A method for extracting nucleic acid based on magnetic resin

Abstract

The present application provides a method for extracting nucleic acid based on magnetic resin, which belongs to the field of biotechnology. The present application can effectively separate impurities in complex samples such as soil and feces by adding a magnetic resin adsorption treatment step during the nucleic acid extraction process, and obtain a high-purity DNA product, which is beneficial to the application of downstream systems.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for extracting nucleic acids based on magnetic resin. Background Art

[0002] The analysis of microbial genomics with the help of second-generation sequencing technology has become the main technical means for microbial diversity analysis. The nucleic acid variation and quantitative analysis of microorganisms in samples such as soil, water and feces can reflect the types and abundance of various microorganisms in the flora, and decode the characteristics and differences of the distribution of flora species. However, samples such as soil, water and feces contain too many impurities (such as humus, humic acid, fulvic acid, etc.). During the nucleic acid extraction process, such impurities usually bind to nucleic acids and remain in the extracted nucleic acid products, which have a significant inhibitory effect on downstream PCR or qPCR. Therefore, when extracting nucleic acids from samples such as soil, water and feces, the interference of impurities becomes an important limiting factor that restricts the quality of nucleic acid extraction.

[0003] For the extraction of nucleic acid from soil and feces, most of the products currently on the market remove impurities through precipitation. For example, US7459548B2 discloses a method of using ammonium acetate as the first flocculant and ammonium aluminum sulfate or ammonium aluminum sulfate dodecahydrate as the second flocculant to precipitate impurities from the sample, thereby improving the purity of nucleic acid extraction. CN113045684A discloses a precipitant prepared by esterification and grafting of ethylenediaminetetraacetic anhydride and dextran, which can remove metal ions in biological samples and improve the activity of nucleic acids in soil samples. However, these impurities are removed by centrifugal precipitation, which is cumbersome to operate and difficult to automate. At the same time, the extraction time for nucleic acid extraction is long, which can easily cause nucleic acid degradation and affect the accuracy of downstream nucleic acid detection. Summary of the invention

[0004] The purpose of this application is to provide a method for extracting nucleic acids based on magnetic resin. The method of this application uses magnetic anion exchange resin to adsorb impurities in soil and fecal samples, and separates the magnetic anion resin bound to impurities from the sample through a magnetic field, which simplifies the process of removing impurities by traditional precipitant centrifugation and shortens the nucleic acid extraction time. The purity of nucleic acids in soil and fecal samples extracted using the method described in this application is significantly improved, which is beneficial for downstream applications.

[0005] The first aspect of the present application provides a method for extracting nucleic acid based on a magnetic resin, the method comprising the following steps:

[0006] (1) Collect biological or environmental samples, add lysis buffer, and release nucleic acids;

[0007] (2) adding magnetic resin for adsorption, magnetic separation to discard the magnetic resin and its adsorbate, and retaining the liquid;

[0008] (3) using a solid phase carrier to absorb the nucleic acid in the liquid;

[0009] (4) washing the solid phase carrier to which the nucleic acid has been adsorbed;

[0010] (5) Elution to obtain the extracted nucleic acid product.

[0011] In some embodiments, the biological sample is a stool sample.

[0012] In some embodiments, the environmental sample is a soil sample or a water sample.

[0013] In some embodiments, the lysate comprises a chaotropic agent, a buffer salt and a surfactant. The chaotropic agent is, for example, one or more of guanidine salt, urea and lithium perchlorate, preferably guanidine salt, more preferably guanidine hydrochloride and / or guanidine isothiocyanate; the buffer salt is one or more of phosphate, borate, citrate and Tris, preferably phosphate, more preferably one or two of NaH2PO3 and Na2HPO4; the surfactant is, for example, one or more of anionic surfactant, cationic surfactant, zwitterionic surfactant and nonionic surfactant, preferably nonionic surfactant, more preferably Tween 20 and / or decyl glycoside.

[0014] In some embodiments, step (1) further comprises using a mechanical lysis method, such as using glass beads, for example, adding glass beads to the sample and vortexing.

[0015] In some embodiments, step (1) further comprises centrifugation after adding the lysate, with a relative centrifugal force of 8000-20000×g, preferably 8000-18000×g, including 8000×g, 8500×g, 9000×g, 9500×g, 10000×g, 11000×g, 12000×g, 13000×g, 14000×g, 15000×g, 16000×g, 17000×g and 18000×g within the range.

[0016] In some embodiments, about 0.1-5 g of magnetic resin is used per gram of stool sample, preferably about 0.5-3 g of magnetic resin, and more preferably about 0.5-2.5 g of magnetic resin, including 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g, 2.1 g, 2.2 g, 2.3 g, 2.4 g and 2.5 g within the range.

[0017] In some embodiments, about 0.1-5 g of magnetic resin is used per gram of soil sample, preferably about 0.2-3 g of magnetic resin, and more preferably about 0.3-2 g of magnetic resin, including 0.3 g, 0.4, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g and 2 g within the range.

[0018] In some embodiments, step (2) comprises adding one or more magnetic resins.

[0019] In some embodiments, the magnetic resin is a magnetic anion exchange resin, and the magnetic anion resin is basic, such as a strongly basic or weakly basic magnetic resin.

[0020] In some embodiments, the matrix (skeleton) type of the magnetic resin is one of styrene, acrylic, acetic, epoxy, vinylpyridine, urea-formaldehyde or vinyl chloride.

[0021] In some embodiments, the pore structure of the magnetic resin is gel-type or macroporous, preferably macroporous.

[0022] In some embodiments, the magnetic core material of the magnetic resin is an iron oxide compound, such as γ-Fe2O3 or Fe3O4.

[0023] In some embodiments, the magnetic resin is a γ-Fe2O3 magnetic macroporous alkaline acrylic anion exchange resin or a Fe3O4 magnetic macroporous alkaline acrylic anion exchange resin, such as a γ-Fe2O3 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a γ-Fe2O3 magnetic macroporous weakly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous weakly alkaline acrylic anion exchange resin.

[0024] In some embodiments, the magnetic resin is MIEX, NDMP-1, or ND100.

[0025] In some embodiments, the magnetic resin binds non-nucleic acid impurities in the sample.

[0026] In some embodiments, the non-nucleic acid impurities are impurities that inhibit the PCR process, such as impurities that inhibit the activity of DNA polymerase.

[0027] In some embodiments, the solid phase carrier is a magnetic adsorption medium, preferably magnetic particles, more preferably magnetic beads.

[0028] In some embodiments, the solid support is a silicon-based adsorption medium, such as silica beads, silicon-based membranes.

[0029] In some embodiments, the solid support is an anion exchange material, such as an anion exchange column.

[0030] In some embodiments, when the solid phase carrier is magnetic beads, the step (3) further comprises adding a lysis binding solution.

