethylene (20) cetyl ether Polyoxyethylene (2) stearyl ether Polyoxyethylene (10) stearyl ether Polyoxyethylene (20) stearyl ether Polyoxyethylene (2) oil-based ether Polyoxyethylene (10) oil-based ether Polyoxyethylene (20) oil-based ether and / or polyoxyethylene (100) stearyl ether

[0104] According to a preferred embodiment of the present invention, the polyoxyethylene fatty alcohol ether comprises a fatty alcohol component having 6-22 carbon atoms and a polyoxyethylene component having 2-150 (CH2CH2O) units.

[0105] In a particularly preferred embodiment of the method, the polyoxyethylene fatty alcohol ether is selected from: polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and / or polyoxyethylene oil ether.

[0106] In a preferred embodiment of method one, the polyoxyethylene fatty alcohol ether is selected from: polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, and / or polyoxyethylene oil-based ether. In this embodiment, it is less preferably included with polyoxyethylene lauryl ether, such as polyoxyethylene (4) lauryl ether. Or polyoxyethylene (23) lauryl ether The cleavage and / or binding agents are preferred. Therefore, the cleavage and / or binding composition is preferably free of any of these substances. In a particularly preferred embodiment, the polyoxyethylene fatty alcohol ether of the cleavage and / or binding agent is not a polyoxyethylene lauryl ether.

[0107] Preferred are polyoxyethylene cetyl alcohol ether, polyoxyethylene oleyl alcohol ether, or polyoxyethylene stearyl alcohol ether, preferably selected from polyoxyethylene (10) cetyl alcohol ether. Polyoxyethylene (20) cetyl ether Polyoxyethylene (20) stearyl ether and / or polyoxyethylene (20) oil-based ether Especially preferred are polyoxyethylene cetyl alcohol ether or polyoxyethylene oleyl alcohol ether, preferably selected from: polyoxyethylene (10) cetyl alcohol ether Polyoxyethylene (20) cetyl ether and / or polyoxyethylene (20) oil-based ether

[0108] Furthermore, suitable options include polyethoxylated lauryl alcohol, cetyl alcohol, stearyl alcohol, or oleyl alcohol, which can be obtained by the INCI names laureth, cetyl alcohol, stearyl alcohol, or oleyl alcohol, respectively.

[0109] A further preferred nonionic surfactant based on polyoxyethylene is a polyoxyethylene alkylphenyl ether. Preferably, the alkyl group comprises 5-15 carbon atoms, more preferably 6-10 carbon atoms. More preferably, it is a branched or unbranched C7- to C6-carbon alkylphenyl ether. 10 -alkyl, more preferably branched or unbranched C8- and C9-alkyl, particularly isooctyl and nonyl. In a preferred embodiment of method one, the polyoxyethylene alkylphenyl ether is selected from: polyoxyethylene nonylphenyl ether and / or polyoxyethylene isooctylphenyl ether. Suitable polyoxyethylene nonylphenyl ether and polyoxyethylene isooctylphenyl ether are, for example, marketed under trade names. Obtained from BASF.

[0110] A more preferred nonionic surfactant based on polyoxyethylene is a polyoxyethylene-polyoxypropylene block copolymer. Polyoxyethylene-polyoxypropylene block copolymers are also known as "poloxamers." The preferred empirical formula is HO(C₂H₄O). a (C3H6O) b (C2H4O)a H is a polyoxyethylene-polyoxypropylene block copolymer, wherein "a" represents the number of polyoxyethylene units and "b" represents the number of polyoxypropylene units, and the a / b weight ratio is preferably in the range of 0.1 to 3.

[0111] "a" is more preferably 2-150, more preferably 4-120, more preferably 8-80, and even more preferably "a" is an integer selected from the following group: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36. 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 12 4, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 and 150, the optimal choice "a" is an integer selected from 2, 4, 10, 20, 23, 40, 55, 70 and 100.

[0112] "b" is more preferably 10-90, more preferably 20-80, more preferably 30-70, and even more preferably "b" is an integer selected from the following group: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47. 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90, the optimal choice "b" is an integer selected from 15, 18, 23, 40, 55, 67, and 75.

