Magnetic bead extraction reagent, kit, and extraction method

Abstract

Provided in the present invention are a magnetic bead extraction reagent, a corresponding kit, and an extraction method. The magnetic bead extraction reagent comprises a magnetic bead solution I and a magnetic bead solution II, wherein the magnetic bead solution I includes magnetic beads I used for performing impurity removal treatment on a nucleic acid-containing biological sample to be extracted, and the magnetic bead solution II includes magnetic beads II used for performing nucleic acid adsorption.

Description

A magnetic bead extraction reagent, kit and extraction method TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, and particularly relates to a magnetic bead extraction reagent, kit and extraction method. BACKGROUND

[0002] Proteins are the basic substances of life, and nucleic acids are the original templates for expressing proteins. High-quality extraction of nucleic acids from complex samples, various pathogens and cells is the cornerstone of high-quality transcription and translation of proteins.

[0003] The manual nucleic acid extraction method is the most common method in the field, and common methods mainly include alkali lysis, boiling lysis, small one-step extraction, etc. From a technical point of view, alkali lysis is a better method for nucleic acid extraction. Currently, nucleic acid extraction kits such as (Tiangen) use this method. Manual extraction of nucleic acid has the following general process: (1) centrifuge, manually remove cells; (2) use P1 suspension to shake the bacteria; (3) use P2 alkali to destroy the cell wall, use P3 acid neutralizing solution to neutralize the extraction combined solution, and centrifuge to remove solid impurities; (4) adsorb and wash away soluble impurities with a silicon-based membrane, and elute.

[0004] However, the manual nucleic acid extraction method has the problem of wasting a large amount of human resources, and is limited by characteristics such as small throughput, high extraction cost, etc. Therefore, automatic extraction is a better choice, which can effectively reduce the cost of human resources.

[0005] Currently, the separation technology using magnetic beads as carriers has become the preferred method for extracting nucleic acids. The magnetic bead extraction method does not require contact with toxic reagents and is simple to operate, easy to automate, and can quickly and efficiently extract high-quality DNA samples in a short time. It is an important development direction for high-throughput nucleic acid extraction in the future, and has advantages that traditional DNA extraction methods cannot match.

[0006] However, during the extraction process, a small amount of residual cell membrane and other insoluble impurities can affect the performance of the magnetic beads, resulting in a failure to guarantee the extraction efficiency and concentration of nucleic acids, and the extracted nucleic acids do not have supercoiled structures or have a low content of supercoiled structures, which affects the quality of nucleic acids.

[0007] Therefore, there is an urgent need to develop a nucleic acid extraction kit and extraction method suitable for high-throughput and automation and capable of ensuring a high proportion of supercoiled structures in the extracted plasmids. SUMMARY

[0008] In view of the deficiencies of the prior art, the present application provides a magnetic bead extraction reagent, kit and extraction method. The kit and nucleic acid extraction method are suitable for full-automatic nucleic acid extraction technology.

[0009] The first aspect of the present application provides a magnetic bead extraction reagent, comprising magnetic bead solution I and magnetic bead solution II components; the magnetic bead solution I comprises magnetic beads I; the magnetic bead solution II comprises magnetic beads II; the magnetic beads I are used for removing impurities from a biological sample containing nucleic acids to be extracted, and the magnetic beads II are used for adsorbing the nucleic acids.

[0010] Further preferably, the magnetic bead solution II comprises magnetic beads II and does not comprise an alcohol solvent.

[0011] Further preferably, the outer surface of the magnetic microsphere body of the magnetic beads I has at least one branched polymer with a functional group; and / or the nucleic acid is DNA or RNA, and further, the DNA is genomic DNA or plasmid DNA.

[0012] Further preferably, one end of the branched polymer is covalently coupled to the outer surface of the magnetic microsphere body, and the other part is free on the outer surface of the magnetic microsphere body; further, the polymer is a linear polymer; and further, the main chain of the polymer is a polyolefin main chain.

[0013] Further preferably, the branched polymer is obtained by polymerization of one of acrylic acid, acrylic acid salt, acrylic acid ester, methacrylic acid, methacrylic acid salt, methacrylic acid ester, other acrylic monomers, or a combination thereof.

[0014] Further preferably, no crosslinking agent is required in the formation of the main chain of the polymer.

[0015] Further preferably, the functional group is one of carboxyl, hydroxyl, amino, sulfydryl, or a combination thereof.

