Reagent combination and kit for extracting nucleic acid by paramagnetic particle method and application of reagent combination and kit

Abstract

The invention belongs to the technical field of biology, and particularly relates to a reagent combination and a kit for extracting nucleic acid through a paramagnetic particle method and application of the reagent combination and the kit. The reagent combination or the kit does not adopt beta-mercaptoethanol with pungent smell or dithiothreitol needing to be stored at low temperature as an antioxidant, but adopts tris (2-formylethyl) phosphine hydrochloride and / or metabisulfite as an antioxidant, belongs to common salt chemical reagent powder, can be stored at normal temperature, and can be used for preparing a reagent for the first time. Manual addition by experimenters before an experiment is not needed, so that nucleic acid (RNA) released by splitting decomposition can be effectively prevented from being oxidized and denatured, and the integrity of extracted nucleic acid (total RNA) fragments is ensured; especially, the metabisulfite is commonly used in food additives, does not cause harm to the body, and is non-toxic and non-volatile.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a reagent combination and a kit for extracting nucleic acid using a magnetic bead method and applications thereof. Background Art

[0002] Nucleic acid is an important material basis of biological inheritance and can be divided into two categories: deoxyribonucleotides (DNA) and ribonucleotides (RNA). RNA ranked first in the top 10 scientific achievements selected by Science in 2002. In all RNA experiments, the most critical step is to isolate high-purity and complete total RNA. However, due to the widespread existence of RNase and its difficulty in inactivation, RNA extraction has various problems such as long time consumption, low purity, low yield and easy extraction failure. So far, the work involving RNA extraction is still the most difficult problem to overcome in molecular biology research.

[0003] Traditional RNA extraction reagents often use volatile toxic reagents or chemical reagents that need to be stored at low temperatures. These defects not only affect the health of operators, but also have a certain impact on the extraction effect of the reagents, thus greatly limiting the applicability and stability of the extraction reagents for multiple types of samples. For example, the mainstream QIAGEN extraction reagent on the market requires the addition of β-mercaptoethanol, an antioxidant with a pungent odor, before use, and the same set of reagents is not compatible with multiple types of samples such as cells, bacteria, whole blood, and tissues.

[0004] At present, the main types of total RNA extraction reagents include phenol / chloroform extraction method, alcohol-free method, alcohol precipitation method, etc. The phenol / chloroform extraction method is specifically: adding phenol or chloroform to the lysate or binding solution to precipitate and protect the RNA, so as to obtain high-purity total RNA. The phenol or chloroform used in the phenol / chloroform extraction reagent is usually harmful to the human body and contains a pungent odor. At the same time, if there is phenol or chloroform residue in the extraction product, the purity of the product will be reduced, which will have a certain impact on the downstream qPCR or library sequencing; the alcohol-free method is specifically: using high-concentration polyethylene glycol instead of isopropanol or ethanol to capture the water in the hydration layer outside the RNA molecule, causing the hydration layer to be destroyed and the RNA molecules to aggregate and precipitate. The alcohol-free extraction reagent requires the use of high-concentration polyethylene glycol. The extraction reagent also contains a high concentration of salts. After being stored at room temperature or low temperature, it may cause the components to precipitate and cannot be dissolved back, which will have an irreversible effect on the effect of the extraction reagent, and the alcohol-free extraction reagent cannot meet the requirements of being applicable to multiple types of samples; the alcohol precipitation method is specifically: using a certain amount of anhydrous ethanol, combined with a high concentration of guanidine salt, to cause the released water-soluble RNA molecules to aggregate and precipitate. At present, most of the alcohol precipitation extraction reagents on the market require the addition of an antioxidant component to protect RNA molecules before extraction, but most of them choose β-mercaptoethanol with a pungent odor or dithiothreitol that needs to be stored at low temperature, which has unknown risks to the performance of the reagent. Therefore, it is very competitive to develop a nucleic acid extraction reagent combination with high total RNA yield, high purity, complete fragments, applicable to multiple types of samples, non-toxic and can be stored at room temperature. Summary of the invention

[0005] The first aspect of the present invention aims to provide a reagent combination or a kit.

[0006] The second aspect of the present invention aims to provide a method for extracting nucleic acid.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a reagent combination or kit, comprising: a lysing solution and a washing solution;

[0009] The lysate comprises: guanidine salt, sodium salt, nonionic surfactant, alcohol, and antioxidant;

[0010] The washing liquid comprises: washing liquid I;

[0011] The washing solution I comprises: sodium salt, nonionic surfactant, alcohol, and antioxidant;

[0012] The antioxidants in the lysate and the washing solution I are independently selected from at least one of tri(2-formylethyl)phosphine hydrochloride and metabisulfite.

[0013] Preferably, the antioxidant in the lysate comprises metabisulfite.

[0014] Preferably, the antioxidant in the washing solution I comprises: metabisulfite.

[0015] Preferably, the metabisulfite comprises at least one of sodium metabisulfite, potassium metabisulfite and magnesium metabisulfite.

[0016] Preferably, the concentration of the antioxidant in the lysate is 1.5-3 mg / mL.

[0017] Preferably, the concentration of the antioxidant in the washing solution I is 1.5-4 mg / mL.

[0018] Preferably, the guanidine salt comprises at least one of guanidine hydrochloride, guanidine nitrate, guanidine carbonate, guanidine acetate, guanidine isothiocyanate and guanidine thiocyanate.

[0019] Preferably, the concentration of the guanidine salt in the lysate is 250-325 mg / mL.

[0020] Preferably, the sodium salt in the lysing solution and the washing solution I is independently selected from at least one of sodium chloride, sodium acetate, sodium sulfate and sodium citrate.

[0021] Preferably, the sodium salt in the lysate comprises: sodium acetate.

[0022] Preferably, the sodium salt in the washing solution I comprises: sodium chloride and sodium acetate.

[0023] Preferably, the concentration of the sodium salt in the lysate is 12.5-24 mg / mL.

[0024] Preferably, the concentration of the sodium salt in the washing solution I is 45-65 mg / mL.

[0025] Preferably, the nonionic surfactants in the lysing solution and the washing solution I are independently selected from at least one of Triton 100, Tween 20, Tween 80, ethylphenyl polyethylene glycol, and polyether alcohol.

[0026] Preferably, the nonionic surfactant in the lysate comprises: Tween 20.

[0027] Preferably, the nonionic surfactant in the washing solution I comprises: Tween 20.

[0028] Preferably, the concentration of the nonionic surfactant in the lysate is 2-3.25 v / v%.

[0029] Preferably, the concentration of the nonionic surfactant in the washing liquid I is 2-4 v / v%.

[0030] Preferably, the alcohols in the lysing solution and the washing solution I are independently selected from at least one of ethanol and isopropanol.

[0031] Preferably, the alcohol in the lysate comprises: ethanol.

[0032] Preferably, the alcohol in the washing solution I comprises: ethanol.

[0033] Preferably, the concentration of the alcohol in the lysate is 40-55 v / v%.

[0034] Preferably, the concentration of the alcohol in the washing liquid I is 30-45 v / v%.

[0035] Preferably, the lysate further comprises: a cationic surfactant.

[0036] Preferably, the cationic surfactant comprises: at least one of dodecyldimethylbenzylammonium chloride, hexadecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, dodecyldimethylbenzylammonium bromide, hexadecyltrimethylammonium bromide and octadecyltrimethylammonium bromide.

[0037] Preferably, the concentration of the cationic surfactant in the lysate is 0.8-2.2 mg / mL.

[0038] Preferably, the lysate further comprises: a chelating agent.

[0039] Preferably, the chelating agent comprises: at least one of ethylenediaminetetraacetic acid and a water-soluble salt of ethylenediaminetetraacetic acid.

[0040] Preferably, the concentration of the chelating agent in the lysate is 1.5-3 mg / mL.

[0041] Preferably, the lysate further comprises: a buffer salt.

[0042] Preferably, the buffer salt comprises at least one of phosphate, borate, citrate and tris(hydroxymethyl)aminomethane.

[0043] Preferably, the concentration of the buffer salt in the lysate is 0.5-1.75 mg / mL.

[0044] Preferably, the washing liquid further comprises: washing liquid II.

[0045] Preferably, the washing liquid II is alcohol; further, 65-90 v / v% alcohol.

[0046] Preferably, the alcohol comprises at least one of ethanol and isopropanol.

[0047] Preferably, the reagent combination or kit further comprises: an eluent.

[0048] Preferably, the eluent comprises at least one of sterile water, enzyme-free water, NF water, and DEPC water.

[0049] Preferably, the reagent combination or kit further comprises: magnetic beads.

[0050] Preferably, the magnetic beads comprise nanomagnetic beads.

[0051] Preferably, the magnetic beads include at least one of hydroxyl magnetic beads, carboxyl magnetic beads, glass magnetic beads, amino magnetic beads, tosyl magnetic beads, and epoxy magnetic beads.