[0031] In some embodiments, the lysis binding solution comprises a chaotropic agent, a buffer salt, a metal chelator, an alcohol and a surfactant; the chaotropic agent is, for example, one or more of guanidine salts, urea and lithium perchlorate, preferably guanidine salts, more preferably guanidine hydrochloride and / or guanidine isothiocyanate; the buffer salt is, for example, Tris and / or Tris-base; the metal chelator is, for example, disodium EDTA; the alcohol is, for example, ethanol and / or isopropanol, preferably isopropanol; the surfactant is, for example, one or more of anionic surfactants, cationic surfactants, zwitterionic surfactants and nonionic surfactants, preferably nonionic surfactants, more preferably nonionic surfactants and anionic surfactants, and also preferably Tween 20, PEG8000 decyl glycoside, TritonX-100 and / or sodium N-lauroyl sarcosine.

[0032] In some embodiments, the lysis-binding solution further comprises polyvinyl pyrrolidone.

[0033] In some embodiments, when the solid phase carrier is magnetic beads, the step (4) further comprises applying an external magnetic field to separate the magnetic beads that have adsorbed the nucleic acid.

[0034] In some embodiments, when the solid phase carrier is a spin column containing a silicon matrix membrane, the step (3) further comprises centrifugation to separate the adsorbed nucleic acid and impurities.

[0035] In some embodiments, the washing comprises adding a wash solution.

[0036] In some embodiments, the washing solution comprises a buffer component, an alcohol and a chaotropic agent; the buffer component is, for example, Tris and / or Tris-base, preferably Tris-base; the alcohol is, for example, ethanol and / or isopropanol, preferably isopropanol, and the chaotropic agent is, for example, one or more of guanidine salts, urea and lithium perchlorate, preferably guanidine salts, more preferably guanidine hydrochloride and / or guanidine isothiocyanate.

[0037] In some embodiments, the washing further comprises adding a rinse solution.

[0038] In some embodiments, the rinse solution comprises alcohol, preferably ethanol and / or isopropanol, more preferably ethanol.

[0039] In some embodiments, the eluting comprises adding an elution buffer, such as water or TE buffer.

[0040] In some embodiments, the method is partially or entirely performed by manual operation. For example, when the solid phase carrier is a spin column containing a silicon matrix membrane, the method is entirely performed by manual operation.

[0041] In some embodiments, the method is partially or entirely performed on an automatic nucleic acid extraction platform, for example, when the solid phase carrier is a magnetic bead, the method is entirely performed on a nucleic acid extraction instrument, which can achieve full process automation. For example, a magnetic stand can also be used for manual operation.

[0042] In some embodiments, the method is performed partially or entirely on a microfluidic platform, such as on a microfluidic chip.

[0043] In some embodiments, the nucleic acid is DNA and / or RNA, preferably, the nucleic acid is DNA.

[0044] The second aspect of the present application provides a method for extracting nucleic acid based on a magnetic resin, the method comprising the following steps:

[0045] (1) Collect biological or environmental samples, add diluent, and release or disperse the microorganisms in the samples;

[0046] (2) adding magnetic resin for adsorption, magnetic separation to discard the magnetic resin and its adsorbate, and retaining the liquid;

[0047] (3) lysing the liquid to release nucleic acid;

[0048] (4) using a solid phase carrier to absorb the nucleic acid in the liquid;

[0049] (5) washing the solid phase carrier to which the nucleic acid has been adsorbed;

[0050] (6) Elution to obtain the extracted nucleic acid product.

[0051] In some embodiments, the biological sample is a stool sample.

[0052] In some embodiments, the environmental sample is a soil sample or a water sample.

[0053] In some embodiments, the microorganism is one or more of a bacterium, a fungus, and a virus.

[0054] In some embodiments, the diluent includes a buffer salt, which is one or more of phosphate, borate, citrate and Tris, preferably phosphate, and more preferably one or two of NaH2PO3 and Na2HPO4.

[0055] In some embodiments, step (1) also includes centrifugation after adding the diluent, with a relative centrifugal force of 500-5000×g, preferably 500-2000×g, including 500×g, 600×g, 700×g, 800×g, 900×g, 1000×g, 1100×g, 1200×g, 1300×g, 1400×g, 1500×g, 1600×g, 1700×g, 1800×g, 1900×g and 2000×g within the range.

[0056] In some embodiments, about 0.1-5 g of magnetic resin is used per gram of stool sample, preferably about 0.5-3 g of magnetic resin, and more preferably about 0.5-2.5 g of magnetic resin, including 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g, 2.1 g, 2.2 g, 2.3 g, 2.4 g and 2.5 g within the range.

[0057] In some embodiments, about 0.1-5 g of magnetic resin is used per gram of soil sample, preferably about 0.2-3 g of magnetic resin, and more preferably about 0.3-2 g of magnetic resin, including 0.3 g, 0.4, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g and 2 g within the range.

[0058] In some embodiments, the magnetic resin is a magnetic anion exchange resin, and the magnetic anion resin is basic, such as a strongly basic or weakly basic magnetic resin.

[0059] In some embodiments, the matrix type of the magnetic resin is one of styrene, acrylic, acetic, epoxy, vinylpyridine, urea-formaldehyde or vinyl chloride.

[0060] In some embodiments, the pore structure of the magnetic resin is gel-type or macroporous, preferably macroporous.

[0061] In some embodiments, the magnetic core material of the magnetic resin is an iron oxide compound, such as γ-Fe2O3 or Fe3O4.

[0062] In some embodiments, the magnetic resin is a γ-Fe2O3 magnetic macroporous alkaline acrylic anion exchange resin or a Fe3O4 magnetic macroporous alkaline acrylic anion exchange resin, such as a γ-Fe2O3 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a γ-Fe2O3 magnetic macroporous weakly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous weakly alkaline acrylic anion exchange resin.

[0063] In some embodiments, the magnetic resin is MIEX, NDMP-1, or ND100.

[0064] In some embodiments, the magnetic resin binds non-nucleic acid impurities in the sample.

[0065] In some embodiments, the non-nucleic acid impurities are impurities that inhibit the PCR process, such as impurities that inhibit the activity of DNA polymerase.

[0066] In some embodiments, the lysis comprises adding a lysis solution, wherein the lysis solution comprises a chaotropic agent, a buffer salt and a surfactant. The chaotropic agent is, for example, one or more of guanidine salts, urea and lithium perchlorate; the buffer salt is, for example, a weak acid salt and / or a weak base salt; and the surfactant is, for example, one or more of an anionic surfactant, a cationic surfactant, a zwitterionic surfactant and a nonionic surfactant.

[0067] In some embodiments, the lysis further comprises using a mechanical lysis method, such as using glass beads, for example, adding glass beads to the sample and vortexing.

[0068] In some embodiments, the solid phase carrier is a magnetic adsorption medium, preferably magnetic particles, more preferably magnetic beads.

[0069] In some embodiments, the solid support is a silicon-based adsorption medium, such as silica beads, silicon-based membranes.

[0070] In some embodiments, the solid support is an anion exchange material, such as an anion exchange column.