[0016] Further preferably, the preparation method of the magnetic beads I is as follows: (1) chemically modifying the magnetic microsphere body to introduce an amino group to the outer surface of the magnetic microsphere body to form a magnetic microsphere A;

[0017] Further, the magnetic microsphere body is chemically modified using a silane coupling agent;

[0018] (2) covalently coupling an acrylic acid molecule to the outer surface of the magnetic microsphere A using a covalent reaction between a carboxyl group and an amino group, thereby introducing a carbon-carbon double bond, to form a magnetic microsphere B;

[0019] (3) under the condition of no crosslinking agent, using a polymerization reaction of the carbon-carbon double bond to polymerize acrylic monomers, covalently coupling the obtained polymer to the outer surface of the magnetic microsphere B, performing solid-liquid separation, removing the liquid phase, and obtaining the magnetic beads I.

[0020] Further preferably, the preparation method of the magnetic beads I is as follows:

[0021] (I) using trimethoxysilane-modified acrylic molecules to chemically modify the magnetic microsphere body, introducing carbon-carbon double bonds to the outer surface of the magnetic microsphere body to form magnetic microspheres B;

[0022] Further, the trimethoxysilane-modified acrylic molecules are preferably γ-methacryloxypropyl trimethoxysilane.

[0023] (II) under the condition of no crosslinking agent, using the polymerization reaction of carbon-carbon double bonds to polymerize acrylic monomer molecules, and the obtained polymer is covalently coupled to the outer surface of the magnetic microspheres B, solid-liquid separation is performed, the liquid phase is removed, and the magnetic beads I are obtained.

[0024] Further preferably, the structure of the magnetic beads II is the same as that of the magnetic beads I, or the outer surface of the magnetic microsphere body of the magnetic beads II is directly modified with a silicon hydroxyl group or a silicon carboxyl group.

[0025] Further preferably, the reagent further comprises any one or more components of a lysis solution, a neutralization solution, a washing solution, and an elution solution.

[0026] Further preferably, the reagent further comprises a suspension component.

[0027] Further preferably, the storage of each component in the extraction reagent comprises any one of the following modes:

[0028] (1) each component in the extraction reagent is stored independently;

[0029] (2) the magnetic bead solution I is stored in the neutralization solution and / or the lysis solution, and the remaining components are stored independently;

[0030] (3) the magnetic bead solution I is stored in the washing solution, and the remaining components are stored independently.

[0031] (4) the magnetic bead solution II is stored in the washing solution 2, and the remaining components are stored independently.

[0032] Further preferably, the alcohol solvent comprises any one or more of an ethanol solvent, a propanol solvent, and an isopropanol solvent.

[0033] Further preferably, the magnetic bead solution II comprises magnetic beads II and water; more preferably, the water is selected from deionized water or distilled water.

[0034] Further preferably, the body of the magnetic beads I and the magnetic beads II is a magnetic particle wrapped with SiO2.

[0035] Further preferably, the chemical composition of the magnetic particles comprises any one or any combination of iron compound, iron alloy, zinc oxide, manganese oxide, gadolinium oxide, cobalt compound, nickel compound, nickel alloy, manganese oxide, manganese alloy, zinc oxide, gadolinium oxide, chromium oxide.

[0036] Further preferably, the chemical composition of the magnetic particles comprises any one or any combination of iron oxide, zinc oxide, manganese oxide, gadolinium oxide, cobalt oxide, nickel oxide, manganese oxide, zinc oxide, gadolinium oxide, chromium oxide.

[0037] Further preferably, the chemical composition of the magnetic particles comprises any one or any combination of iron oxide, iron, cobalt, Co 2+ , iron nitride, Mn3O4, GdO, nickel, Ni 2+ ; wherein the iron oxide is preferably Fe3O4, γ-Fe2O3, or a combination thereof.

[0038] Further preferably, the chemical composition of the magnetic particles comprises any one or any combination of Fe3O4, γ-Fe2O3, iron nitride, Mn3O4, AlNi(Co), FeCr(Co), FeCrMo, FeAlC, AlNi(Co), FeCrCo, ReCo, ReFe, PtCo, MnAlC, CuNiFe, AlMnAg, MnBi, FeNi(Mo), FeSi, FeAl, FeNi(Mo), FeSiAl, BaO-6Fe2O3, SrO-6Fe2O3, PbO-6Fe2O3, GdO.