[0052] Preferably, the reagent combination or kit further comprises: proteinase K.

[0053] Preferably, the reagent combination or kit further comprises: DNA enzyme.

[0054] Preferably, the DNase comprises DNase I.

[0055] Preferably, the reagent combination or kit is used for extracting nucleic acids using a magnetic bead method.

[0056] Preferably, the nucleic acid comprises at least one of DNA and RNA.

[0057] Preferably, the nucleic acid is from any one or more of the following biological samples: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, and nasal swabs.

[0058] Preferably, the body fluid comprises at least one of tissue fluid, lymph fluid, blood and cerebrospinal fluid.

[0059] Preferably, the microorganism comprises at least one of bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, and spirochetes.

[0060] Preferably, the nucleic acid is used for PCR, enzyme digestion, molecular hybridization, library construction or Southern hybridization.

[0061] The second aspect of the present invention provides a method for extracting nucleic acid, comprising the steps of using the reagent combination or kit of the first aspect of the present invention.

[0062] Preferably, the method is A1) or A2):

[0063] When the nucleic acid does not contain DNA, the method is A1), which comprises the following steps:

[0064] a1) mixing the sample, magnetic beads, proteinase K, and the lysate of the first aspect of the present invention, and separating the magnetic beads;

[0065] a2) mixing the magnetic beads with the washing solution I in the first aspect of the present invention to separate the magnetic beads;

[0066] a3) mixing the magnetic beads with DNA enzyme, and mixing the obtained mixture with the washing solution I in the first aspect of the present invention to separate the magnetic beads;

[0067] a4) mixing the magnetic beads with washing solution II to separate the magnetic beads;

[0068] a5) mixing the magnetic beads with the eluent, separating the magnetic beads, and obtaining the nucleic acid;

[0069] When the nucleic acid comprises DNA, the method is A2), which comprises the following steps:

[0070] b1) mixing the sample, magnetic beads, proteinase K, and the lysate of the first aspect of the present invention, and separating the magnetic beads;

[0071] b2) mixing the magnetic beads with the washing solution I in the first aspect of the present invention to separate the magnetic beads;

[0072] b3) mixing the magnetic beads with washing solution II to separate the magnetic beads;

[0073] b4) mixing the magnetic beads with the elution solution, separating the magnetic beads, and obtaining the nucleic acid.

[0074] Preferably, the magnetic beads, proteinase K, DNA enzyme, washing solution II and / or elution solution are the magnetic beads, proteinase K, DNA enzyme, washing solution II and / or elution solution in the first aspect of the present invention.

[0075] The beneficial effects of the present invention are:

[0076] The invention provides a reagent combination or a kit. The reagent combination or the kit does not use beta-mercaptoethanol with a pungent odor or dithiothreitol that needs to be stored at a low temperature as an antioxidant, but uses tri(2-formylethyl)phosphine hydrochloride and / or metabisulfite as an antioxidant. The tri(2-formylethyl)phosphine hydrochloride and / or metabisulfite are common salt chemical reagent powders, can be stored at room temperature, do not need to be manually added by experimenters before an experiment, can effectively prevent nucleic acids (RNA) released by cleavage from being oxidized and denatured, and ensure the integrity of the extracted nucleic acid (total RNA) fragments. In particular, metabisulfite is commonly used in food additives, does not cause harm to the body, is non-toxic, and is non-volatile.

[0077] The reagent combination or kit provided by the present invention can fully destroy the cell membrane of samples such as cells, tissues, and bacteria, release nucleic acids, and at the same time destroy the three-dimensional structure of proteins, converting the proteins into a random curled state to denature them, and inhibiting the activity of nucleases to prevent nucleic acids (RNA) from being degraded. At the same time, nucleic acids (RNA) of different sizes can be aggregated together, thereby increasing the yield.

[0078] The reagent combination or kit provided by the present invention can fully remove impurities such as proteins, lipids, and inorganic salts, and can effectively reduce the loss of nucleic acids (RNA) during the washing process. The recovery rate is verified by recovery experiments to be more than 90%, and its purity meets the requirements of downstream qPCR, library construction and sequencing, etc.

[0079] The reagent combination or kit provided by the present invention can simultaneously meet the extraction requirements of different types of samples such as cells, tissues, whole blood, bacteria, etc., and its extraction effect is not inferior to mainstream kits such as QIAGEN, Tiangen, OMEGA, etc. on the market.

[0080] The reagent combination or kit provided by the present invention has high yield, high purity, complete fragments, is applicable to multiple types of samples, is non-toxic and can be stored at room temperature. By using magnetic beads, in a lysis solution (the guanidine salt in the lysis solution is combined with sodium salt and a non-ionic surfactant to lyse and precipitate the sample in an alcohol system, and the nucleic acid (total RNA) with complete fragments is extracted under the action of an antioxidant), the nucleic acid (total RNA) in samples such as blood, tissue, bacteria, and cells is adsorbed, and then a washing solution I (the sodium salt in the washing solution I is combined with an antioxidant and a non-ionic surfactant to remove protein and lipids from the extracted nucleic acid (RNA) in an alcohol system) is used for washing to remove impurities such as proteins and lipids, and then a washing solution II is used to remove impurities of inorganic salts, and finally an elution solution is used for elution. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 It is a result diagram of detecting the integrity of the extracted products of Example 4 and Comparative Examples 1 and 5 by using agarose gel electrophoresis.

[0082] Figure 2 It is a result diagram of detecting the integrity of the extracted products of Example 5 and Comparative Examples 2 and 6 by using agarose gel electrophoresis.

[0083] Figure 3 It is a result diagram of detecting the integrity of the extracted products of Example 7 and Comparative Examples 4 and 8 by using agarose gel electrophoresis. DETAILED DESCRIPTION

[0084] The first aspect of the present invention provides a reagent combination or kit for extracting nucleic acid using a magnetic bead method, comprising: a lysis solution and a washing solution;

[0085] The lysate comprises: guanidine salt, sodium salt, nonionic surfactant, alcohol, and antioxidant;

[0086] The washing liquid comprises: washing liquid I;

[0087] The washing solution I comprises: sodium salt, nonionic surfactant, alcohol, and antioxidant;

[0088] The antioxidants in the lysate and the washing solution I are independently selected from at least one of tri(2-formylethyl)phosphine hydrochloride and metabisulfite.

[0089] Preferably, the antioxidant in the lysate comprises metabisulfite.

[0090] Preferably, the antioxidant in the washing solution I comprises: metabisulfite.

[0091] Preferably, the metabisulfite comprises: at least one of sodium metabisulfite, potassium metabisulfite, and magnesium metabisulfite; and further comprises: sodium metabisulfite.

[0092] Preferably, the concentration of the antioxidant in the lysate is 1.5 to 3 mg / mL; further 2 to 2.5 mg / mL.

[0093] Preferably, the concentration of the antioxidant in the washing solution I is 1.5 to 4 mg / mL; further 1.8 to 3.4 mg / mL.

[0094] Preferably, the guanidine salt comprises: at least one of guanidine hydrochloride, guanidine nitrate, guanidine carbonate, guanidine acetate, guanidine isothiocyanate, and guanidine thiocyanate; further comprises: at least one of guanidine hydrochloride and guanidine isothiocyanate; further comprises: guanidine isothiocyanate.

[0095] Preferably, the concentration of the guanidine salt in the lysate is 250-325 mg / mL; further 275-300 mg / mL.

[0096] Preferably, the sodium salt in the lysate and the washing solution I is independently selected from at least one of sodium chloride, sodium acetate, sodium sulfate and sodium citrate; further preferably, the sodium salt in the lysate and the washing solution I is independently selected from at least one of sodium chloride, sodium acetate and sodium citrate; further preferably, the sodium salt in the lysate and the washing solution I is independently selected from at least one of sodium chloride and sodium acetate.

[0097] Preferably, the sodium salt in the lysate comprises: sodium acetate.

[0098] Preferably, the sodium salt in the washing solution I comprises: sodium chloride and sodium acetate.

[0099] Preferably, the sodium acetate is anhydrous sodium acetate.

[0100] Preferably, the mass ratio of sodium chloride to sodium acetate in the sodium salt in the washing solution I is (25-45):20.

[0101] Preferably, the concentration of the sodium salt in the lysate is 12.5 to 24 mg / mL; further 15 to 22.5 mg / mL.

[0102] Preferably, the concentration of the sodium salt in the washing solution I is 45 to 65 mg / mL; further 50 to 60 mg / mL.

[0103] Preferably, the non-ionic surfactants in the lysate and the washing solution I are each independently selected from: at least one of Triton 100, Tween 20, Tween 80, ethylphenyl polyethylene glycol, and polyether alcohol; further preferably, the non-ionic surfactants in the lysate and the washing solution I are each independently selected from: at least one of Triton 100, Tween 20, ethylphenyl polyethylene glycol, and polyether alcohol.