[0071] In some embodiments, when the solid support is magnetic beads, the lysis comprises adding a lysis binding solution.

[0072] In some embodiments, the lysis binding solution comprises a chaotropic agent, a buffer salt, a metal chelator, an alcohol and a surfactant; the chaotropic agent is, for example, one or more of guanidine salts, urea and lithium perchlorate, preferably guanidine salts, more preferably guanidine hydrochloride and / or guanidine isothiocyanate; the buffer salt is, for example, Tris and / or Tris-base; the metal chelator is, for example, disodium EDTA; the alcohol is, for example, ethanol and / or isopropanol, preferably isopropanol; the surfactant is, for example, one or more of anionic surfactants, cationic surfactants, zwitterionic surfactants and nonionic surfactants, preferably nonionic surfactants, more preferably nonionic surfactants and anionic surfactants, and also preferably Tween 20, PEG8000 decyl glycoside, TritonX-100 and / or sodium N-lauroyl sarcosine.

[0073] In some embodiments, the lysis-binding solution further comprises polyvinyl pyrrolidone.

[0074] In some embodiments, when the solid phase carrier is magnetic beads, the step (4) further comprises applying an external magnetic field to separate the magnetic beads that have adsorbed the nucleic acid.

[0075] In some embodiments, when the solid phase carrier is a spin column containing a silicon matrix membrane, the step (3) further comprises centrifugation to separate the adsorbed nucleic acid and impurities.

[0076] In some embodiments, the washing comprises adding a wash solution.

[0077] In some embodiments, the washing solution comprises a buffer component, an alcohol and a chaotropic agent; the buffer component is, for example, Tris and / or Tris-base, preferably Tris-base; the alcohol is, for example, ethanol and / or isopropanol, preferably isopropanol, and the chaotropic agent is, for example, one or more of guanidine salts, urea and lithium perchlorate, preferably guanidine salts, more preferably guanidine hydrochloride and / or guanidine isothiocyanate.

[0078] In some embodiments, the washing further comprises adding a rinse solution.

[0079] In some embodiments, the rinse solution comprises alcohol, preferably ethanol and / or isopropanol, more preferably ethanol.

[0080] In some embodiments, the eluting comprises adding an elution buffer, such as water or TE buffer.

[0081] In some embodiments, the method is partially or entirely performed by manual operation. For example, when the solid phase carrier is a spin column containing a silicon matrix membrane, the method is entirely performed by manual operation.

[0082] In some embodiments, the method is partially or entirely performed on an automatic nucleic acid extraction platform, for example, when the solid phase carrier is a magnetic bead, the method is entirely performed on a nucleic acid extraction instrument, which can achieve full process automation. For example, a magnetic stand can also be used for manual operation.

[0083] In some embodiments, the method is performed partially or entirely on a microfluidic platform, such as on a microfluidic chip.

[0084] In some embodiments, the nucleic acid is DNA and / or RNA, preferably, the nucleic acid is DNA.

[0085] The third aspect of the present application provides a method for extracting nucleic acid based on a magnetic resin, the method comprising the following steps:

[0086] (1) Collect biological or environmental samples, add lysis buffer, and release nucleic acids;

[0087] (2) adding magnetic resin for adsorption, magnetic separation to discard the magnetic resin and its adsorbate, and retaining the liquid;

[0088] (3) adding phenol-chloroform solution, mixing, and centrifuging to obtain the supernatant;

[0089] (4) adding isopropanol / ethanol to the supernatant and centrifuging to obtain DNA precipitate;

[0090] (5) Wash the DNA precipitate and dissolve it with water or TE buffer to obtain the DNA product.

[0091] The fourth aspect of the present application provides a method for extracting nucleic acid based on a magnetic resin, the method comprising the following steps:

[0092] (1) Collect biological or environmental samples, add lysis buffer, and release nucleic acids;

[0093] (2) adding magnetic resin for adsorption, magnetic separation to discard the magnetic resin and its adsorbate, and retaining the liquid;

[0094] (3) Add saturated NaCl solution or a solution containing 5-6M NaCl, mix well, and centrifuge to obtain the supernatant.

[0095] (4) adding isopropanol / ethanol to the supernatant and centrifuging to obtain DNA precipitate;

[0096] (5) Wash the DNA precipitate and dissolve it with water or TE buffer to obtain the DNA product.

[0097] In some embodiments, the phenol-chloroform solution further comprises isoamyl alcohol, i.e., a phenol-chloroform-isoamyl alcohol solution, and the volume ratio of the phenol, chloroform and isoamyl alcohol is 25:24:1.

[0098] In some embodiments, the ethanol is 95-100% (volume fraction) ethanol.

[0099] In some embodiments, washing the DNA precipitate comprises washing the DNA precipitate with ethanol, preferably 75-95% (volume fraction) ethanol.

[0100] In some embodiments, the method further comprises drying the residual ethanol and adding ddH2O or TE buffer to dissolve the DNA.

[0101] In some embodiments, the biological sample is a stool sample.

[0102] In some embodiments, the environmental sample is a soil sample or a water sample.

[0103] In some embodiments, the lysis buffer comprises a chaotropic agent, a buffer salt, and a surfactant.

[0104] In some embodiments, step (1) of the third aspect and the fourth aspect further comprises using a mechanical lysis method, such as using glass beads, for example, adding glass beads to the sample and vortexing.

[0105] In some embodiments, step (1) of the third aspect and the fourth aspect further includes centrifugation after adding the lysate, with a relative centrifugal force of 8000-20000×g, preferably 8000-18000×g, including 8000×g, 8500×g, 9000×g, 9500×g, 10000×g, 11000×g, 12000×g, 13000×g, 14000×g, 15000×g, 16000×g, 17000×g and 18000×g within the range.

[0106] In some embodiments, about 0.1-5 g of magnetic resin is used per gram of stool sample, preferably about 0.5-3 g of magnetic resin, and more preferably about 0.5-2.5 g of magnetic resin, including 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g, 2.1 g, 2.2 g, 2.3 g, 2.4 g and 2.5 g within the range.

[0107] In some embodiments, about 0.1-5 g of magnetic resin is used per gram of soil sample, preferably about 0.2-3 g of magnetic resin, and more preferably about 0.3-2 g of magnetic resin, including 0.3 g, 0.4, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g and 2 g within the range.

[0108] In some embodiments, the saturated NaCl solution or the 5-6 M NaCl solution further comprises chloroform.

[0109] In some embodiments, the magnetic resin is a magnetic anion exchange resin, and the magnetic anion resin is basic, such as a strongly basic or weakly basic magnetic resin.

[0110] In some embodiments, the matrix type of the magnetic resin is one of styrene, acrylic, acetic, epoxy, vinylpyridine, urea-formaldehyde or vinyl chloride.

[0111] In some embodiments, the pore structure of the magnetic resin is gel-type or macroporous, preferably macroporous.

[0112] In some embodiments, the magnetic core material of the magnetic resin is an iron oxide compound, such as γ-Fe2O3 or Fe3O4.