[0039] Further preferably, the lysis solution comprises 200-500 mM, 200-250 mM, 250-300 mM, 300-350 mM, 350-400 mM, 400-450 mM, or 450-500 mM of the strong base and 20-100 mM, 20-30 mM, 30-40 mM, 40-50 mM, 50-60 mM, 60-70 mM, 70-80 mM, 80-90 mM, or 90-100 mM of SDS. Still further, the concentration of the strong base is 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, or 450 mM; and the concentration of SDS is 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM.

[0040] Further preferably, the neutralization solution comprises 0.5-1 M, 0.5-0.8 M, 0.6-1.0 M, 0.7-1.0 M, 0.8-0.9 M or 0.9-1 M potassium acetate / acetic acid buffer solution and 3-5 M, 3-4 M, 3.5-4.5 M or 4-5 M guanidine hydrochloride. Still further, the concentration of the potassium acetate / acetic acid buffer solution is 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M or 1 M; and the concentration of the guanidine hydrochloride is 3 M, 3.5 M, 4 M, 4.5 M or 5 M.

[0041] Further preferably, the washing solution 2 comprises 60%-90%, 60%-80%, 70%-90%, 75%-90% or 65%-85% of an alcohol solvent; still further preferably, the alcohol solvent is ethanol. The concentration of the ethanol is 60%, 65%, 70%, 75%, 80%, 85% or 90%.

[0042] Further preferably, the elution solution comprises 5-15 mM, 5-10 mM, 7-12 mM, 9-11 mM, 11-13 mM or 10-15 mM Tris-HCl and 0.2-2 mM, 0.3-0.5 mM, 0.5-0.8 mM, 0.8-1.0 mM, 1.0-1.2 mM, 1.2-1.5 mM, 1.5-1.8 mM or 1.2-2.0 mM EDTA. Still further, the concentration of Tris-HCl is 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM or 15 mM; and the concentration of EDTA is 0.2 mM, 0.4 mM, 0.6 mM, 0.7 mM, 0.6 mM, 1.0 mM, 1.2 mM, 1.5 mM, 1.8 mM or 2 mM.

[0043] Further preferably, the magnetic bead solution I has a concentration of 5-25% V / V, 5-15% V / V, 5-10% V / V, 5-20% V / V, 10-20% V / V or 25-25% V / V.

[0044] Further preferably, the magnetic bead solution II has a concentration of 5-35% V / V, 5-15% V / V, 10-20% V / V, 15-25% V / V, 20-25% V / V, 25-30% V / V or 30-35% V / V.

[0045] Preferably, the volumes of the lysis solution, the neutralization solution, the washing solution, the magnetic bead solution I and the magnetic bead solution II are 1-10 portions, 5-10 portions, 1-10 portions, 0.1-1 portion, 0.1-1 portion, respectively.

[0046] More preferably, the volume of the lysis solution is 1-10 parts, 5-10 parts, or 3-8 parts; the volume of the neutralization solution is 5-10 parts, 5-8 parts, or 6-8 parts; the volume of the washing solution is 1-10 parts, 5-10 parts, 3-8 parts, or 6-8 parts; the volume of the elution solution is 0.5-2 parts, 0.5-1 part, 0.5-1.5 parts, 0.8-1.5 parts, 1.2-2 parts, or 1.5-2 parts; the volume of the magnetic bead solution I is 0.1-1 part, 0.3-1 part, 0.3-0.5 part, 0.5-0.8 part, or 0.8-1 part; and the volume of the magnetic bead solution II is 0.1-1 part, 0.3-1 part, 0.3-0.5 part, 0.5-0.8 part, or 0.8-1 part.

[0047] More preferably, the washing solution further comprises a washing solution 1, which comprises 1-3 M, 1-2 M, 2-3 M, 1.5-2.5 M, or 2.5-3 M guanidine hydrochloride; and the volume of the washing solution 1 is 5-10 parts or 6-8 parts. Further, the volumes of the lysis solution, the neutralization solution, the washing solution 1, the washing solution 2, the elution solution, the magnetic bead solution I, and the magnetic bead solution II are 3-8 parts, 10 parts, 5-10 parts, 5-10 parts, 0.5-2 parts, 0.2-2 parts, and 0.2-2 parts, respectively.

[0048] The second aspect of the present application provides a magnetic bead extraction kit, which comprises the magnetic bead extraction reagent provided in the first aspect of the present application.