[0104] Preferably, the nonionic surfactant in the lysate comprises: Tween 20.

[0105] Preferably, the nonionic surfactant in the washing solution I comprises: Tween 20.

[0106] Preferably, the concentration of the nonionic surfactant in the lysate is 2 to 3.25 v / v%; further 2.25 to 3 v / v%.

[0107] Preferably, the concentration of the nonionic surfactant in the washing liquid I is 2 to 4 v / v%; further 2.5 to 3.5 v / v%.

[0108] Preferably, the alcohols in the lysing solution and the washing solution I are independently selected from at least one of ethanol and isopropanol.

[0109] Preferably, the alcohols are anhydrous alcohols.

[0110] Preferably, the alcohols in the lysate include: ethanol; and further include: anhydrous ethanol.

[0111] Preferably, the alcohols in the washing solution I include: ethanol; and further include: anhydrous ethanol.

[0112] Preferably, the concentration of the alcohol in the lysate is 40 to 55 v / v%; further 45 to 50 v / v%.

[0113] Preferably, the concentration of the alcohol in the washing liquid I is 30 to 45 v / v%; further 35 to 44 v / v%.

[0114] Preferably, the lysate further comprises: a cationic surfactant.

[0115] Preferably, the cationic surfactant comprises: at least one of dodecyldimethylbenzylammonium chloride, hexadecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, dodecyldimethylbenzylammonium bromide, hexadecyltrimethylammonium bromide, and octadecyltrimethylammonium bromide; and further comprises: hexadecyltrimethylammonium bromide.

[0116] Preferably, the concentration of the cationic surfactant in the lysate is 0.8 to 2.2 mg / mL; further preferably 1 to 2 mg / mL.

[0117] Preferably, the lysate further comprises: a chelating agent.

[0118] Preferably, the chelating agent comprises: at least one of ethylenediaminetetraacetic acid and a water-soluble salt of ethylenediaminetetraacetic acid; further comprises: at least one of ethylenediaminetetraacetic acid and disodium ethylenediaminetetraacetate; further comprises: disodium ethylenediaminetetraacetate.

[0119] Preferably, the concentration of the chelating agent in the lysate is 1.5 to 3 mg / mL; further preferably 1.65 to 2.5 mg / mL.

[0120] Preferably, the lysate further comprises: a buffer salt.

[0121] Preferably, the buffer salt comprises: at least one of phosphate, borate, citrate and tris(hydroxymethyl)aminomethane; and further comprises: tris(hydroxymethyl)aminomethane.

[0122] Preferably, the concentration of the buffer salt in the lysate is 0.5 to 1.75 mg / mL; further 0.75 to 1.5 mg / mL.

[0123] Preferably, the washing liquid further comprises: washing liquid II.

[0124] Preferably, the washing liquid II is alcohol; further, 65-90 v / v% alcohol.

[0125] Preferably, the alcohol comprises at least one of ethanol and isopropanol; further comprises ethanol.

[0126] Preferably, the reagent combination or kit further comprises: an eluent.

[0127] Preferably, the eluent comprises: at least one of sterile water, enzyme-free water, NF water, and DEPC water; and further comprises: DEPC water.

[0128] Preferably, the reagent combination or kit further comprises: magnetic beads.

[0129] Preferably, the magnetic beads comprise nanomagnetic beads.

[0130] Preferably, the magnetic beads comprise: at least one of hydroxyl magnetic beads, carboxyl magnetic beads, glass magnetic beads, amino magnetic beads, tosyl magnetic beads, and epoxy magnetic beads; further comprise: at least one of hydroxyl magnetic beads and carboxyl magnetic beads; further comprise: carboxyl magnetic beads.

[0131] Preferably, the magnetic beads are a magnetic bead suspension; further, a magnetic bead suspension with a solid content of 20 to 30 mg / mL.

[0132] Preferably, the reagent combination or kit further comprises: proteinase K.

[0133] Preferably, the proteinase K is a proteinase K solution.

[0134] Preferably, the proteinase K solution comprises: proteinase K.

[0135] Preferably, the proteinase K solution further comprises: at least one of glycerol, buffer salt, and calcium chloride; and further comprises: glycerol, buffer salt, and calcium chloride.

[0136] Preferably, the buffer salt comprises: at least one of phosphate, borate, citrate and tris(hydroxymethyl)aminomethane hydrochloride; and further comprises: tris(hydroxymethyl)aminomethane hydrochloride.

[0137] Preferably, the calcium chloride comprises: calcium chloride dihydrate.

[0138] Preferably, the concentration of the proteinase K in the proteinase K solution is 30 to 55 mg / mL; further 32 to 50 mg / mL.

[0139] Preferably, the concentration of the glycerol in the proteinase K solution is 35 to 50 v / v%; further 40 to 45 v / v%.

[0140] Preferably, the concentration of the buffer salt in the proteinase K solution is 0.04-0.10M; further 0.05-0.09M.

[0141] Preferably, the concentration of calcium chloride in the proteinase K solution is 0.4-0.8 mg / mL; further 0.5-0.7 mg / mL.

[0142] Preferably, the reagent combination or kit further comprises: DNA enzyme.

[0143] Preferably, the DNA enzyme comprises DNA enzyme I; and further comprises a DNA enzyme I solution.

[0144] Preferably, the DNase I solution comprises: DNase I.

[0145] Preferably, the DNase I solution also contains: at least one of calcium chloride, magnesium chloride, and buffer salt; and further contains: calcium chloride, magnesium chloride, and buffer salt.

[0146] Preferably, the buffer salt comprises: at least one of phosphate, borate, citrate and tris(hydroxymethyl)aminomethane hydrochloride; and further comprises: tris(hydroxymethyl)aminomethane hydrochloride.

[0147] Preferably, the concentration of the DNase I in the DNase I solution is 0.4 to 0.7 mg / mL; further 0.5 to 0.6 mg / mL.

[0148] Preferably, the concentration of calcium chloride in the DNase I solution is 0.6 to 1.5 mg / mL; further 0.8 to 1.4 mg / mL.

[0149] Preferably, the concentration of magnesium chloride in the DNase I solution is 3.5 to 5.5 mg / mL; further 4 to 5 mg / mL.

[0150] Preferably, the concentration of the buffer salt in the DNase I solution is 0.005-0.025M; further 0.01-0.02M.

[0151] Preferably, the lysate, washing solution, magnetic bead suspension, proteinase K solution and DNase I solution further contain a solvent.

[0152] Preferably, the solvent comprises: at least one of sterile water, enzyme-free water, NF water, and DEPC water; and further comprises: DEPC water.

[0153] Preferably, the amount of the solvent used is the balance.

[0154] Preferably, the nucleic acid comprises: at least one of DNA and RNA; further RNA; further total RNA.

[0155] Preferably, when the nucleic acid comprises DNA, the reagent combination or kit does not comprise DNase.

[0156] Preferably, when the nucleic acid does not comprise DNA, the reagent combination or kit comprises DNA enzyme.

[0157] Preferably, the nucleic acid is from any one or more of the following biological samples: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, and nasal swabs.

[0158] Preferably, the body fluid comprises at least one of tissue fluid, lymph fluid, blood and cerebrospinal fluid.

[0159] Preferably, the microorganism comprises at least one of bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, and spirochetes.

[0160] Preferably, the nucleic acid is used for PCR, enzyme digestion, molecular hybridization, library construction or Southern hybridization.

[0161] The second aspect of the present invention provides a system comprising a nucleic acid extractor and the reagent combination or kit of the first aspect of the present invention.

[0162] The third aspect of the present invention provides use of the reagent combination or kit of the first aspect of the present invention and / or the system of the second aspect of the present invention in extracting nucleic acids.

[0163] Preferably, the nucleic acid comprises: at least one of DNA and RNA; further RNA; further total RNA.

[0164] Preferably, the nucleic acid is from any one or more of the following biological samples: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, and nasal swabs.

[0165] Preferably, the body fluid comprises at least one of tissue fluid, lymph fluid, blood and cerebrospinal fluid.

[0166] Preferably, the microorganism comprises at least one of bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, and spirochetes.

[0167] Preferably, the nucleic acid is used for PCR, enzyme digestion, molecular hybridization, library construction or Southern hybridization.

[0168] The fourth aspect of the present invention provides a method for extracting nucleic acid, comprising the steps of using the reagent combination or kit of the first aspect of the present invention and / or the system of the second aspect of the present invention.