[0113] In some embodiments, the magnetic resin is a γ-Fe2O3 magnetic macroporous alkaline acrylic anion exchange resin or a Fe3O4 magnetic macroporous alkaline acrylic anion exchange resin, such as a γ-Fe2O3 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a γ-Fe2O3 magnetic macroporous weakly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous weakly alkaline acrylic anion exchange resin.

[0114] In some embodiments, the magnetic resin is MIEX, NDMP-1, or ND100.

[0115] In some embodiments, the magnetic resin binds non-nucleic acid impurities in the sample.

[0116] In some embodiments, the non-nucleic acid impurities are impurities that inhibit the PCR process, such as impurities that inhibit the activity of DNA polymerase.

[0117] The fifth aspect of the present application provides a method for extracting nucleic acid based on a magnetic resin, the method comprising the following steps:

[0118] (1) Collect biological or environmental samples, add diluent, and release or disperse the microorganisms in the samples;

[0119] (2) adding magnetic resin for adsorption, magnetic separation to discard the magnetic resin and its adsorbate, and retaining the liquid;

[0120] (3) lysing the liquid to release nucleic acid;

[0121] (4) adding phenol-chloroform solution, mixing, and centrifuging to obtain the supernatant;

[0122] (5) adding isopropanol / ethanol to the supernatant and centrifuging to obtain DNA precipitate;

[0123] (6) Wash the DNA precipitate and dissolve it with water or TE buffer to obtain the DNA product.

[0124] The sixth aspect of the present application provides a method for extracting nucleic acid based on a magnetic resin, the method comprising the following steps:

[0125] (1) Collect biological or environmental samples, add diluent, and release or disperse the microorganisms in the samples;

[0126] (2) adding magnetic resin for adsorption, magnetic separation to discard the magnetic resin and its adsorbate, and retaining the liquid;

[0127] (3) lysing the liquid to release nucleic acid;

[0128] (4) adding a saturated NaCl solution or a solution containing 5-6 M NaCl, mixing, and centrifuging to obtain the supernatant;

[0129] (5) adding isopropanol / ethanol to the supernatant and centrifuging to obtain DNA precipitate;

[0130] (6) Wash the DNA precipitate and dissolve it with water or TE buffer to obtain the DNA product.

[0131] In some embodiments, the phenol-chloroform solution further comprises isoamyl alcohol, i.e., a phenol-chloroform-isoamyl alcohol solution, and the volume ratio of the phenol, chloroform and isoamyl alcohol is 25:24:1.

[0132] In some embodiments, the ethanol is 95-100% (volume fraction) ethanol.

[0133] In some embodiments, washing the DNA precipitate comprises washing the DNA precipitate with ethanol, preferably 75-95% (volume fraction) ethanol.

[0134] In some embodiments, the method further comprises drying the residual ethanol and adding ddH2O or TE buffer to dissolve the DNA.

[0135] In some embodiments, the biological sample is a stool sample.

[0136] In some embodiments, the environmental sample is a soil sample or a water sample.

[0137] In some embodiments, the microorganism is one or more of a bacterium, a fungus, and a virus.

[0138] In some embodiments, the lysing comprises adding a lysis solution comprising a chaotropic agent, a buffer salt and a surfactant.

[0139] In some embodiments, the lysis further comprises using a mechanical lysis method, such as using glass beads, for example, adding glass beads to the sample and vortexing.

[0140] In some embodiments, step (1) of the fifth aspect and the sixth aspect also includes centrifugation after adding the diluent, with a relative centrifugal force of 500-5000×g, preferably 500-2000×g, including 500×g, 600×g, 700×g, 800×g, 900×g, 1000×g, 1100×g, 1200×g, 1300×g, 1400×g, 1500×g, 1600×g, 1700×g, 1800×g, 1900×g and 2000×g within the range.

[0141] In some embodiments, about 0.1-5 g of magnetic resin is used per gram of stool sample, preferably about 0.5-3 g of magnetic resin, and more preferably about 0.5-2.5 g of magnetic resin, including 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g, 2.1 g, 2.2 g, 2.3 g, 2.4 g and 2.5 g within the range.

[0142] In some embodiments, about 0.1-5 g of magnetic resin is used per gram of soil sample, preferably about 0.2-3 g of magnetic resin, and more preferably about 0.3-2 g of magnetic resin, including 0.3 g, 0.4, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g and 2 g within the range.

[0143] In some embodiments, the saturated NaCl solution or the 5-6 M NaCl solution further comprises chloroform.

[0144] In some embodiments, the magnetic resin is a magnetic anion exchange resin, and the magnetic anion resin is basic, such as a strongly basic or weakly basic magnetic resin.

[0145] In some embodiments, the matrix type of the magnetic resin is one of styrene, acrylic, acetic, epoxy, vinylpyridine, urea-formaldehyde or vinyl chloride.

[0146] In some embodiments, the pore structure of the magnetic resin is gel-type or macroporous, preferably macroporous.

[0147] In some embodiments, the magnetic core material of the magnetic resin is an iron oxide compound, such as γ-Fe2O3 or Fe3O4.

[0148] In some embodiments, the magnetic resin is a γ-Fe2O3 magnetic macroporous alkaline acrylic anion exchange resin or a Fe3O4 magnetic macroporous alkaline acrylic anion exchange resin, such as a γ-Fe2O3 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a γ-Fe2O3 magnetic macroporous weakly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous weakly alkaline acrylic anion exchange resin.

[0149] In some embodiments, the magnetic resin is MIEX, NDMP-1, or ND100.

[0150] In some embodiments, the magnetic resin binds non-nucleic acid impurities in the sample.

[0151] In some embodiments, the non-nucleic acid impurities are impurities that inhibit the PCR process, such as impurities that inhibit the activity of DNA polymerase.

[0152] A seventh aspect of the present application provides a kit, which includes a lysis solution, a washing solution, an elution solution and a magnetic resin.

[0153] In some embodiments, the kit is a nucleic acid extraction kit, preferably a DNA extraction kit.

[0154] In some embodiments, the magnetic resin is a magnetic anion exchange resin, and the magnetic anion resin is basic, such as a strongly basic or weakly basic magnetic resin.

[0155] In some embodiments, the matrix type of the magnetic resin is one of styrene, acrylic, acetic, epoxy, vinylpyridine, urea-formaldehyde or vinyl chloride.

[0156] In some embodiments, the pore structure of the magnetic resin is gel-type or macroporous, preferably macroporous.

[0157] In some embodiments, the magnetic core material of the magnetic resin is an iron oxide compound, such as γ-Fe2O3 or Fe3O4.

[0158] In some embodiments, the magnetic resin is a γ-Fe2O3 magnetic macroporous alkaline acrylic anion exchange resin or a Fe3O4 magnetic macroporous alkaline acrylic anion exchange resin, such as a γ-Fe2O3 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a γ-Fe2O3 magnetic macroporous weakly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous weakly alkaline acrylic anion exchange resin.