[0113] More preferably, a polyoxyethylene-polyoxypropylene block copolymer having polyoxyethylene and polyoxypropylene blocks of different lengths, wherein a polyoxypropylene block having 15-67 polypropylene units is surrounded by two polyoxyethylene blocks, each having 2-130 polyethylene units, which are independent of each other.

[0114] Suitable polyoxyethylene-polyoxypropylene block copolymers can be traded under the name or For example, it can be obtained from BASF.

[0115] According to a preferred embodiment of Method 1, the cleavage and / or binding composition comprises, by total volume of the composition, ≥0.2% (weight / volume) to ≤30% (weight / volume) of a nonionic surfactant, preferably ≥3% (weight / volume) to ≤10% (weight / volume), and more preferably ≥3.2% (weight / volume) to ≤8% (weight / volume).

[0116] This is particularly beneficial for pyrolysis compositions and mixtures of pyrolysis and binding compositions.

[0117] According to a preferred embodiment of Method 1, the liquid-dissociating compound of the cleavage and / or binding composition is a sodium salt or a guanidine salt, preferably selected from: sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, and / or a mixture of two or more salts. The liquid-dissociating compound is preferably a guanidine salt, preferably selected from: guanidine hydrochloride, guanidine thiocyanate, and / or guanidine isothiocyanate.

[0118] Specifically, the combination of the aforementioned liquid-dissolving compound and the polyoxyethylene-based nonionic surfactant is beneficial for lysing viral cells and isolating nucleic acids from viral cells.

[0119] It is advantageous for the concentration of the ionized and / or bound compound in the composition to be from ≥0.1M to ≤10M. Preferably, the concentration of the ionized compound is from ≥1M to ≤8M, more preferably from ≥3M to ≤7M, and particularly preferably from ≥4M to ≤6M.

[0120] According to a preferred embodiment of Method 1, the cleavage and / or binding composition comprises at least one buffer compound selected from: tris(hydroxymethyl)aminomethane (TRIS), N-(tris(hydroxymethyl)methyl)glycine (TRICINE), N,N-bis(2-hydroxyethyl)glycine (BICINE), N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), and / or a phosphate buffer.

[0121] According to a particularly preferred embodiment of Method 1, the cleavage and / or binding composition comprises at least one buffer compound selected from: tris(hydroxymethyl)aminomethane (TRIS) and / or N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) and / or a phosphate buffer. According to yet another more preferred embodiment of the method, the cleavage and / or binding composition comprises at least one buffer compound selected from: tris(hydroxymethyl)aminomethane (TRIS) and / or N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES).

[0122] Biological samples can be lysed at room temperature, such as between 15°C and 25°C, or at elevated temperatures, such as from ≥37°C to ≤75°C.

[0123] In a preferred embodiment, the lysis composition may further comprise enzymes such as proteinase K, proteases (e.g., QIAGEN protease), digestive enzymes, lysozymes, colorless peptidases, lysozymes, lysozymes, and, depending on the application, nucleases such as DNases and / or RNases.

[0124] Released nucleic acids are immobilized onto a matrix based on one or more silica compounds in the presence of ionizing compounds and / or branched or unbranched alkanols.

[0125] Preferably, the combination composition comprises branched or unbranched alkanols. According to a preferred embodiment, the branched or unbranched alkanol is an alcohol having 1-5 carbon atoms, preferably selected from: methanol, ethanol, isopropanol, n-propanol, n-butanol, isobutanol, sec-butanol, tert-butanol, n-pentanol, isopentanol and / or mixtures thereof.

[0126] According to a preferred embodiment, the combined composition comprises, by volume, branched or unbranched alkanols from ≥1% to ≤80%, preferably ≥5% to ≤70%, more preferably ≥10% to ≤60%, and more preferably ≥15% to ≤50%.

[0127] According to a preferred embodiment of the invention, the mixture of the combined composition, based on the total volume of the mixture, contains a cleavage agent and optionally one or more other additives, preferably branched or unbranched alkanols, wherein the volume content of the branched or unbranched alkanols is from ≥1% to ≤80%, preferably ≥5% to ≤70%, and preferably ≥15% to ≤50%.