[0169] Preferably, the method is A1) or A2):

[0170] When the nucleic acid does not contain DNA, the method is A1), which comprises the following steps:

[0171] a1) mixing the sample, magnetic beads, proteinase K, and the lysate of the first aspect of the present invention, and separating the magnetic beads;

[0172] a2) mixing the magnetic beads obtained in step a1) with the washing solution I in the first aspect of the present invention to separate the magnetic beads;

[0173] a3) mixing the magnetic beads obtained in step a2) with DNA enzyme, and mixing the obtained mixture with the washing solution I in the first aspect of the present invention to separate the magnetic beads;

[0174] a4) mixing the magnetic beads obtained in step a3) with washing solution II to separate the magnetic beads;

[0175] a5) mixing the magnetic beads obtained in step a4) with an eluent, separating the magnetic beads, and obtaining the nucleic acid;

[0176] When the nucleic acid comprises DNA, the method is A2), which comprises the following steps:

[0177] b1) mixing the sample, magnetic beads, proteinase K, and the lysate of the first aspect of the present invention, and separating the magnetic beads;

[0178] b2) mixing the magnetic beads obtained in step b1) with the washing solution I in the first aspect of the present invention to separate the magnetic beads;

[0179] b3) mixing the magnetic beads obtained in step b2) with washing solution II to separate the magnetic beads;

[0180] b4) mixing the magnetic beads obtained in step b3) with an elution solution, separating the magnetic beads, and obtaining the nucleic acid.

[0181] Preferably, the magnetic beads, proteinase K, DNA enzyme, washing solution II and / or elution solution are the magnetic beads, proteinase K, DNA enzyme, washing solution II and / or elution solution in the first aspect of the present invention.

[0182] Preferably, the sample comprises at least one of cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, and nasal swabs.

[0183] Preferably, the body fluid comprises at least one of tissue fluid, lymph fluid, blood and cerebrospinal fluid.

[0184] Preferably, the microorganism comprises at least one of bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, and spirochetes.

[0185] Preferably, the nucleic acid is used for PCR, enzyme digestion, molecular hybridization, library construction or Southern hybridization.

[0186] Preferably, the mixing in a1) and b1) is vortex mixing; further, the mixing is vortex mixing for 0.5 to 1.5 minutes, and then allowed to stand for 8 to 12 minutes, during which time the mixture is vortexed for 5 to 15 seconds every 4 to 6 minutes.

[0187] Preferably, the separation of magnetic beads in a1), a2), a3), a4), a5), b1), b2), b3), and b4) is carried out on a magnetic stand.

[0188] Preferably, the ratio of the sample to the magnetic beads in a1) and b1) is calculated as (1x10 4 ~1x10 6 ): 60 μL (for example, when the sample is cells); or

[0189] The ratio of the sample to the magnetic beads in a1) and b1) is (2-8) mg: 60 μL by mass-to-volume ratio (for example, when the sample is tissue); or

[0190] The volume ratio of the sample to the magnetic beads in a1) and b1) is (100-300):60 (for example, when the sample is blood); or

[0191] The ratio of sample to magnetic beads in a1) and b1) is calculated as (2x10 6 ~2x10 8 ): 60μL (for example, when the sample is bacteria).

[0192] Preferably, the volume ratio of the magnetic beads, proteinase K and lysis solution in a1) and b1) is 60:(10-30):(500-700).

[0193] Preferably, the mixing in a2) and b2) is vortex mixing; further, the vortex mixing is performed for 0.5 to 1.5 minutes.

[0194] Preferably, the amount (volume) of the washing solution I in a2) and b2) is 8 to 12 times that of the magnetic beads in a1) and b1).

[0195] Preferably, a drying step is further included between a2) and a3); the drying step is further performed for 8 to 12 minutes.

[0196] Preferably, the magnetic beads and DNA enzyme are mixed in a3) by vortex mixing; further, the mixture is vortex mixed for 0.5 to 1.5 min, allowed to stand for 8 to 12 min, and vortexed for 5 to 15 s every 2 to 4 min.

[0197] Preferably, the mixture obtained in a3) is mixed with the washing solution I by vortex mixing; further, the vortex mixing is performed for 0.5 to 1.5 min.

[0198] Preferably, the amount (volume) of the DNA enzyme in a3) is 1 to 2 times that of the magnetic beads in a1).

[0199] Preferably, the amount (volume) of the washing solution I in a3) is 8 to 14 times that of the magnetic beads in a1).

[0200] Preferably, the mixing in a4) is vortex mixing; further, the vortex mixing is performed for 0.5 to 1.5 minutes.

[0201] Preferably, a4) is repeated 1 to 3 times.

[0202] Preferably, the amount (volume) of the washing solution II in a4) is 8 to 14 times that of the magnetic beads in a1).

[0203] Preferably, the step of drying is included before a5); further drying is performed for 8 to 12 minutes.

[0204] Preferably, the mixing in a5) is vortex mixing; further, the vortex mixing is performed for 0.5 to 1.5 minutes.

[0205] Preferably, the amount (volume) of the eluent in a5) is 1 to 2 times that of the magnetic beads in a1).

[0206] Preferably, the mixing in a5) further comprises standing still; further standing still for 8 to 12 minutes.

[0207] Preferably, the mixing in b3) is vortex mixing; further, the vortex mixing is performed for 0.5 to 1.5 minutes.

[0208] Preferably, b3) is repeated 1 to 3 times.

[0209] Preferably, the amount (volume) of the washing solution II in b3) is 8 to 14 times that of the magnetic beads in b1).

[0210] Preferably, the step of drying is included before b4); further, the drying time is 8 to 12 minutes.

[0211] Preferably, the mixing in b4) is vortex mixing; further, the vortex mixing is performed for 0.5 to 1.5 minutes.

[0212] Preferably, the amount (volume) of the eluent in b4) is 1 to 2 times that of the magnetic beads in b1).

[0213] Preferably, the mixing in b4) further comprises standing still; further standing still for 8 to 12 minutes.

[0214] The present invention is further described in detail below through specific examples.

[0215] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0216] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0217] The human colon cancer cells used in this example were HCT116 cell lines purchased from Bio-Tech, mouse liver tissues were from experimental BALB / c mice, EDTA anticoagulated SD rat blood was purchased from Nanjing Senbega Biotechnology Co., Ltd., and Escherichia coli was purchased from Ningbo Mingzhou Biotechnology.

[0218] Example 1 A reagent combination for extracting nucleic acid (total RNA) by magnetic bead method

[0219] A reagent combination for extracting nucleic acid (total RNA) by magnetic bead method, comprising: a lysis solution, a washing solution, an eluent, a magnetic bead suspension, a proteinase K solution and a DNase I solution;

[0220] The lysate contains the following components: 50 v / v% anhydrous ethanol, 1.75 mg / mL disodium ethylenediaminetetraacetate, 300 mg / mL guanidine isothiocyanate, 0.75 mg / mL tris(hydroxymethyl)aminomethane, 2.5 mg / mL sodium metabisulfite, 15 mg / mL anhydrous sodium acetate, 1.5 mg / mL hexadecyltrimethylammonium bromide and 2.5 v / v% Tween20;

[0221] The washing solution comprises washing solution I and washing solution II;

[0222] Washing solution I contains the following components: 40 v / v% anhydrous ethanol, 36 mg / mL sodium chloride, 20 mg / mL anhydrous sodium acetate, 1.8 mg / mL sodium metabisulfite, and 3 v / v% Tween20;

[0223] Washing solution II was 80 v / v% ethanol;

[0224] The eluent was DEPC water;

[0225] The concentration of the magnetic bead suspension was 25 mg / mL (carboxyl magnetic beads TQ01, purchased from MGI, product number 940-000121-00, model 100 mL / bottle);

[0226] The proteinase K solution contains the following components: 40 v / v% glycerol, 0.05 M tris(hydroxymethyl)aminomethane hydrochloride, 0.5 mg / mL calcium chloride dihydrate, and 50 mg / mL proteinase K;

[0227] The DNase I solution contained the following components: 1 mg / mL calcium chloride, 5 mg / mL magnesium chloride, 0.01 M tris hydrochloride, and 0.5 mg / mL DNase I;

[0228] The preparation method of the above lysate is as follows:

[0229] 1) First, wash the rotor, measuring cylinder and beaker with purified water for 3 times, wash the measuring cylinder and beaker by rotating, and then wash the above consumables with DEPC water for 3 times. After washing, pour out the remaining water and set aside. After washing, the rotor needs to be placed in a beaker or wrapped in a disposable fresh-keeping bag for storage;

[0230] 2) Place the rinsed 3L beaker on a magnetic stirrer and add 450mL DEPC water and 1000mL anhydrous ethanol;

[0231] 3) Place the rotor in the beaker, turn on the magnetic stirrer, and set the speed to 800 rpm;

[0232] 4) Take 3.5g of disodium ethylenediaminetetraacetate and add it to a beaker, put the rinsed magnetic stirrer rotor into the beaker, and start stirring;