[0159] In some embodiments, the magnetic resin is MIEX, NDMP-1, or ND100.

[0160] In some embodiments, the lysate comprises a chaotropic agent, a buffer salt and a surfactant. The chaotropic agent is, for example, one or more of guanidine salt, urea and lithium perchlorate, preferably guanidine salt, more preferably guanidine hydrochloride and / or guanidine isothiocyanate; the buffer salt is one or more of phosphate, borate, citrate and Tris, preferably phosphate, more preferably one or two of NaH2PO3 and Na2HPO4; the surfactant is, for example, one or more of anionic surfactant, cationic surfactant, zwitterionic surfactant and nonionic surfactant, preferably nonionic surfactant, more preferably Tween 20 and decyl glycoside.

[0161] In some embodiments, the washing solution comprises a buffer component and a chaotropic agent, wherein the buffer component is, for example, Tris and / or Tris-base, preferably Tris-base; the chaotropic agent is, for example, one or more of guanidine salts, urea and lithium perchlorate, preferably guanidine salts, more preferably guanidine hydrochloride and / or guanidine isothiocyanate.

[0162] In some embodiments, the washing liquid further comprises an alcohol, such as ethanol and / or isopropanol, preferably isopropanol.

[0163] In some embodiments, the kit further comprises a rinse solution.

[0164] In some embodiments, the rinse solution comprises alcohol, preferably ethanol and / or isopropanol, more preferably ethanol.

[0165] In some embodiments, the kit further comprises an elution solution.

[0166] In some embodiments, the eluent is, for example, water or TE buffer.

[0167] In some embodiments, the kit further comprises a lysis and binding solution.

[0168] In some embodiments, the lysis binding solution comprises a chaotropic agent, a buffer salt, a metal chelator, an alcohol and a surfactant, wherein the chaotropic agent is, for example, one or more of guanidine salts, urea and lithium perchlorate, preferably guanidine salts, more preferably guanidine hydrochloride and / or guanidine isothiocyanate; the buffer salt is, for example, Tris and / or Tris-base; the metal chelator is, for example, disodium EDTA; the alcohol is, for example, ethanol and / or isopropanol, preferably isopropanol; the surfactant is, for example, one or more of anionic surfactants, cationic surfactants, zwitterionic surfactants and nonionic surfactants, preferably nonionic surfactants, more preferably nonionic surfactants and anionic surfactants, more preferably Tween 20, PEG8000 decyl glycoside, TritonX-100 and sodium N-lauroyl sarcosine.

[0169] In some embodiments, the lysis-binding solution further comprises polyvinyl pyrrolidone.

[0170] In some embodiments, the kit further comprises a solid phase carrier, which is at least one of magnetic beads, silica beads, spin columns and anion exchange columns.

[0171] In some embodiments, the kit further comprises a sample lysis tube comprising glass beads, preferably acid-washed glass beads.

[0172] In some embodiments, the kit further comprises a diluent.

[0173] In some embodiments, the diluent comprises a buffer salt, which is one or more of phosphate, borate, citrate and Tris, preferably phosphate, and more preferably one or two of NaH2PO3 and Na2HPO4.

[0174] In some embodiments, the kit is used to perform the method described in the first aspect or the second aspect.

[0175] An eighth aspect of the present application provides a kit, comprising a lysis solution, a magnetic resin and a phenol-chloroform solution.

[0176] In some embodiments, the kit is a nucleic acid extraction kit, preferably a DNA extraction kit.

[0177] In some embodiments, the magnetic resin is a magnetic anion exchange resin, and the magnetic anion resin is basic, such as a strongly basic or weakly basic magnetic resin.

[0178] In some embodiments, the matrix type of the magnetic resin is one of styrene, acrylic, acetic, epoxy, vinylpyridine, urea-formaldehyde or vinyl chloride.

[0179] In some embodiments, the pore structure of the magnetic resin is gel-type or macroporous, preferably macroporous.

[0180] In some embodiments, the magnetic core material of the magnetic resin is an iron oxide compound, such as γ-Fe2O3 or Fe3O4.

[0181] In some embodiments, the magnetic resin is a γ-Fe2O3 magnetic macroporous alkaline acrylic anion exchange resin or a Fe3O4 magnetic macroporous alkaline acrylic anion exchange resin, such as a γ-Fe2O3 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a γ-Fe2O3 magnetic macroporous weakly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous weakly alkaline acrylic anion exchange resin.

[0182] In some embodiments, the magnetic resin is MIEX, NDMP-1, or ND100.

[0183] In some embodiments, the lysis buffer comprises a chaotropic agent, a buffer salt, and a surfactant.

[0184] In some embodiments, the phenol-chloroform solution further comprises isoamyl alcohol, i.e., a phenol-chloroform-isoamyl alcohol solution, and the volume ratio of the phenol, chloroform and isoamyl alcohol is 25:24:1.

[0185] In some embodiments, the kit further comprises ethanol and / or isopropanol.

[0186] In some embodiments, the kit further comprises a sample lysis tube comprising glass beads, preferably acid-washed glass beads.

[0187] In some embodiments, the kit further comprises a diluent.

[0188] In some embodiments, the diluent comprises a buffer salt, which is one or more of phosphate, borate, citrate and Tris, preferably phosphate, and more preferably one or two of NaH2PO3 and Na2HPO4.

[0189] In some embodiments, the kit is used to implement the method described in the third aspect or the fifth aspect.

[0190] A ninth aspect of the present application provides a kit, comprising a lysis solution, a magnetic resin, and a saturated NaCl solution or a 5-6 M NaCl solution.

[0191] In some embodiments, the lysis buffer comprises a chaotropic agent, a buffer salt, and a surfactant.

[0192] In some embodiments, the saturated NaCl solution or the 5-6 M NaCl solution further comprises chloroform.

[0193] In some embodiments, the magnetic resin is a magnetic anion exchange resin, and the magnetic anion resin is basic, such as a strongly basic or weakly basic magnetic resin.

[0194] In some embodiments, the matrix type of the magnetic resin is one of styrene, acrylic, acetic, epoxy, vinylpyridine, urea-formaldehyde or vinyl chloride.

[0195] In some embodiments, the pore structure of the magnetic resin is gel-type or macroporous, preferably macroporous.

[0196] In some embodiments, the magnetic core material of the magnetic resin is an iron oxide compound, such as γ-Fe2O3 or Fe3O4.

[0197] In some embodiments, the magnetic resin is a γ-Fe2O3 magnetic macroporous alkaline acrylic anion exchange resin or a Fe3O4 magnetic macroporous alkaline acrylic anion exchange resin, such as a γ-Fe2O3 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a γ-Fe2O3 magnetic macroporous weakly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous strongly alkaline acrylic anion exchange resin, or a Fe3O4 magnetic macroporous weakly alkaline acrylic anion exchange resin.

[0198] In some embodiments, the magnetic resin is MIEX, NDMP-1, or ND100.

[0199] In some embodiments, the kit is a nucleic acid extraction kit, preferably a DNA extraction kit.