[0049] Further preferably, the storage of each component in the extraction reagent comprises any one of the following modes:

[0050] (1) each component in the extraction reagent is stored independently;

[0051] (2) the magnetic bead solution I is stored in the neutralization solution and / or the lysis solution, and the remaining components are stored independently;

[0052] (3) the magnetic bead solution I is stored in the washing solution, and the remaining components are stored independently;

[0053] (4) the magnetic bead solution II is stored in the washing solution 2, and the remaining components are stored independently.

[0054] Further preferably, the kit is provided with a sample addition plate comprising at least one set of first, second, third, and fourth sample addition positions.

[0055] Further preferably, the first sample addition position comprises at least two reaction solution cavities completely separated by a separation layer, i.e., a neutralization solution cavity and a lysis solution cavity, for containing the neutralization solution and the lysis solution, respectively.

[0056] The separation layer is easily broken;

[0057] The second hole position contains magnetic bead solution II;

[0058] The third hole position contains magnetic bead solution I, and further, the third hole position also contains washing solution;

[0059] The fourth hole position contains elution solution.

[0060] Further preferably, the first hole position comprises at least two reaction liquid cavities separated by a separation layer: a magnetic bead solution I cavity and a lysis solution cavity, for containing the magnetic bead solution I and the lysis solution, respectively, and further, the magnetic bead solution I cavity also contains neutralizing solution;

[0061] The second hole position contains magnetic bead solution II;

[0062] The third hole position contains washing solution;

[0063] The fourth hole position contains elution solution; or

[0064] The third scheme is:

[0065] The first hole position comprises two reaction cavities separated by a separation layer: a lysis solution cavity and a neutralizing solution cavity, for containing the lysis solution and the neutralizing solution, respectively;

[0066] The separation layer is easily broken;

[0067] The second hole position contains magnetic bead solution I, and further, the second hole position also contains washing solution 1;

[0068] The third hole position contains magnetic bead solution II, and further, the third hole position also contains washing solution 2;

[0069] The fourth hole position contains elution solution.

[0070] Further preferably, the length of the first hole position is at least 2 times, more preferably 3 times, the length of the other hole positions, and further preferably, the length of the other hole positions is a standard hole.

[0071] Further preferably, the sample loading plate is provided with two groups of the first hole position, the second hole position, the third hole position and the fourth hole position.

[0072] The third aspect of the present application provides a method for extracting nucleic acid using the magnetic bead extraction reagent of the first aspect of the present application and the magnetic bead extraction kit of the second aspect of the present application, characterized in that it comprises the following steps:

[0073] (1) Bacterial solution lysis: adding lysis solution to the bacterial solution to be extracted to release nucleic acid to obtain a reaction liquid;

[0074] Further, after adding the lysis solution, RNase can also be selectively added according to requirements.

[0075] After mixing, a reaction solution in which the released nucleic acid is dissolved is obtained;

[0076] (2.1) Decontamination treatment: the magnetic beads I are transferred from the third hole site to the first hole site for adsorption decontamination, the magnetic beads II in the second hole site are transferred to the third hole site for temporary storage, and then the magnetic beads I after decontamination treatment are transferred to the second hole site, and the magnetic beads I are discarded by magnetic separation;

[0077] (3.1) Adsorption treatment: the magnetic beads II temporarily stored in the third hole site are transferred to the first hole site for adsorption of nucleic acid, and then transferred to the third hole site and washed with the washing solution to wash the magnetic beads II adsorbing nucleic acid;

[0078] (4.1) Elution: the magnetic beads II adsorbing the nucleic acid are transferred from the third hole site to the fourth hole site for elution, and the target nucleic acid is stored in the elution solution.

[0079] Further preferably, the method for extracting nucleic acid can further comprise

[0080] (2.2) Decontamination treatment: the magnetic beads I are adsorbed in the first hole site for decontamination, the magnetic beads II in the second hole site are transferred to the third hole site for temporary storage, and then the magnetic beads I after decontamination are transferred to the second hole site, and the magnetic beads I are discarded by magnetic separation;

[0081] (3.2) Adsorption treatment: the magnetic beads II temporarily stored in the third hole site are transferred to the first hole site for adsorption of nucleic acid, and then transferred to the third hole site and washed with the washing solution to wash the magnetic beads II adsorbing nucleic acid;

[0082] (4.2) Elution: the magnetic beads II adsorbing the nucleic acid are transferred from the third hole site to the fourth hole site for elution, and the target nucleic acid is stored in the elution solution.