[0233] 5) After the disodium ethylenediaminetetraacetic acid is dissolved, take out the guanidine isothiocyanate reagent, shake each bottle vigorously to break up the guanidine isothiocyanate lumps in the bottle before opening, and weigh 600g of guanidine isothiocyanate using an electronic balance and add it to a 3L beaker;

[0234] 6) Add 1.5 g tris(hydroxymethyl)aminomethane, 5.0 g sodium metabisulfite, 30 g anhydrous sodium acetate, and 3.0 g hexadecyltrimethylammonium bromide into a beaker;

[0235] 7) Use an electric pipette to measure 50 mL of Tween 20 and add it to the beaker and continue stirring. After the reagent addition is completed, a total volume of about 2000 mL of lysate is obtained;

[0236] The preparation method of above-mentioned washing solution I is as follows:

[0237] 1) First, wash the rotor, measuring cylinder and beaker with purified water for 3 times, wash the measuring cylinder and beaker by rotating, and then wash the above consumables with DEPC water for 3 times. After washing, pour out the remaining water and set aside. After washing, the rotor needs to be placed in a beaker or wrapped in a disposable fresh-keeping bag for storage;

[0238] 2) Place the rinsed 2L beaker on a magnetic stirrer and add 550mL DEPC water and 400mL anhydrous ethanol;

[0239] 3) Place the rotor in the beaker, turn on the magnetic stirrer, and set the speed to 800 rpm;

[0240] 4) Add 36g sodium chloride, 20g anhydrous sodium acetate and 1.8g sodium metabisulfite into a beaker;

[0241] 5) Use an electric pipette to measure 30 mL of Tween 20 and add it to the beaker and continue stirring. After the reagent addition is completed, a total volume of about 1000 mL of washing solution I is obtained;

[0242] The preparation method of the above-mentioned washing solution II is as follows:

[0243] Take a certain amount of DEPC water, add 80% anhydrous ethanol by volume, and mix well to complete the preparation;

[0244] The preparation method of the above proteinase K solution is as follows:

[0245] 1) First, clean the measuring cylinder, rotor and beaker with purified water respectively. Clean the beaker and measuring cylinder by rotating. Then, clean the above consumables with DEPC water for 3 times. After cleaning, drain the remaining water and set aside.

[0246] 2) Take a clean 500mL measuring cylinder and measure 500mL of DEPC water. After measuring the solution, place the measuring cylinder on the table. After the liquid level stabilizes, keep your line of sight flush with the liquid surface and read the reading indicated by the position of the concave liquid surface, which is the measured volume.

[0247] 3) Use a clean 500mL graduated cylinder to measure 400mL of glycerol and pour it into the beaker;

[0248] 4) Take 50 mL of 1 M Tris(2-hydroxymethyl)aminomethane hydrochloride solution with a 50 mL pipette and add it to the beaker;

[0249] 5) Weigh 0.5 g of calcium chloride dihydrate and add it into a beaker;

[0250] 6) Weigh 50 g of proteinase K (purchased from: Jinpu Nuoan; model: 50 g / bottle; item number: GPK003001 / 2-50g) and add it into a beaker. Mix well to obtain a proteinase K solution with a total volume of about 1000 mL.

[0251] The preparation method of the above-mentioned DNase I solution is as follows:

[0252] 1) First, clean the measuring cylinder, rotor and beaker with purified water respectively. Clean the beaker and measuring cylinder by rotating. Then, clean the above consumables with DEPC water for 3 times. After cleaning, drain the remaining water and set aside.

[0253] 2) Take a clean 500mL measuring cylinder and measure 492mL of DEPC-treated water. After measuring the solution, place the measuring cylinder on the table. When the liquid level is stable, read the concave liquid surface with your line of sight flush with the liquid surface, which is the measured volume.

[0254] 3) Weigh 0.5 g of calcium chloride and 2.5 g of magnesium chloride respectively and add them into a beaker;

[0255] 4) Take 5 mL of 1 M Tris(2-hydroxymethyl)aminomethane hydrochloride solution with a 5 mL pipette and add it to the beaker;

[0256] 5) Weigh 0.25 g of DNA enzyme I (Tian Gen (RT411)) and add it into a beaker. Mix well to complete the preparation, and obtain a DNA enzyme I solution with a total volume of about 500 mL.

[0257] Example 2 A reagent combination for extracting nucleic acid (total RNA) by magnetic bead method

[0258] A reagent combination for extracting nucleic acid (total RNA) by magnetic bead method, comprising: a lysis solution, a washing solution, an eluent, a magnetic bead suspension, a proteinase K solution and a DNase I solution;

[0259] The lysate contains the following components: 45 v / v% anhydrous ethanol, 2.5 mg / mL disodium ethylenediaminetetraacetate, 275 mg / mL guanidine isothiocyanate, 1.5 mg / mL tris(hydroxymethyl)aminomethane, 2 mg / mL sodium metabisulfite, 20 mg / mL anhydrous sodium acetate, 2 mg / mL hexadecyltrimethylammonium bromide and 3 v / v% Tween20;

[0260] The washing solution comprises washing solution I and washing solution II;

[0261] Washing solution I contains the following components: 44 v / v% anhydrous ethanol, 30 mg / mL sodium chloride, 20 mg / mL anhydrous sodium acetate, 3.4 mg / mL sodium metabisulfite, and 3.5 v / v% Tween20;

[0262] Washing solution II was 80 v / v% ethanol;

[0263] The eluent was DEPC water;

[0264] The concentration of the magnetic bead suspension was 25 mg / mL (carboxyl magnetic beads TQ01, purchased from MGI, product number 940-000121-00, model 100 mL / bottle);

[0265] The proteinase K solution contains the following components: 40 v / v% glycerol, 0.055 M tris(hydroxymethyl)aminomethane hydrochloride, 0.6 mg / mL calcium chloride dihydrate, and 45 mg / mL proteinase K;

[0266] The DNase I solution contained the following components: 0.8 mg / mL calcium chloride, 4.4 mg / mL magnesium chloride, 0.016 M tris hydrochloride, and 0.6 mg / mL DNase I;

[0267] The preparation method of the above lysate is as follows:

[0268] 1) First, wash the rotor, measuring cylinder and beaker with purified water for 3 times, wash the measuring cylinder and beaker by rotating, and then wash the above consumables with DEPC water for 3 times. After washing, pour out the remaining water and set aside. After washing, the rotor needs to be placed in a beaker or wrapped in a disposable fresh-keeping bag for storage;

[0269] 2) Place the rinsed 3L beaker on a magnetic stirrer and add 550mL DEPC water and 900mL anhydrous ethanol;

[0270] 3) Place the rotor in the beaker, turn on the magnetic stirrer, and set the speed to 800 rpm;

[0271] 4) Take 5g of disodium ethylenediaminetetraacetate and add it to a beaker, put the rinsed magnetic stirrer rotor into the beaker, and start stirring;

[0272] 5) After the disodium ethylenediaminetetraacetic acid is dissolved, take out the guanidine isothiocyanate reagent, shake each bottle vigorously to break up the guanidine isothiocyanate lumps in the bottle before opening, and weigh 550g of guanidine isothiocyanate using an electronic balance and add it to a 3L beaker;

[0273] 6) Add 3 g of tris(hydroxymethyl)aminomethane, 4.0 g of sodium metabisulfite, 40 g of anhydrous sodium acetate, and 4.0 g of hexadecyltrimethylammonium bromide into a beaker;

[0274] 7) Use an electric pipette to measure 60 mL of Tween 20 and add it to the beaker and continue stirring. After the reagent addition is completed, a total volume of about 2000 mL of lysate is obtained;

[0275] The preparation method of above-mentioned washing solution I is as follows:

[0276] 1) First, wash the rotor, measuring cylinder and beaker with purified water for 3 times, wash the measuring cylinder and beaker by rotating, and then wash the above consumables with DEPC water for 3 times. After washing, pour out the remaining water and set aside. After washing, the rotor needs to be placed in a beaker or wrapped in a disposable fresh-keeping bag for storage;

[0277] 2) Place the rinsed 2L beaker on a magnetic stirrer and add 500mL DEPC water and 440mL anhydrous ethanol;

[0278] 3) Place the rotor in the beaker, turn on the magnetic stirrer, and set the speed to 800 rpm;

[0279] 4) Add 30g sodium chloride, 20g anhydrous sodium acetate and 3.4g sodium metabisulfite into a beaker;

[0280] 5) Use an electric pipette to measure 35 mL of Tween 20 and add it to the beaker and continue stirring. After the reagent addition is completed, a total volume of about 1000 mL of washing solution I is obtained;

[0281] The preparation method of the above-mentioned washing solution II is as follows:

[0282] Take a certain amount of DEPC water, add 80% anhydrous ethanol by volume, and mix well to complete the preparation;

[0283] The preparation method of the above proteinase K solution is as follows:

[0284] 1) First, clean the measuring cylinder, rotor and beaker with purified water respectively. Clean the beaker and measuring cylinder by rotating. Then, clean the above consumables with DEPC water for 3 times. After cleaning, drain the remaining water and set aside.