[0128] Nucleic acids are isolated by contacting and incubating a sample with a matrix based on one or more silica compounds, such as silica, silicates, glass, and / or silica gel, for a sufficient time to achieve binding. The matrix can be of conventional designs known in the art, such as in particulate, membrane, or filter form. Magnetic particles are preferred for ease of removal. Incubation times between 10 seconds and 30 minutes are convenient for nucleic acids. Incubation times from 1 minute to 20 minutes, specifically approximately 10 minutes, are advantageous.

[0129] Magnetic particles with a colloidal silica coating are preferably used to separate nucleic acids. More preferably, magnetic particles with a colloidal silica coating and an average particle size from ≥1 μm to ≤25 μm, more preferably from ≥5 μm to ≤15 μm, particularly preferably from ≥6 μm to ≤10 μm, and preferably with a narrow particle size distribution are used to separate nucleic acids. More preferably, magnetic particles with a colloidal silica coating and an average particle size from ≥1 μm to ≥5 μm, preferably with a narrow particle size distribution, are used to separate nucleic acids.

[0130] In yet another preferred embodiment, the magnetic or magnetically attractive particles are particles having an iron oxide-based magnetic core, preferably selected from magnetite (Fe3O4), maghemite (γ-Fe2-O3), and / or ferrite.

[0131] Magnetic silica particles that can be used in a beneficial manner can be found, for example, in international application WO 01 / 71732, the contents of which are incorporated herein by reference.

[0132] In a preferred embodiment, a matrix based on one or more silica compounds in the form of magnetic or magnetically attractive particles having a silica surface can be used.

[0133] The bonding is preferably carried out at a temperature of ≥15°C to ≤75°C, more preferably ≥20°C to ≤70°C, particularly preferably ≥46°C to ≤65°C, and most preferably ≥50°C to ≤60°C. The bonding can also be carried out at room temperature, for example, ≥15°C to ≤28°C.

[0134] Following incubation, nucleic acids bound to a matrix based on one or more silica compounds are removed from the lysis and / or binding composition. When magnetic silica particles are used, this can be achieved with the aid of a magnetic field. For example, magnetic particles can be dragged against the wall of the container in which incubation takes place by applying a magnetic field, collected at a suitable pipette tip, or attached to a magnetic rod protected by a plastic coating. Examples of suitable method steps for removing the lysis and / or binding composition include removal by aspirating or suctioning liquid, or by raising the magnetic particles at a pipette tip or magnetic rod, or by lowering the lysis and / or binding mixture, wherein the separated magnetic particles remain at the same level.

[0135] Optionally, the nucleic acids immobilized on the matrix can be washed before removal. The washing step is preferably performed by incubating a washing solution with the loaded particles, preferably involving the resuspension of the particles, for example by shaking or applying a magnetic field. The washing solution is preferably used to remove contaminating components, i.e., the cleavage and / or binding compositions remaining after binding, specifically a mixture of cleavage and / or binding compositions.

[0136] The washing agent used can be a conventional washing buffer or any other suitable medium. Generally, a washing agent with low to moderate ionic strength is preferred, such as a 10 mM solution of tris(hydroxymethyl)aminomethane (TRIS). Washing buffers with higher salt concentrations, such as 4-6 M guanidine hydrochloride solutions, can also be used. As described above, the washing agent of the present invention is similarly a suitable washing agent.

[0137] Furthermore, alcohol-containing washing agents can also be used, such as aqueous solutions of alcohols having 1-5 carbon atoms, preferably aqueous solutions of ethanol, specifically aqueous solutions of 50-100% strong ethanol.

[0138] Preferably, the nucleic acids immobilized to the matrix are washed several times, for example, 2-4 times, preferably with different washing reagents. In a preferred embodiment, washing is first performed with a washing reagent with low to medium ionic strength, followed by an aqueous ethanol solution with a strength of 70-100%.

[0139] More specifically, magnetic particles are used, which facilitate separation and / or washing steps due to their magnetic aggregation.

[0140] After the final washing step or water washing, the preferred magnetic particles can be dried, for example by vacuum drying or by evaporating the liquid or allowing the liquid to evaporate.

[0141] According to step d) of the method, the bound nucleic acids can be removed from the matrix. Removal of nucleic acids is also known as elution.

[0142] It is also preferable to use nucleic acids that are bound to a matrix (specifically magnetic particles) without the need for removal steps, for example, for PCR or other amplification methods, DNA detection methods or DNA identification methods.