[0200] In some embodiments, the kit further comprises ethanol and / or isopropanol.

[0201] In some embodiments, the kit further comprises a sample lysis tube comprising glass beads, preferably acid-washed glass beads.

[0202] In some embodiments, the kit further comprises a diluent.

[0203] In some embodiments, the diluent comprises a buffer salt, which is one or more of phosphate, borate, citrate and Tris, preferably phosphate, and more preferably one or two of NaH2PO3 and Na2HPO4.

[0204] In some embodiments, the kit is used to implement the method described in the fourth aspect or the sixth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0205] Figure 1 :Technical roadmap for nucleic acid extraction method based on magnetic resin and first release bacteria;

[0206] Figure 2 :Technical roadmap for nucleic acid extraction method based on magnetic resin with nucleic acid release first;

[0207] Figure 3 : Electrophoresis of DNA extraction products from feces and soil samples based on technical route 1;

[0208] Figure 4 : Electrophoresis of PCR amplification products of bacterial 16S rRNA loci of DNA extraction products from feces and soil samples based on technical route 1;

[0209] Figure 5 : Gel electrophoresis of DNA amplification products obtained by treating feces and soil samples with different concentrations of NDMP-1 magnetic anion exchange resin;

[0210] Figure 6 : Electrophoresis of DNA extraction products from feces and soil samples based on technical route 2;

[0211] Figure 7 : Electrophoresis of bacterial 16S rRNA site PCR amplification products of DNA extraction products from feces and soil samples based on technical route 2.

[0212] Specific implementation (embodiment)

[0213] The technical solution of the present application is further explained below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are merely simple examples of the present application and do not represent or limit the scope of protection of the present application. The scope of protection of the present application shall be subject to the claims.

[0214] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0215] Reagent preparation

[0216] Sample lysis tube: contains glass beads (Sigma, acid-washed glass beads), with a diameter of 0.4-0.6 mm and a mass of 0.6 g / tube;

[0217] Dilution 1: 200 mM NaH2PO3; 200 mM Na2HPO4; pH 7.0;

[0218] Lysis and binding solution: 75mM Tris-base; 10mM EDTA·2Na·2H2O; 0.1% PEG8000; 3M guanidine thiocyanate; 25% isopropanol; 10mM sodium N-lauroyl sarcosinate; 10% Tween20; 0.01% Triton X-100; 1.5% polyvinyl pyrrolidone; pH 7.0 of lysis and binding solution;

[0219] Dilution 2 (lysis buffer): 200 mM NaH2PO3; 200 mM Na2HPO4; 3 M guanidine thiocyanate; 5% decyl glucoside; pH 7.0;

[0220] Magnetic anion resin solution: NDMP-1 magnetic resin (Jiangsu Guoxin Innovation Materials Research Center Co., Ltd., a strong basic anion exchange resin loaded with Fe3O4, chloride type, particle size range 80μm to 200μm, Fe3O4 loading is 10%, see Selection of magnetic anion exchange resins for the removal of dissolved organic and inorganic matters [J]. Journal of Environmental Sciences, 2012, 24 (11): 1972-1980.), 1-400 mg / reaction; MIEX magnetic resin (Orica, MIEX, acrylic acid quaternary ammonium type ion exchange resin, strong basic anion resin, 150-180μm, magnet is γ-Fe2O3, and the ion exchanged with the dissolved organic matter is chloride ion (Cl － ), which can be easily regenerated by contact with a regeneration solution containing chloride ions) 1-400 mg / reaction; ND100 magnetic resin (Suzhou Bojie Resin Technology Co., Ltd., ND100, Fe3O4 magnetic macroporous acrylic weakly basic anion exchange resin) 1-400 mg / reaction;

[0221] Magnetic bead solution: superparamagnetic silicon oxide hydroxy nanoparticles (Rebecil, MagOH 60), with a diameter of 20-1000 nm and a concentration of 50 mg / mL;

[0222] Washing solution: 20 mM Tris-base; 60% isopropanol; 2 M guanidine hydrochloride; washing solution pH 7.5;

[0223] Rinsing solution: 10 mM sodium chloride; 80% ethanol;

[0224] Eluent: Nuclease-free Water.

[0225] Example 1

[0226] The nucleic acid was extracted using the technique of releasing bacteria first and then removing impurities 1 ( Figure 1 ), the specific steps are as follows:

[0227] 1. Sample pretreatment

[0228] Fecal sample pretreatment: Weigh 150 mg of mouse fecal sample into a sample lysis tube, add 1200 μl of diluent 1, and place the sample lysis tube on a vortexer and vortex at maximum speed for 2 minutes. After vortexing, centrifuge at 1,000×g for 2 minutes to obtain supernatant 1.

[0229] Soil sample pretreatment: Weigh 250 mg of wheat surface soil sample into a sample lysis tube, add 800 μl of diluent 1, and place the sample lysis tube on a vortexer and vortex at maximum speed for 2 min. After vortexing, centrifuge at 1,000 × g for 2 min to obtain supernatant 2.

[0230] 2. Take 500 μl / well of supernatant 1 and add it to a 96-well plate. Then add 120 mg / well magnetic resins (NDMP-1, MIEX and ND100, three magnetic resins corresponding to three 96-well plates) to the plate. Place the 96-well plate on the first plate position of the VNP-96 automatic nucleic acid extractor of Novazonic Biotech Co., Ltd., place an empty 96-well plate on the second plate position, install the magnetic rod cover, and confirm that it is in place. Run the pretreatment Pre program in Table 1. The control group without magnetic resin does not have this step.

[0231] Table 1 Pre program

[0232]

[0233] 3. After the Pre program is finished, take out the 96-well plate in the first plate position, add 500μl lysis solution (diluent 2) to each well, and define it as the experimental group lysis plate; for the control group without magnetic resin, take 500μl supernatant 1 in step 1 and add it to a 96-well plate, add 500μl lysis binding solution to each well, and define it as the control group lysis plate. Dispense 700μl magnetic bead solution, 700μl washing solution, 700μl rinsing solution, and 70μl elution solution into different 96-well plates, and define them as magnetic bead plate, washing plate, rinsing plate 1, rinsing plate 2, and elution plate respectively;

[0234] Place the magnetic bead plate, washing plate, rinse plate 1, rinse plate 2, and elution plate in the corresponding different plate positions of the VNP-96 nucleic acid automatic extractor, install the magnetic rod cover, and confirm that it is installed in place, run the nucleic acid extraction Ext program in Table 2, perform automated extraction, and obtain fecal DNA. Among them,

[0235] Plate position 1: experimental group lysis plate / control group lysis plate;

[0236] The second board position: magnetic bead board;

[0237] Plate position 3, washing plate;

[0238] Plate position 4, rinsing plate 1;

[0239] Plate position 5, rinsing plate 2;

[0240] Plate position 6: elution plate.