[0285] 2) Take a clean 500mL measuring cylinder and measure 500mL of DEPC water. After measuring the solution, place the measuring cylinder on the table. After the liquid level stabilizes, keep your line of sight flush with the liquid surface and read the reading indicated by the position of the concave liquid surface, which is the measured volume.

[0286] 3) Use a clean 500mL graduated cylinder to measure 400mL of glycerol and pour it into the beaker;

[0287] 4) Take 55 mL of 1 M Tris(2-hydroxymethyl)aminomethane hydrochloride solution with a 50 mL pipette and add it to the beaker;

[0288] 5) Weigh 0.6 g of calcium chloride dihydrate and add it into the beaker;

[0289] 6) Weigh 45 g of proteinase K (purchased from: Jinpu Nuoan; model: 50 g / bottle; item number: GPK003001 / 2-50g) and add it into a beaker. Mix well to obtain a proteinase K solution with a total volume of about 1000 mL.

[0290] The preparation method of the above-mentioned DNase I solution is as follows:

[0291] 1) First, clean the measuring cylinder, rotor and beaker with purified water respectively. Clean the beaker and measuring cylinder by rotating. Then, clean the above consumables with DEPC water for 3 times. After cleaning, drain the remaining water and set aside.

[0292] 2) Take a clean 500mL measuring cylinder and measure 490mL of DEPC-treated water. After measuring the solution, place the measuring cylinder on the table. When the liquid level is stable, read the concave liquid surface with your line of sight flush with the liquid surface, which is the measured volume.

[0293] 3) Weigh 0.4 g of calcium chloride and 2.2 g of magnesium chloride respectively and add them into a beaker;

[0294] 4) Use a 5 mL pipette to draw up 8 mL of 1 M Tris(2-hydroxymethyl)aminomethane hydrochloride solution and add it to the beaker;

[0295] 5) Weigh 0.3 g of DNase I and add it into a beaker. Mix well to complete the preparation, and obtain a DNase I solution with a total volume of about 500 mL.

[0296] Example 3 A reagent combination for extracting nucleic acid (total RNA) by magnetic bead method

[0297] A reagent combination for extracting nucleic acid (total RNA) by magnetic bead method, comprising: a lysis solution, a washing solution, an eluent, a magnetic bead suspension, a proteinase K solution and a DNase I solution;

[0298] The lysate contains the following components: 48.5 v / v% anhydrous ethanol, 1.65 mg / mL disodium ethylenediaminetetraacetate, 290 mg / mL guanidine isothiocyanate, 0.9 mg / mL tris(hydroxymethyl)aminomethane, 2 mg / mL sodium metabisulfite, 22.5 mg / mL anhydrous sodium acetate, 1 mg / mL hexadecyltrimethylammonium bromide and 2.25 v / v% Tween20;

[0299] The washing solution comprises washing solution I and washing solution II;

[0300] Washing solution I contains the following components: 35 v / v% anhydrous ethanol, 40 mg / mL sodium chloride, 20 mg / mL anhydrous sodium acetate, 2 mg / mL sodium metabisulfite, and 2.5 v / v% Tween20;

[0301] Washing solution II was 80 v / v% ethanol;

[0302] The eluent was DEPC water;

[0303] The concentration of the magnetic bead suspension was 25 mg / mL (carboxyl magnetic beads TQ01, purchased from MGI, product number 940-000121-00, model 100 mL / bottle);

[0304] The proteinase K solution contains the following components: 40 v / v% glycerol, 0.09 M tris(hydroxymethyl)aminomethane hydrochloride, 0.7 mg / mL calcium chloride dihydrate, and 32 mg / mL proteinase K;

[0305] The DNase I solution contained the following components: 1.4 mg / mL calcium chloride, 4 mg / mL magnesium chloride, 0.02 M tris hydrochloride, and 0.6 mg / mL DNase I;

[0306] The preparation method of the above lysate is as follows:

[0307] 1) First, wash the rotor, measuring cylinder and beaker with purified water for 3 times, wash the measuring cylinder and beaker by rotating, and then wash the above consumables with DEPC water for 3 times. After washing, pour out the remaining water and set aside. After washing, the rotor needs to be placed in a beaker or wrapped in a disposable fresh-keeping bag for storage;

[0308] 2) Place the rinsed 3L beaker on a magnetic stirrer and add 480mL DEPC water and 970mL anhydrous ethanol;

[0309] 3) Place the rotor in the beaker, turn on the magnetic stirrer, and set the speed to 800 rpm;

[0310] 4) Take 3.3 g of disodium ethylenediaminetetraacetate and add it to a beaker. Place the rinsed magnetic stirrer rotor into the beaker and start stirring.

[0311] 5) After the disodium ethylenediaminetetraacetic acid is dissolved, take out the guanidine isothiocyanate reagent, shake each bottle vigorously to break up the guanidine isothiocyanate lumps in the bottle before opening, and weigh 580g of guanidine isothiocyanate using an electronic balance and add it to a 3L beaker;

[0312] 6) Add 1.8 g tris(hydroxymethyl)aminomethane, 4.0 g sodium metabisulfite, 45 g anhydrous sodium acetate, and 2.0 g hexadecyltrimethylammonium bromide into a beaker;

[0313] 7) Use an electric pipette to measure 45 mL of Tween 20 and add it to the beaker and continue stirring. After the reagent addition is completed, a total volume of about 2000 mL of lysate is obtained;

[0314] The preparation method of above-mentioned washing solution I is as follows:

[0315] 1) First, wash the rotor, measuring cylinder and beaker with purified water for 3 times, wash the measuring cylinder and beaker by rotating, and then wash the above consumables with DEPC water for 3 times. After washing, pour out the remaining water and set aside. After washing, the rotor needs to be placed in a beaker or wrapped in a disposable fresh-keeping bag for storage;

[0316] 2) Place the rinsed 2L beaker on a magnetic stirrer and add 600mL DEPC water and 350mL anhydrous ethanol;

[0317] 3) Place the rotor in the beaker, turn on the magnetic stirrer, and set the speed to 800 rpm;

[0318] 4) Add 40g sodium chloride, 20g anhydrous sodium acetate and 2g sodium metabisulfite into a beaker;

[0319] 5) Use an electric pipette to measure 25 mL of Tween 20 and add it to the beaker and continue stirring. After the reagent addition is completed, a total volume of about 1000 mL of washing solution I is obtained;

[0320] The preparation method of the above-mentioned washing solution II is as follows:

[0321] Take a certain amount of DEPC water, add 80% anhydrous ethanol by volume, and mix well to complete the preparation;

[0322] The preparation method of the above proteinase K solution is as follows:

[0323] 1) First, clean the measuring cylinder, rotor and beaker with purified water respectively. Clean the beaker and measuring cylinder by rotating. Then, clean the above consumables with DEPC water for 3 times. After cleaning, drain the remaining water and set aside.

[0324] 2) Take a clean 500mL measuring cylinder and measure 480mL of DEPC water. After measuring the solution, place the measuring cylinder on the table. After the liquid level stabilizes, keep your line of sight flush with the liquid surface and read the reading indicated by the position of the concave liquid surface, which is the measured volume.

[0325] 3) Use a clean 500mL graduated cylinder to measure 400mL of glycerol and pour it into the beaker;

[0326] 4) Take 90 mL of 1 M Tris(2-hydroxymethyl)aminomethane hydrochloride solution with a 50 mL pipette and add it to the beaker;

[0327] 5) Weigh 0.7 g of calcium chloride dihydrate and add it into a beaker;

[0328] 6) Weigh 32 g of proteinase K (purchased from: Jinpu Nuoan; model: 50 g / bottle; item number: GPK003001 / 2-50g) and add it into a beaker. Mix well to obtain a proteinase K solution with a total volume of about 1000 mL.

[0329] The preparation method of the above-mentioned DNase I solution is as follows:

[0330] 1) First, clean the measuring cylinder, rotor and beaker with purified water respectively. Clean the beaker and measuring cylinder by rotating. Then, clean the above consumables with DEPC water for 3 times. After cleaning, drain the remaining water and set aside.

[0331] 2) Take a clean 500mL measuring cylinder and measure 488mL of DEPC-treated water. After measuring the solution, place the measuring cylinder on the table. When the liquid level is stable, read the concave liquid surface at the same level as the liquid level. This is the measured volume.

[0332] 3) Weigh 0.7 g of calcium chloride and 2.0 g of magnesium chloride respectively and add them into a beaker;

[0333] 4) Use a 5 mL pipette to draw up 10 mL of 1 M Tris(2-hydroxymethyl)aminomethane hydrochloride solution and add it to the beaker;

[0334] 5) Weigh 0.3 g of DNase I and add it into a beaker. Mix well to complete the preparation, and obtain a DNase I solution with a total volume of about 500 mL.