[0143] The bound nucleic acids can be removed from the particles using a low-salt elution reagent. More specifically, a reagent with a salt content of less than 0.1 mol / L can be used as a low-salt elution reagent. Elution reagents containing the buffer compound tris(hydroxymethyl)aminomethane (TRIS) are particularly preferred. Softened water, optionally containing one or more additives, is also particularly suitable for elution, such as complexing agents like ethylenediaminetetraacetic acid (EDTA), azide compounds, and / or buffer compounds like tris(hydroxymethyl)aminomethane (TRIS).

[0144] Specifically, the use of lysis and / or binding compositions has produced a particularly advantageous method for isolating nucleic acids from biological samples, especially viral DNA.

[0145] Even after storage, the advantage of cleavage and / or binding reagents lies particularly in the excellent yield that can be obtained.

[0146] The present invention also relates to a kit for isolating and / or purifying nucleic acids from biological samples containing nucleic acids, the kit comprising the lysis, binding and / or washing reagents of the present invention.

[0147] In a preferred embodiment, the kit further comprises a matrix based on one or more silica compounds, specifically a matrix based on one or more silica compounds in the form of magnetic or magnetically attractive particles having a silica surface. Examples of magnetic silica particles preferably included in the kit can be found in International Application WO 01 / 71732, the entire contents of which are incorporated herein by reference.

[0148] In yet another preferred embodiment, the kit may also contain suitable washing and / or elution reagents, specifically the washing reagents of the present invention.

[0149] In another preferred embodiment, the kit may contain a silanized carrier material other than magnetic silica particles, preferably a centrifuge column with a silica membrane.

[0150] This invention also relates to the application of polyoxyethylene-based nonionic surfactants in dissolving lipids in biological samples, wherein the polyoxyethylene-based nonionic surfactants are selected from: polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers and / or polyoxyethylene-polyoxypropylene block copolymers, specifically polyoxyethylene fatty alcohol ethers selected from polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and / or polyoxyethylene oil-based ether.

[0151] For the purposes of this invention, the term "lipid" refers to a naturally occurring substance that is insoluble in water or at least largely insoluble in water. For the purposes of this invention, the term "lipid" includes triglycerides, including fats and oils, waxes, phospholipids, sphingolipids, lipid sugars, and isopentenylenes, including steroids and carotenoids. More specifically, the term "lipid" refers to lipid components or structural components of the cell membranes of organisms, such as phospholipids and sphingolipids.

[0152] In methods for isolating and / or purifying nucleic acids from biological samples containing nucleic acids, it is preferable to use a polyoxyethylene-based nonionic surfactant to dissolve the lipids in the biological sample, wherein the polyoxyethylene-based nonionic surfactant is selected from: polyoxyethylene fatty alcohol ethers, polyoxyethylene alkyl phenyl ethers, and / or polyoxyethylene-polyoxypropylene block copolymers.

[0153] For information on nonionic surfactants based on polyoxyethylene, please refer to the full contents of the instruction manual.

[0154] In methods for isolating and / or purifying nucleic acids from biological samples containing nucleic acids, polyoxyethylene fatty alcohol ethers are particularly preferred for dissolving lipids in the biological samples, wherein the polyoxyethylene fatty alcohol ethers are selected from polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and / or polyoxyethylene oil ether.

[0155] In methods for separating and / or purifying nucleic acids using a matrix based on one or more silica compounds, preferably a matrix in the form of magnetic or magnetically attractive particles with a silica surface, it is preferable to use a nonionic surfactant based on polyoxyethylene, specifically polyoxyethylene fatty alcohol ethers, to dissolve lipids in biological samples.

[0156] Advantageously, in methods for separating and / or purifying nucleic acids using a matrix based on one or more silica compounds, preferably a matrix in the form of magnetic or magnetically attractive particles having a silica surface, when using a polyoxyethylene-based nonionic surfactant, specifically a polyoxyethylene fatty alcohol ether, it has been found that the eluent is free of contaminants or contains at least significantly fewer contaminants.