[0241] Table 2Ext program

[0242]

[0243] 4. Take 500 μl of supernatant 2 and add it to a 96-well plate. The subsequent extraction process is the same as the above steps 2-3 to obtain soil DNA.

[0244] 5. Detection and analysis of extracted products

[0245] First, the concentration and purity of DNA extracted from feces and soil samples were measured on a NanoDrop micro-volume UV / Vis spectrophotometer, and the results were shown in Table 3. Then, 5 μL of the extracted product was electrophoresed in a 1% agarose gel at 110 V, and then imaged and observed in a UV gel imaging system. The gel electrophoresis results of the extracted product were shown in Table 3. Figure 3 shown.

[0246] Table 3 DNA concentration and purity of soil and feces DNA extraction products

[0247]

[0248]

[0249] 2 μL of the extracted product was taken as template DNA, and the bacterial V3-V4 variable region 16S sequencing PCR amplification was performed according to the reaction system and procedure in Table 4 and Table 5, wherein Table 6 shows the specific sequences of the upstream and downstream amplification primers, and 5 μL of the PCR amplification product was taken and electrophoresed in 1% agarose gel electrophoresis at a voltage of 110 V, and then imaged and observed in a UV gel imaging system, and the gel electrophoresis results of the PCR amplification product were obtained as shown in Figure 4 shown.

[0250] The configuration of the PCR detection reaction system was based on the 2×Phanta Max Master Mix (VAZYME, P515) kit.

[0251] Table 4 2×Phanta Max Master Mix reaction system

[0252] Components volume 2×Phanta Max Master Mix 25μl Upstream primer (10 μM) 2μl Downstream primer (10 μM) 2μl Template DNA 2μl <![CDATA[ddH2O]]> Make up to 50 μl

[0253] Table 5 PCR reaction program:

[0254]

[0255] Table 6 Bacterial V3-V4 variable region 16S sequencing amplification primer sequences

[0256] 338F SEQ ID NO.1 ACTCCTACGGGAGGCAGCA 806R SEQ ID NO.2 GGACTACHVGGGTWTCTAAT

[0257] 6. Results Analysis

[0258] Analysis of DNA extraction results from feces and soil:

[0259] As shown in Table 3 and Figure 3 As shown, the method provided in the present application can extract DNA from feces and soil samples. For the purity analysis of DNA from feces and soil samples, after treatment with three different magnetic anion exchange resins NDMP-1, MIEX and ND100, the purity of DNA was significantly increased, specifically, A260 / A280 and A260 / A230 were significantly increased, indicating that after treatment with the three magnetic anion exchange resins, the impurities in the soil and feces DNA extraction products were significantly reduced.

[0260] like Figure 4 The DNA extracted from soil and feces samples contains bacterial DNA. PCR amplification of the bacterial 16SrRNA variable region V3-V4 is performed to identify bacterial diversity. When there are too many impurities in the DNA extraction product, PCR amplification will be inhibited. Figure 4 The results showed that when there was no magnetic resin treatment, the DNA extraction products of feces and soil samples could not be PCR amplified by 338F / 806R primers. After the soil and feces samples were treated with three magnetic anion exchange resins NDMP-1, MIEX and ND100, the DNA extraction products could be effectively PCR amplified by 338F / 806R primers. This shows that NDMP-1, MIEX and ND100 magnetic resin treatment can not only effectively reduce impurities in feces and soil samples, improve the amplification ability of DNA extraction products, but also realize full-process automated extraction.

[0261] Example 2

[0262] Different concentrations of NDMP-1 magnetic anion exchange resin were set, which were 75 mg, 100 mg, 125 mg and 150 mg respectively. The sample treatment and reaction process were the same as in Example 1, and the DNA extraction products after 250 mg of soil and 150 mg of feces samples were treated with different concentrations of NDMP-1 magnetic resin were obtained. The bacterial 16S rRNA site 338F / 806R primers were used for PCR amplification. The amplification system and procedure were the same as in Example 1. The amplified products were subjected to agarose gel electrophoresis to obtain the results as shown in the following figure. Figure 5 shown.

[0263] Figure 5 The results showed that when 150 mg of NDMP-1 magnetic anion exchange resin was used to treat soil and feces samples, the PCR amplification ability of 338F / 806R was significantly stronger than that of 75-125 mg of resin, indicating that for 150 mg of feces samples and 250 mg of soil samples, the processing ability of 150 mg of NDMP-1 magnetic anion exchange resin for soil and feces samples is more suitable.

[0264] Example 3

[0265] The nucleic acid was extracted using the technique 2 of releasing nucleic acid first and then removing impurities ( Figure 2 ), the specific steps are as follows:

[0266] 1. Sample pretreatment

[0267] Fecal sample pretreatment: Weigh 150 mg of mouse fecal sample into a sample lysis tube, add 1200 μl of diluent 2, and place the sample lysis tube on a vortexer and vortex at maximum speed for 2 min. After vortexing, heat in a water bath at 65°C for 5 min, and centrifuge at 12,000×g for 2 min to obtain supernatant 1.

[0268] Soil sample pretreatment: Weigh 250 mg of wheat surface soil sample into a sample lysis tube, add 800 μl of diluent 2, and place the sample lysis tube on a vortexer and vortex at maximum speed for 2 min. After vortexing, centrifuge at 12,000 × g for 2 min to obtain supernatant 2.

[0269] 2. The subsequent magnetic resin (NDMP-1) impurity removal and nucleic acid extraction process were the same as in Example 1, and the DNA extraction product after 250 mg of soil and 150 mg of feces samples were treated with NDMP-1 magnetic resin was obtained. The concentration and purity of the DNA product were determined, as shown in Table 7. The DNA extraction product was subjected to agarose gel electrophoresis to obtain the gel image as shown in Table 7. Figure 6 PCR amplification was performed using the bacterial 16S rRNA site 338F / 806R primers, and the amplification system and procedure were the same as in Example 1. The amplified product was subjected to agarose gel electrophoresis to obtain the result as shown in FIG. Figure 7 shown.

[0270] Table 7 Concentration and purity of soil and feces DNA extraction products

[0271]

[0272] 3. Results Analysis

[0273] The data in Table 7 show the purity and concentration of DNA extraction products from feces and soil samples. After treatment with the magnetic anion exchange resin NDMP-1, the purity of DNA was significantly increased, as shown by the significant increase in A260 / A280 and A260 / A230, indicating that after treatment with the NDMP-1 magnetic anion exchange resin, the impurities in soil and feces DNA were significantly reduced.

[0274] Figure 7 The results showed that when there was no magnetic resin treatment, the DNA extraction products of feces and soil samples could not be PCR amplified by 338F / 806R primers. After the soil and feces samples were treated with NDMP-1 magnetic anion exchange resin, the DNA extraction products could be effectively PCR amplified by 338F / 806R primers. This shows that NDMP-1 treatment can not only effectively reduce impurities in feces and soil samples, improve the amplification ability of DNA extraction products, but also realize full-process automated extraction.