[0335] Example 4 A method for extracting nucleic acid (total RNA) from human colon cancer cells

[0336] A method for extracting nucleic acid (total RNA) from human colon cancer cells comprises the following steps using the reagent combination of Example 1:

[0337] 1) Add 50 μL of 1x10 5 Add 60 μL of magnetic bead suspension, 20 μL of proteinase K solution, and 600 μL of lysis buffer to the centrifuge tubes of human colon cancer cells, vortex mix for 1 minute, and leave at room temperature for 10 minutes, vortexing for 10 seconds every 5 minutes;

[0338] 2) Place the centrifuge tube on the magnetic rack and let it stand for 2 minutes. During this period, slowly turn the centrifuge tube upside down on the magnetic rack (the main purpose is to let it stand, and the upside down during this period is to wash off the magnetic beads on the tube wall. The lack of this step may cause the loss of magnetic beads and affect the yield). Wash off the magnetic beads on the centrifuge tube wall and the tube cover. After the magnetic beads are completely adsorbed, aspirate and discard the supernatant;

[0339] 3) Add 600 μL of washing solution I, vortex mix for 1 minute, and place on the magnetic stand for 2 minutes. During this period, keep the centrifuge tube on the magnetic stand and slowly turn it upside down. After the magnetic beads are completely adsorbed, aspirate and discard the supernatant;

[0340] 4) Open the lid and let it dry for 10 minutes;

[0341] 5) Add 80 μL of DNase I solution, vortex mix for 1 minute, and leave at room temperature for 10 minutes, vortexing for 10 seconds every 3 minutes;

[0342] 6) Add 700 μL of washing solution I, vortex mix for 1 minute, let stand at room temperature for 5 minutes, and place on the magnetic stand for 2 minutes. During this period, keep the centrifuge tube slowly upside down on the magnetic stand. After the magnetic beads are completely adsorbed, aspirate and discard the supernatant;

[0343] 7) Add 700 μL of washing solution II, vortex mix for 1 minute, place on the magnetic stand for 2 minutes, and after the magnetic beads are completely adsorbed, aspirate and discard the supernatant;

[0344] 8) Repeat step 7) once;

[0345] 9) Open the lid and let it dry for 10 minutes;

[0346] 10) Add 100 μL of elution buffer, vortex mix for 1 minute, let stand at room temperature for 10 minutes, then place on a magnetic rack. After the magnetic beads are completely adsorbed, aspirate the supernatant to obtain the desired product.

[0347] Example 5 A method for extracting nucleic acid (total RNA) from mouse liver tissue

[0348] A method for extracting nucleic acid (total RNA) from mouse liver tissue is the same as that of Example 4, except that the sample is 5 mg of mouse liver tissue.

[0349] Example 6 A method for extracting nucleic acid (total RNA) from SD rat blood

[0350] A method for extracting nucleic acid (total RNA) from SD rat blood is the same as that of Example 4, except that the sample is 200 μL of SD rat blood.

[0351] Example 7 A method for extracting nucleic acid (total RNA) from Escherichia coli

[0352] A method for extracting nucleic acid (total RNA) from Escherichia coli is the same as that in Example 4, except that the sample is 50 μL and the amount is 2×10 7 CFU of Escherichia coli.

[0353] Example 8 A method for extracting nucleic acid (total RNA) from human colon cancer cells

[0354] A method for extracting nucleic acid (total RNA) from human colon cancer cells is the same as that in Example 4, except that the sample is 50 μL and the amount is 1.5×10 5 of human colon cancer cells.

[0355] Example 9 A method for extracting nucleic acid (total RNA) from human colon cancer cells

[0356] A method for extracting nucleic acid (total RNA) from human colon cancer cells is the same as that of Example 8, except that the reagent combination of Example 2 is used.

[0357] Example 10 A method for extracting nucleic acid (total RNA) from human colon cancer cells

[0358] A method for extracting nucleic acid (total RNA) from human colon cancer cells is the same as that of Example 8, except that the reagent combination of Example 3 is used.

[0359] Example 11 A method for extracting nucleic acid (total RNA) from human colon cancer cells

[0360] A method for extracting nucleic acid (total RNA) from human colon cancer cells is the same as Example 8, except that the content of one component in the lysate or washing solution I of the reagent combination in Example 1 is different, as shown in Table 7.

[0361] Comparative Example 1 A method for extracting nucleic acid (total RNA) from human colon cancer cells

[0362] A method for extracting nucleic acid (total RNA) from human colon cancer cells. The sample dosage is the same as that in Example 4, except that QIAGEN (RNeasy Mini Kit (50), Catalog No. 74104) is used. The method is described in the instruction manual.

[0363] Comparative Example 2 A method for extracting nucleic acid (total RNA) from mouse liver tissue

[0364] A method for extracting nucleic acid (total RNA) from mouse liver tissue, the sample amount is the same as that in Example 5, the difference is that QIAGEN (RNeasy Mini Kit (50), catalog number 74104) is used, and the method is referred to the instruction manual.

[0365] Comparative Example 3 A method for extracting nucleic acid (total RNA) from SD rat blood

[0366] A method for extracting nucleic acid (total RNA) from SD rat blood. The sample amount is the same as that in Example 6, except that QIAGEN (RNeasy Mini Kit (50), Catalog No. 74104) is used. The method is described in the instruction manual.

[0367] Comparative Example 4: A method for extracting nucleic acid (total RNA) from Escherichia coli

[0368] A method for extracting nucleic acid (total RNA) from Escherichia coli, the sample dosage is the same as that in Example 7, except that QIAGEN (RNeasy Mini Kit (50), Catalog No. 74104) is used, and the method is referred to the instruction manual.

[0369] Comparative Example 5 A method for extracting nucleic acid (total RNA) from human colon cancer cells

[0370] A method for extracting nucleic acid (total RNA) from human colon cancer cells. The sample dosage is the same as that in Example 4, except that Tiangen (magnetic bead method tissue / cell / blood total RNA extraction kit (DP761)) is used. The method is described in the instructions.

[0371] Comparative Example 6 A method for extracting nucleic acid (total RNA) from mouse liver tissue

[0372] A method for extracting nucleic acid (total RNA) from mouse liver tissue. The sample dosage is the same as that in Example 5, except that Tiangen (magnetic bead tissue / cell / blood total RNA extraction kit (DP761)) is used. The method is described in the instructions.

[0373] Comparative Example 7 A method for extracting nucleic acid (total RNA) from SD rat blood

[0374] A method for extracting nucleic acid (total RNA) from SD rat blood. The sample dosage is the same as that in Example 6, except that Tiangen (magnetic bead method tissue / cell / blood total RNA extraction kit (DP761)) is used. The method is described in the instructions.

[0375] Comparative Example 8 A method for extracting nucleic acid (total RNA) from Escherichia coli

[0376] A method for extracting nucleic acid (total RNA) of Escherichia coli, the sample amount is the same as that of Example 7, the difference is that OMEGA ( Total RNA Kit, model M6930), refer to the instructions.

[0377] Effect Example Performance Verification of the Reagent Combination for Extracting Nucleic Acid (Total RNA) by Magnetic Bead Method

[0378] The concentration and purity of the extracted products of Examples 4 to 7 and Comparative Examples 1 to 8 were tested by NanoDrop 8000, and the fragment integrity of the extracted products of Examples 4 to 5, 7, Comparative Examples 1 to 2, 4 to 6, and 8 were tested by agarose gel electrophoresis. The results are shown in Tables 1 to 4, Figures 1 to 3 As shown:

[0379] As can be seen from Table 1, the concentration of the extraction reagent combination provided by the present invention (Example 1) for extracting human colon cancer cells is higher than that of QIAGEN and Tiangen kits, with a CV value of <10%; the purity A260 / 280 ≥1.8, A260 / 230 ≥1.7, which meets the requirements of downstream qPCR and library sequencing; Figure 1 It can be seen that the extraction reagent combination provided by the present invention (Example 1) has clear bands at 28s, 18s, and 5s for the products extracted from human colon cancer cells, and is overall superior to the competing QIAGEN and Tiangen kits;

[0380] As can be seen from Table 2, the concentration of the extraction reagent combination provided by the present invention (Example 1) for mouse liver tissue extraction is higher than that of the QIAGEN kit and slightly lower than that of the Tiangen kit, with a CV value of <10%; the purity A260 / 280 ≥1.8 and A260 / 230 ≥1.8 meet the requirements of downstream qPCR and library construction and sequencing, and are better than Tiangen; Figure 2 It can be seen that the extraction reagent combination provided by the present invention (Example 1) has clear bands at 28s, 18s, and 5s for the product extracted from mouse liver tissue, and is overall superior to the competing QIAGEN and Tiangen kits;

[0381] As can be seen from Table 3, the concentration of the extraction reagent combination provided by the present invention (Example 1) for SD rat blood extraction is higher than that of QIAGEN and Tiangen kits as a whole, and the CV value is <10%; the purity A260 / 280 ≥1.8, A260 / 230 ≥1.7, which is better than QIAGEN and Tiangen, and meets the requirements of downstream qPCR and library construction and sequencing;

[0382] As can be seen from Table 4, the concentration of the extraction reagent combination provided by the present invention (Example 1) for Escherichia coli extraction is higher than OMEGA and slightly lower than QIAGEN, and the CV value is <10%; the purity A260 / 280 ≥1.8, A260 / 230 ≥1.7, which is better than OMEGA and QIAGEN, and meets the requirements of downstream qPCR and library construction and sequencing; Figure 3 It can be seen that the extraction reagent combination provided by the present invention (Example 1) has clear bands at 28s, 18s, and 5s for the product extracted from E. coli, and is overall superior to the competing QIAGEN and OMEGA kits.