[0083] Further preferably, in the third scheme, the second hole site also stores washing solution 1, and the third hole site also stores washing solution 2; at this time, the subsequent steps further comprise:

[0084] (2.3) The magnetic beads I are transferred from the second hole site to the first hole site for adsorption decontamination, and then transferred to the second hole site to discard the magnetic beads I by magnetic separation;

[0085] (3.3) The magnetic beads II are transferred from the third hole site to the first hole site for adsorption of nucleic acid, and then transferred to the third hole site to wash the magnetic beads II with the washing solution 2;

[0086] (4.3) transferring the magnetic beads II adsorbing the nucleic acid from the third hole site to the fourth hole site for elution; discarding the magnetic beads II by magnetic separation in the fourth hole site, and the target nucleic acid is retained in the eluent;

[0087] Further preferably, the method does not need centrifugation.

[0088] Further preferably, the method is a fully automatic method.

[0089] The present application has the following advantages:

[0090] (1) The reagent and the corresponding kit of the present application can ensure the activity of the magnetic beads I for removing impurities, so as to effectively ensure the extraction quality of the bacterial liquid for extracting nucleic acid without centrifugation. After adding the bacterial liquid running program, high-quality nucleic acid can be obtained by direct extraction.

[0091] (2) The magnetic beads I for removing impurities are modified by the branched polymer chain with a functional group, which can better ensure the effect of removing impurities compared with directly modified magnetic beads, and further ensure that the subsequent use of the magnetic beads II for extracting nucleic acid can meet the needs without centrifugation, and more effectively replace the centrifugation step in the traditional process, which is more efficient, fast, suitable for full automation and high-throughput demand. Moreover, compared with the conventional one-time magnetic bead method, only one cleaning is needed to achieve better results.

[0092] (3) The throughput is large, and at most 32 samples of nucleic acid can be extracted at one time: 16 groups of samples in one kit, and two kits at one time.

[0093] (4) The activity of the hydroxyl magnetic beads is further ensured by using the program matched with the reagent and the kit of the present application, and high-quality nucleic acid is extracted, which is suitable for full automation.

[0094] (5) The present application can adsorb impurities by the magnetic beads I after bacterial lysis, and the magnetic beads II can further combine and enrich, so that the extracted nucleic acid has high purity and good quality. BRIEF DESCRIPTION OF DRAWINGS

[0095] Figure 1 shows the agarose gel electrophoresis diagram of the plasmids obtained in Examples 1-4;

[0096] Figure 2 is the characterization result of the agarose gel electrophoresis of the plasmid DNA in the eluent in Examples 5, 6 and 7 of the present application;

[0097] Figure 3 is the characterization result of the downstream protein synthesis of the plasmid DNA in the eluent in Examples 5, 6 and 7 of the present application. DETAILED DESCRIPTION

[0098] The following is further illustrated by taking plasmid extraction as an example in combination with the specific embodiments and examples, and reference should be made to the accompanying drawings. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. In the following examples, the experimental methods not specified in the specific conditions are preferentially performed according to the conditions indicated in the foregoing specific embodiments, and then can be performed according to the conventional conditions, for example, the experimental conditions described in the literatures such as "Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)", "Cell-free Protein Synthesis: Methods and Protocols" Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis: methods and protocols [M]. 2008", or the conditions suggested by the manufacturers.

[0099] Unless otherwise specified, the materials and reagents used in the embodiments of the present application are commercially available products.

[0100] The temperature units in the present application are Celsius (℃) unless otherwise specified.

[0101] Nouns and terms

[0102] The following is an explanation or description of the meanings of some relevant "nouns" and "terms" used in the present application, in order to better understand the present application. The corresponding explanation or description is applicable throughout the present application, both in the following and in the foregoing. When the present application refers to the definition of relevant terms, nouns, phrases in the cited literature, the definition of the cited literature is also cited. However, the definition in the present application is the priority. When the definition in the cited literature conflicts with the definition in the present application, the components, substances, compositions, materials, systems, formulations, species, methods, devices, etc. cited in the cited literature are determined according to the cited literature.