[0157] Furthermore, the present invention relates to the use of polyoxyethylene-based nonionic surfactants in the preparation of storage-stable binding, cleavage, and / or detergent reagents, wherein the polyoxyethylene-based nonionic surfactants are selected from: polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, and / or polyoxyethylene-polyoxypropylene block copolymers, preferably polyoxyethylene fatty alcohol ethers, selected from polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, and / or polyoxyethylene oil-based ether.

[0158] For information on nonionic surfactants based on polyoxyethylene, please refer to the full contents of the instruction manual.

[0159] For the purposes of this invention, "storage stability" preferably refers, in relation to a specific application, to the property of a cleavage, binding, or washing agent that does not change in a manner that significantly impairs the application during a storage period of 3 months, preferably 6 months, more preferably at least 8 months. In a preferred embodiment, storage stability is exhibited at room temperature, more preferably at an elevated temperature, for example, 50°C.

[0160] pH is one of the properties of a reagent relevant to its application. Therefore, preferably, the pH of the reagent does not change significantly during storage, and preferably decreases by less than 1 during storage.

[0161] Further details, features, and advantages of the subject matter of this invention can be found in the dependent claims and the following description of the drawings and embodiments, which illustrate exemplary implementations of the invention through examples.

[0162] Figure 1a , 1b They depicted the conditions at 25℃ ( Figure 1a ) and 50℃ Figure 1b During storage for 33 weeks, the lysis reagent B (represented in blank column) of the present invention and containing Tween... pH changes of lysis reagent A (represented by solid column).

[0163] Figure 2 The lysis reagent B of the present invention and containing... are described. After preparing viral DNA with lysis reagent A at 20%, the mean CT value of HBV-specific real-time PCR was obtained for HBV-DNA.

[0164] Figure 3 The invention's lysis reagent B, after being stored at 50°C for 10 weeks, is described. The mean CT value after HBV-specific real-time PCR was performed on HBV-DNA following preparation of viral DNA with lysis reagent A at 20 μL. Lysis reagent A stored at room temperature for approximately 4 weeks was used as a reference. In each case, real-time PCR was performed using 6 μl and 24 μl of elution buffer; results with 6 μl elution buffer are presented as blank columns, and results with 24 μl elution buffer are presented as solid columns.

[0165] The present invention will now be described based on embodiments. It should be understood that the following embodiments are merely illustrative and should not be construed as limiting the invention.

[0166] Example 1: Stability Test

[0167] Freshly prepared with double-distilled water, containing 20% ​​(w / v) 20 (Fluka), guanidine isothiocyanate, tris(hydroxymethyl)aminomethane cleavage reagent A and with 20% (w / v) 58 (Sigma) instead The lysis reagent B was 20% and stored separately in sealed containers at 25°C and 50°C for 33 weeks.

[0168] At the start of storage and at weekly intervals, the pH of each solution was measured using a pH meter (Metrohm) at a temperature of 20°C–28°C.

[0169] Figure 1a The plotted bar graph shows that the pH of lysis reagent A, stored at 25°C for 33 cycles, decreased slightly from approximately pH 7.8 to pH 7.2, while... Figure 1b The results showed that the pH decreased from approximately pH 7.8 to approximately pH 5.9 during storage at 50°C for 33 weeks. In contrast, the pH of lysis reagent B remained stable at approximately pH 8 during storage at both 25°C and 50°C for 33 weeks.

[0170] Example 2: Extraction of viral DNA

[0171] The plasma of a negative, i.e., HBV-free person was mixed with 10 4 sgU / ml hepatitis B virus (HBV) mixture. Utilizing a commercially available automated platform. Qiagen Corporation has developed an automated process for purifying viral nucleic acids from plasma samples, extracting viral DNA from individual 1000 μl plasma samples.