[0383] Table 1 Human colon cancer cells

[0384]

[0385] Table 2 Mouse liver tissue

[0386]

[0387]

[0388] Table 3 SD rat blood

[0389]

[0390] Table 4 Escherichia coli

[0391]

[0392]

[0393] The reagent combination of Example 1 was used to extract and verify 100 μL of a pure RNA standard solution with a concentration of 232 ng / μL to 244 ng / μL (as shown in Table 5), and the recovery volume was 80 μL. The results are shown in Table 5: The recovery rate of the extraction reagent combination (Example 1) provided by the present invention reached more than 90%.

[0394] Table 5 Recovery

[0395]

[0396] The concentration and purity of the extracted products of Examples 8 to 10 were tested using NanoDrop 8000. The results are shown in Table 6: The effects of the extraction reagent combinations of Examples 1 to 3 are comparable.

[0397] Table 6 Verification data of different embodiments

[0398]

[0399]

[0400] The concentration and purity of the extraction products of Examples 8 and 11 were detected by NanoDrop8000, and the results are shown in Table 7: the concentration and purity of the extraction product of Example 11 are equivalent to those of Example 8, and adjusting the content of one component in the lysis solution or washing solution I in the reagent combination of Example 1 within a certain range does not affect the effect of the reagent combination.

[0401] Table 7 Average values ​​of validation data of extraction reagents with different content components (repeated 3 times)

[0402]

[0403] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A reagent combination or kit comprising: a lysis solution and a washing solution; The lysate comprises: guanidine salt, sodium salt, nonionic surfactant, alcohol, and antioxidant; The washing liquid comprises: washing liquid I; The washing solution I comprises: sodium salt, nonionic surfactant, alcohol, and antioxidant; The antioxidants in the lysate and the washing solution I are independently selected from at least one of tri(2-formylethyl)phosphine hydrochloride and metabisulfite.

2. The reagent combination or kit according to claim 1, characterized in that: The antioxidant in the lysate comprises: metabisulfite; and / or The antioxidant in the washing liquid I comprises: metabisulfite; Preferably, the metabisulfite comprises at least one of sodium metabisulfite, potassium metabisulfite and magnesium metabisulfite.

3. The reagent combination or kit according to claim 1, characterized in that: The guanidine salt comprises: at least one of guanidine hydrochloride, guanidine nitrate, guanidine carbonate, guanidine acetate, guanidine isothiocyanate, and guanidine thiocyanate; and / or The sodium salt in the lysing solution and the washing solution I is independently selected from at least one of sodium chloride, sodium acetate, sodium sulfate and sodium citrate; and / or The nonionic surfactants in the lysing solution and the washing solution I are independently selected from at least one of Triton 100, Tween 20, Tween 80, ethylphenyl polyethylene glycol, and polyether alcohol; and / or The alcohols in the lysate and the washing solution I are independently selected from at least one of ethanol and isopropanol.

4. The reagent combination or kit according to claim 1, characterized in that: The lysate further comprises: a cationic surfactant; and / or The lysate further comprises: a chelating agent; and / or The lysate further comprises: a buffer salt; Preferably, the cationic surfactant comprises: at least one of dodecyldimethylbenzylammonium chloride, hexadecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, dodecyldimethylbenzylammonium bromide, hexadecyltrimethylammonium bromide and octadecyltrimethylammonium bromide; Preferably, the chelating agent comprises: at least one of ethylenediaminetetraacetic acid and a water-soluble salt of ethylenediaminetetraacetic acid; Preferably, the buffer salt comprises at least one of phosphate, borate, citrate and tris(hydroxymethyl)aminomethane.

5. The reagent combination or kit according to claim 1, characterized in that: The washing liquid further comprises: washing liquid II; and / or The reagent combination or kit further comprises: an eluent; and / or The reagent combination or kit further comprises: magnetic beads; and / or The reagent combination or kit further comprises: proteinase K; and / or The reagent combination or kit further comprises: DNA enzyme.

6. The reagent combination or kit according to claim 5, characterized in that: The washing liquid II is an alcohol; further, 65 to 90 v / v% of alcohol; Preferably, the alcohol comprises at least one of ethanol and isopropanol; Preferably, the eluent comprises: at least one of sterile water, enzyme-free water, NF water, and DEPC water; Preferably, the magnetic beads comprise nanomagnetic beads; Preferably, the magnetic beads include: at least one of hydroxy magnetic beads, carboxyl magnetic beads, glass magnetic beads, amino magnetic beads, tosyl magnetic beads, and epoxy magnetic beads; Preferably, the DNase comprises DNase I.

7. The reagent combination or kit according to any one of claims 1 to 6, characterized in that: The concentration of the antioxidant in the lysate is 1.5 to 3 mg / mL; and / or The concentration of the antioxidant in the washing solution I is 1.5-4 mg / mL; and / or The concentration of the guanidine salt in the lysate is 250-325 mg / mL; and / or The concentration of the sodium salt in the lysate is 12.5 to 24 mg / mL; and / or The concentration of the sodium salt in the washing solution I is 45-65 mg / mL; and / or The concentration of the nonionic surfactant in the lysate is 2 to 3.25 v / v%; and / or The concentration of the nonionic surfactant in the washing liquid I is 2 to 4 v / v%; and / or The concentration of the alcohol in the lysate is 40-55 v / v%; and / or The concentration of the alcohol in the washing liquid I is 30 to 45 v / v%; Preferably, the reagent combination or kit is used for extracting nucleic acid by magnetic bead method; Preferably, the nucleic acid comprises: at least one of DNA and RNA; Preferably, the nucleic acid is from any one or more of the following biological samples: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, nasal swabs; Preferably, the body fluid comprises at least one of tissue fluid, lymph fluid, blood, and cerebrospinal fluid; Preferably, the microorganism comprises at least one of bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, and spirochetes; Preferably, the nucleic acid is used for PCR, enzyme digestion, molecular hybridization, library construction or Southern hybridization.

8. The reagent combination or kit according to claim 4, characterized in that: The concentration of the cationic surfactant in the lysate is 0.8 to 2.2 mg / mL; and / or The concentration of the chelating agent in the lysate is 1.5 to 3 mg / mL; and / or The concentration of the buffer salt in the lysate is 0.5-1.75 mg / mL.

9. A method for extracting nucleic acid, comprising the step of using the reagent combination or kit according to any one of claims 1 to 8.

10. The method according to claim 9, characterized in that: The method is A1) or A2): When the nucleic acid does not contain DNA, the method is A1), which comprises the following steps: a1) mixing the sample, magnetic beads, proteinase K, and the lysis solution described in any one of claims 1 to 8, and separating the magnetic beads; a2) mixing the magnetic beads with the washing solution I described in any one of claims 1 to 8, and separating the magnetic beads; a3) mixing the magnetic beads with DNA enzyme, mixing the obtained mixture with the washing solution I described in any one of claims 1 to 8, and separating the magnetic beads; a4) mixing the magnetic beads with washing solution II to separate the magnetic beads; a5) mixing the magnetic beads with the eluent, separating the magnetic beads, and obtaining the nucleic acid; When the nucleic acid comprises DNA, the method is A2), which comprises the following steps: b1) mixing the sample, magnetic beads, proteinase K, and the lysis solution described in any one of claims 1 to 8, and separating the magnetic beads; b2) mixing the magnetic beads with the washing solution I described in any one of claims 1 to 8, and separating the magnetic beads; b3) mixing the magnetic beads with washing solution II to separate the magnetic beads; b4) mixing the magnetic beads with the elution solution, separating the magnetic beads, and obtaining the nucleic acid Preferably, the magnetic beads, proteinase K, DNA enzyme, washing solution II and / or eluent are the magnetic beads, proteinase K, DNA enzyme, washing solution II and / or eluent described in claim 5 or 6.

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