[0103] In the description of the present application, the preferred mode, the preferred embodiment, the preferred example, the preferred example, the preferred example, in a preferred embodiment, in some preferred examples, in some preferred modes, preferably, preferably, preferably, more preferably, more preferably, further preferably, most preferably, one of the embodiments, one of the modes, examples, specific examples, for example, as an example, for example, such as, like, and the like, do not constitute any limitation on the scope of coverage and protection of the invention, and the specific features described in each mode are included in at least one specific embodiment of the present application. In the present application, the specific features described in each mode can be combined in any one or more specific embodiments in a suitable manner. In the present application, the technical features or technical solutions corresponding to each preferred mode can also be combined in any suitable manner.

[0104] In the present application, "any combination thereof" means "more than 1" in terms of quantity and "a group consisting of the following cases" in terms of coverage: "optionally one or a group consisting of at least two".

[0105] In the present application, the description of "one or more" and "one or more" of "one or more" has the same meaning as "at least one", "at least one", "combination", "or combination", "and combination", "or any combination thereof", "and any combination thereof", and can be used interchangeably, indicating that the number is equal to "1" or "more than 1".

[0106] In the present application, "or / and", "and / or" means "optionally one or a combination thereof", and also means at least one of them.

[0107] The prior art means described in the present application, such as "usually", "conventionally", "generally", "often", "often", etc., are also cited as references for the content of the present application, and without special instructions, it can be regarded as one of the preferred modes of the technical features of the present application, and it should be noted that it does not constitute any limitation on the scope of coverage and protection of the invention.

[0108] All documents mentioned in the present application and documents directly or indirectly cited by these documents are cited as references in the present application, as if each document is cited as a reference.

[0109] It should be understood that within the scope of the present application, the above-mentioned technical features of the present application and the technical features specifically described in the following (including but not limited to examples) can be combined with each other to form new or preferred technical solutions, as long as they can be used to implement the present application. Limited to the length, it will not be listed one by one.

[0110] Three-in-one hole refers to the structure that three standard holes in a standard multi-well plate are integrated and the three holes can be sequentially connected, that is, the length of the three-in-one hole is 3 times the length of the standard hole.

[0111] The standard multi-well plate in this paper refers to a flat plate with 96 holes, which is commonly used in chemical experiments, biological experiments or other applications that require multiple locations to perform reactions or tests simultaneously. The design of the multi-well plate allows multiple samples to be processed at the same time, greatly improving the efficiency and accuracy of experiments. Multi-well plates are commonly used in high-throughput experiments. The standard size and design of multi-well plates make them easy to use in various laboratory equipment and instruments, whether manually operated or automated equipment, multi-well plates can be easily used for experiments. Each hole of the standard multi-well plate is a standard hole.

[0112] In the embodiments of the present application, the specific structure and preparation method of magnetic beads I are the magnetic microspheres C in patent CN 112111042 A. The contents related to magnetic microspheres C in patent CN 112111042 A are all introduced into this application. More specifically, the preparation method in the specific embodiments of patent CN 112111042 A is used to prepare the magnetic beads I required in the following embodiments.

[0113] In the following embodiments, the magnetic beads II (conventional magnetic beads) are all directly modified with silicon hydroxyl on the outer surface of the magnetic microsphere body.

[0114] In the following embodiments, protein synthesis uses IVTT reaction (in vitro transcription and translation reaction). IVTT reaction, corresponding to IVTT system, is the process of in vitro transcription and translation of DNA into protein. Specifically, exogenous DNA, mRNA or their combination is used as the nucleic acid template for protein synthesis. By artificially adding substrates and transcription and translation related protein factors required for protein synthesis, the in vitro synthesis of target protein is realized. Refer to the specific instructions in previous patents CN111378707B, CN111378708B, WO2024051855A1, etc.

[0115] In the following embodiments, the preparation of the solution is involved. The solvent is deionized water unless otherwise specified.

[0116] Basic operation process

[0117] Experimental materials: plasmid extraction kit, magnetic rod sleeve, freshly shaken E. coli OD = 2.3 or so.

[0118] Experimental procedure: tear open the sealing film of the kit, take 1.5 mL of the bacterial liquid sample in turn, and add it to the three-in-one hole (sample hole) in column 1 of the kit. Then add 60 μL of the appropriate RNase to each sample hole, put it into the instrument, add a magnetic bar sleeve, and run the program.

[0119] Experimental results: label the centrifuge tube, take the corresponding plasmid, centrifuge at 12000g for 5 min, take the supernatant, and use nanordrop to measure the concentration of DNA,

[0120] Check the DNA concentration value. The reasonable value is greater than 50 ng / ul, and the DNA purity 260 / 280 is between 1.7-2.0.