[0172] According to the adopted protocol, the sample contains guanidine isothiocyanate, tris(hydroxymethyl)aminomethane, and 20% (w / v) as specified in the protocol. 58 (Sigma-Aldrich) and proteinase K lysis reagent B, along with a solution containing vector RNA (AVE), were contacted. The mixture was then incubated at 65°C to lyse the sample. The specified volume of guanidine isothiocyanate, tris(hydroxymethyl)aminomethane, and 9% (w / v) [a specific compound] was then added to the sample mixture. 58 (Sigma-Aldrich) and isopropanol binding reagent C. After incubation for another 3 minutes, add MagAttract suspension containing magnetic silica particles as specified in the protocol and mix. During this time, nucleic acids bind to the silica particles. Then separate the magnetic silica particles and remove the liquid phase. Then, add the specified volume of wash solution containing guanidine thiocyanate and ethanol to the silica particles, suspending the particles in the wash solution. Remove the supernatant again and add the specified volume of wash solution containing Tris, NaCl, and ethanol for a second washing step. After separation and removal of the liquid phase, wash the particles with the specified volume of aqueous 80% ethanol. After separation of the particles, remove the supernatant and air-dry the particles for 8 minutes. To elute the DNA, add the specified volume of elution solution E, suspending the particles in it for 3 minutes. Then remove the particles to obtain the eluent.

[0173] According to the described protocol, viral DNA was then extracted from a 1000 μl plasma sample, with the change being the use of a solution containing 20% ​​(w / v) viral DNA. 20 (Frucca Corporation) lysis reagent A and containing 9% (w / v) 20 (Fruka Company) binding reagent D.

[0174] Each of the resulting eluents was subjected to HBV-specific real-time (RT-) PCR, using 24 μl of eluent for each. Figure 2 As shown, the average CT value (threshold cycle number) describing the cycle at which fluorescence begins to increase logarithmically indicates that high yields were achieved using the lysis reagent B and binding reagent C of the present invention.

[0175] Example 3: Extraction of viral DNA after storage of lysis reagent

[0176] Contains guanidine isothiocyanate, tris(hydroxymethyl)aminomethane, and 20% (w / v) 58 (Sigma Corporation) lysis reagent B and 20% (w / v) 20 (Fruca Company) replaced The lysis reagent A of 58 was stored in a sealed container at 50°C for 10 weeks.

[0177] Then, use a commercially available automation platform. (Chagan Corporation) extracts viral nucleic acids using an automated process for purifying viral nucleic acids from plasma samples.

[0178] According to the solution described in Example 2, a commercially available automation platform is used. (Chagan) extracted viral DNA from 1000 μl plasma samples, for different mixtures, containing guanidine isothiocyanate, tris(hydroxymethyl)aminomethane, and 20% (w / v). 58 (Sigma Corporation) cleavage reagent B and 58 by 20% (w / v) The lysis reagent A replaced by 20 (Fluka Corporation) was stored at 50°C for 10 weeks. The reference used was lysis reagent A stored at room temperature for approximately 4 weeks.

[0179] We found that the eluent obtained using lysis reagent A, stored at 50°C, was very turbid, while the eluent obtained using lysis reagent B was clear.

[0180] In each case, HBV-specific real-time (RT-) PCR was performed on the obtained 6 μl and 24 μl eluates. Figure 3 As shown, the average CT value indicates that high yields were achieved using the lysis reagent B and binding reagent C of this invention.

Claims

1. A cleavage, binding, and / or washing agent, said agent being an aqueous solution having a pH of ≥6 and ≤9, and comprising: - At least one liquid-dissociating compound, said liquid-dissociating compound being a guanidine salt; - At least one buffering compound for buffering or stabilizing the pH of an aqueous solution; and - At least one nonionic surfactant based on polyoxyethylene, selected from polyoxyethylene nonylphenyl ether, polyoxyethylene (5) isooctylphenyl ether and polyoxyethylene (12) isooctylphenyl ether, used in an amount from ≥ 8% by weight / volume to ≤ 30% by weight / volume based on the total volume of the reagent, wherein the buffering compound is selected from: tris(hydroxymethyl)aminomethane, N-(tris(hydroxymethyl)methyl)glycine, N,N-bis(2-hydroxyethyl)glycine, 3-(N-morpholino)propanesulfonic acid, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), piperazine-1,4-bis(2-ethanesulfonic acid), N-cyclohexyl-2-aminoethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid and / or phosphate buffer.

2. The cleavage, binding, and / or washing reagent as described in claim 1, characterized in that, The polyoxyethylene nonylphenyl ether is selected from: polyoxyethylene (2) nonylphenyl ether, polyoxyethylene (5) nonylphenyl ether, polyoxyethylene (9) nonylphenyl ether, polyoxyethylene (12) nonylphenyl ether and polyoxyethylene (100) nonylphenyl ether (Igepal® CO-990).