Claims

1. A method for extracting nucleic acid based on magnetic resin, the method comprising the following steps: (1) Collecting biological samples or environmental samples, adding lysis solution, and releasing nucleic acid, wherein the biological sample is a fecal sample and the environmental sample is a soil sample; (2) adding magnetic resin for adsorption, magnetic separation and discarding the magnetic resin and its adsorbent, retaining the liquid, using 0.1-1 g of magnetic resin per gram of the stool sample, and using 0.1-0.6 g of magnetic resin per gram of the soil sample, and the magnetic resin is MIEX, NDMP-1 or ND100; (3) using a solid phase carrier to adsorb the nucleic acid in the liquid; (4) Cleaning the solid phase carrier that has adsorbed nucleic acid; (5) Elution to obtain nucleic acid products.

2. A method for extracting nucleic acid based on magnetic resin, the method comprising the following steps: (1) Collecting a biological sample or an environmental sample, adding a diluent to release or disperse the microorganisms in the sample, wherein the diluent includes a buffer salt, the biological sample is a fecal sample, and the environmental sample is a soil sample; (2) adding magnetic resin for adsorption, magnetic separation and discarding the magnetic resin and its adsorbent, retaining the liquid, using 0.1-1 g of magnetic resin per gram of the stool sample, and using 0.1-0.6 g of magnetic resin per gram of the soil sample, and the magnetic resin is MIEX, NDMP-1 or ND100; (3) lysing the liquid to release nucleic acid; (4) using a solid phase carrier to absorb the nucleic acid in the liquid; (5) Cleaning the solid phase carrier that has adsorbed nucleic acid; (6) Elution to obtain nucleic acid products.

3. A method for extracting nucleic acid based on magnetic resin, the method comprising the following steps: (1) Collecting biological samples or environmental samples, adding lysis solution, and releasing nucleic acid, wherein the biological sample is a fecal sample and the environmental sample is a soil sample; (2) adding magnetic resin for adsorption, magnetic separation and discarding the magnetic resin and its adsorbent, retaining the liquid, using 0.1-1 g of magnetic resin per gram of the stool sample, and using 0.1-0.6 g of magnetic resin per gram of the soil sample, and the magnetic resin is MIEX, NDMP-1 or ND100; (3) Add phenol-chloroform solution, mix well, and centrifuge to obtain the supernatant; (4) adding isopropanol and / or ethanol to the supernatant and centrifuging to obtain DNA precipitate; (5) Wash the DNA precipitate and dissolve it with water or TE buffer to obtain the DNA product.

4. A method for extracting nucleic acid based on magnetic resin, the method comprising the following steps: (1) Collecting biological samples or environmental samples, adding lysis solution, and releasing nucleic acid, wherein the biological sample is a fecal sample and the environmental sample is a soil sample; (2) adding magnetic resin for adsorption, magnetic separation and discarding the magnetic resin and its adsorbent, retaining the liquid, using 0.1-1 g of magnetic resin per gram of the stool sample, and using 0.1-0.6 g of magnetic resin per gram of the soil sample, and the magnetic resin is MIEX, NDMP-1 or ND100; (3) Add saturated NaCl solution or a solution containing 5-6M NaCl, mix well, and centrifuge to obtain the supernatant. (4) adding isopropanol and / or ethanol to the supernatant and centrifuging to obtain DNA precipitate; (5) Wash the DNA precipitate and dissolve it with water or TE buffer to obtain the DNA product.

5. A method for extracting nucleic acid based on magnetic resin, the method comprising the following steps: (1) Collecting a biological sample or an environmental sample, adding a diluent to release or disperse the microorganisms in the sample, wherein the diluent includes a buffer salt, the biological sample is a fecal sample, and the environmental sample is a soil sample; (2) adding magnetic resin for adsorption, magnetic separation and discarding the magnetic resin and its adsorbent, retaining the liquid, using 0.1-1 g of magnetic resin per gram of the stool sample, and using 0.1-0.6 g of magnetic resin per gram of the soil sample, and the magnetic resin is MIEX, NDMP-1 or ND100; (3) lysing the liquid to release nucleic acid; (4) Add phenol-chloroform solution, mix well, and centrifuge to obtain the supernatant; (5) Add isopropanol and / or ethanol to the supernatant and centrifuge to obtain DNA precipitate; (6) Wash the DNA precipitate and dissolve it with water or TE buffer to obtain the DNA product.

6. A method for extracting nucleic acid based on magnetic resin, the method comprising the following steps: (1) Collecting a biological sample or an environmental sample, adding a diluent to release or disperse the microorganisms in the sample, wherein the diluent includes a buffer salt, the biological sample is a fecal sample, and the environmental sample is a soil sample; (2) adding magnetic resin for adsorption, magnetic separation and discarding the magnetic resin and its adsorbent, retaining the liquid, using 0.1-1 g of magnetic resin per gram of the stool sample, and using 0.1-0.6 g of magnetic resin per gram of the soil sample, and the magnetic resin is MIEX, NDMP-1 or ND100; (3) lysing the liquid to release nucleic acid; (4) Add saturated NaCl solution or a solution containing 5-6 M NaCl, mix well, and centrifuge to obtain the supernatant; (5) Add isopropanol and / or ethanol to the supernatant and centrifuge to obtain DNA precipitate; (6) Wash the DNA precipitate and dissolve it with water or TE buffer to obtain the DNA product.

7. The method according to any one of claims 1 and 3 to 4, wherein the lysate comprises a chaotropic agent, a buffer salt and a surfactant.

8. The method according to any one of claims 1 and 3 to 4, wherein step (1) further comprises centrifugation after adding the lysate.

9. The method according to any one of claims 2 and 5 to 6, wherein the buffer salt is phosphate.

10. The method of claim 9, wherein the phosphate is one or both of NaH2PO3 and Na2HPO4.

11. The method according to any one of claims 2 and 5 to 6, wherein step (1) further comprises centrifugation after adding the diluent.

12. The method according to any one of claims 1 to 2, wherein the solid phase carrier is a magnetic adsorption medium, or the solid phase carrier is a silicon-based adsorption medium, or the solid phase carrier is an anion exchange material.

13. The method according to claim 12, wherein the solid phase carrier is magnetic beads, and the step (3) further comprises adding a lysis and binding solution, wherein the lysis and binding solution comprises a chaotropic agent, a buffer salt, a metal chelating agent, an alcohol and a surfactant.

14. The method according to any one of claims 1-2, wherein the washing comprises adding a washing solution, wherein the washing solution comprises a buffer component, an alcohol and a chaotropic agent.

15. The method of claim 14, wherein the cleaning further comprises adding a rinsing solution, wherein the rinsing solution comprises ethanol.

16. The method according to any one of claims 1-2, wherein the elution comprises adding an eluent, and the eluent is water or TE buffer.

17. The method according to any one of claims 1 to 6, wherein the method is partially or entirely performed by manual operation, or the method is partially or entirely performed on an automatic nucleic acid extraction platform, or the method is partially or entirely performed on a microfluidic platform.

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