3. The cleavage, binding, and / or washing agent as described in claim 1, characterized in that, The polyoxyethylene nonylphenyl ether is selected from: polyoxyethylene (5) nonylphenyl ether, polyoxyethylene (9) nonylphenyl ether and polyoxyethylene (12) nonylphenyl ether.

4. The cleavage, binding, and / or washing agent as described in claim 1, characterized in that, The cleavage, binding, and / or washing reagents comprise nonionic surfactants, used in amounts ranging from ≥ 9% by weight / volume to ≤ 30% by weight / volume, based on the total volume of the reagents.

5. The cleavage, binding, and / or washing agent of claim 4, wherein the nonionic surfactant is used in an amount from ≥ 10% by weight / volume to ≤ 30% by weight / volume, based on the total volume of the agent.

6. The cleavage, binding and / or washing agent of claim 4, wherein the nonionic surfactant is used in an amount from ≥ 15% by weight / volume to ≤ 20% by weight / volume based on the total volume of the agent.

7. The cleavage, binding, and / or washing agent as described in claim 1, characterized in that, The ionized compound is selected from: guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate and / or a mixture of two or more of these salts.

8. The cleavage, binding, and / or washing agent as described in claim 1, characterized in that, The binding agent comprises branched or unbranched alkanols.

9. The cleavage, binding, and / or washing agent as described in claim 8, characterized in that, The branched or unbranched alkanols are branched or unbranched alcohols having 1-5 carbon atoms.

10. The cleavage, binding, and / or washing agent as described in claim 8, characterized in that, The branched or unbranched alkanols are selected from: methanol, ethanol, isopropanol, n-propanol, n-butanol, branched or unbranched butanol or pentanol and / or mixtures thereof.

11. The use of the lysis, binding and / or washing reagents as described in any one of claims 1-10 in the isolation and / or purification of nucleic acids.

12. A method for isolating and / or purifying nucleic acids from a biological sample containing nucleic acids, the method comprising the following steps: a) Lysing biological samples, b) Immobilizing released nucleic acids onto a matrix based on one or more silica compounds, wherein the liquid-dissolving compound is a guanidine salt, in the presence of a liquid-dissolving compound and / or branched or unbranched alkanols. c) Optionally wash the nucleic acids immobilized on the matrix. d) Optional removal of bound nucleic acids, The cleavage and / or fixation are carried out in the presence of a cleavage and / or binding composition comprising the following components: - The cleavage and / or binding agent as claimed in claim 1, and wherein the cleavage and / or binding composition comprises at least one polyoxyethylene-based nonionic surfactant selected from polyoxyethylene nonylphenyl ether, polyoxyethylene (5) isooctylphenyl ether and polyoxyethylene (12) isooctylphenyl ether, in an amount from ≥ 8% by weight / volume to ≤ 30% by weight / volume based on the total volume of the composition.

13. The method as described in claim 12, characterized in that, The polyoxyethylene nonylphenyl ether is selected from: polyoxyethylene (2) nonylphenyl ether, polyoxyethylene (5) nonylphenyl ether, polyoxyethylene (9) nonylphenyl ether, polyoxyethylene (12) nonylphenyl ether and polyoxyethylene (100) nonylphenyl ether.

14. The method as described in claim 13, characterized in that, The polyoxyethylene nonylphenyl ether is selected from: polyoxyethylene (5) nonylphenyl ether, polyoxyethylene (9) nonylphenyl ether and polyoxyethylene (12) nonylphenyl ether.

15. The method as described in claim 12, characterized in that, The nonionic surfactant is used in an amount ranging from ≥ 8% by weight / volume to ≤ 10% by weight / volume, based on the total volume of the composition.

16. A kit for isolating and / or purifying nucleic acids from biological samples containing nucleic acids, the kit comprising lysis, binding, and / or washing reagents as described in any one of claims 1-10.

17. The use of a polyoxyethylene-based nonionic surfactant in the preparation of a storage-stable binding, cleavage, and / or washing agent as described in any one of claims 1-10, wherein the polyoxyethylene-based nonionic surfactant is selected from: polyoxyethylene nonylphenyl ether, polyoxyethylene (5) isooctylphenyl ether, and polyoxyethylene (12) isooctylphenyl ether.

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