[0121] Take 5 ul of the extracted plasmid to run the gel and observe whether the plasmid band is normal:

[0122] A better plasmid has three bands. The lowermost one is the supercoiled band, the middle one is the half-open plasmid, and the upper one is the open plasmid. The higher the proportion of supercoiled plasmid, the better the plasmid band, which proves that the quality of the plasmid is higher.

[0123] The carboxyl magnetic beads I and hydroxyl magnetic beads II added in the following examples respectively refer to the magnetic bead liquid I and magnetic bead liquid II described in the application. In the following examples, the concentration of the magnetic bead liquid involved refers to the original concentration, that is, the concentration before being divided into the kit. In examples 1-4, the original concentration of the magnetic bead liquid I and the magnetic bead liquid II is 10%.

[0124] Example 1

[0125] 1. Reagents required for the experiment: lysis solution (200-500 mM strong base and 20-100 mM SDS), neutralization solution (0.5-1 M potassium acetate / acetic acid buffer solution and 3-5 M guanidine hydrochloride), deionized water, 80% ethanol, hydroxyl magnetic beads II (magnetic bead liquid II, original concentration 10%), carboxyl magnetic beads I (magnetic bead liquid I, original concentration 10%), eluent (5-15 mM Tris-HCl and 0.2-2 mM EDTA).

[0126] 2. Pre-packaging of the kit

[0127] Preload the neutralizing liquid 950 μl + 50 μl carboxyl magnetic beads I into the three-in-one hole of the multi-well plate (i.e. contains 1, 2, 3 standard hole, corresponding to the first hole of the application), heat the wax to form a liquid wax, drop to the surface of the neutralizing liquid, cool and solidify to form a wax layer, and preload 600 μl of lysis liquid above the wax layer; Preload 700 μl of deionized water + 50 μl of hydroxyl magnetic beads II (MB) into the 4th standard hole (corresponding to the second hole of the application); Preload 700 μl of 80% ethanol into the 5th standard hole (corresponding to the third hole of the application); Preload 100 μl of eluent into the 6th standard hole (corresponding to the fourth hole of the application).

[0128] 3. Specific operation program (fully automatic operation): After placing the reagent kit into the extraction device, align the magnetic bar and the magnetic bar sleeve with the hole:

[0129] (1) In the 2nd standard hole, mix the bacterial solution and the lysis solution uniformly by moving the magnetic bar sleeve horizontally, so that the cell lysis is more complete.

[0130] (2) Break the wax layer by moving the magnetic bar with the sleeve vertically through the wax, so that the neutralizing liquid contacts the solution above the wax layer.

[0131] (3) Mix horizontally to better mix the neutralizing liquid with the solution above, to neutralize the mixed solution in the three-in-one hole.

[0132] (4) Disperse the carboxyl magnetic beads, so that the dispersed carboxyl magnetic beads better adsorb the solid impurities in the three-in-one hole.

[0133] (5) In the 4th standard hole, adsorb the dispersed hydroxyl magnetic beads II onto the magnetic bar, and then transfer to the 5th standard hole for temporary storage.

[0134] (6) Transfer the carboxyl magnetic beads I adsorbed with impurities from the three-in-one hole to the deionized water in the 4th standard hole.

[0135] (7) Adsorb the hydroxyl magnetic beads II from the 5th standard hole.

[0136] (8) Transfer the hydroxyl magnetic beads II from the 5th hole to the three-in-one hole, so that the plasmid better binds to the hydroxyl magnetic beads II.

[0137] (9) Transfer the hydroxyl magnetic beads II carrying the plasmid to the ethanol in the 5th standard hole for cleaning.

[0138] (10) Adsorb the hydroxyl magnetic beads II cleaned with ethanol, and evaporate the residual ethanol on the hydroxyl magnetic beads II in the air.

[0139] (11) Perform plasmid elution of the cleaned hydroxyl magnetic beads II with plasmid in the 6th standard hole, and dissolve the plasmid into the TE buffer.

[0140] (12) Transfer the extracted hydroxyl magnetic beads II to the 5th standard well.

[0141] Example 2

[0142] 1. Reagents required for the experiment: same as in Example 1.

[0143] 2. Pre-dispensing of the kit

[0144] Pre-dispense 950 μl of neutralizing solution into the three-in-one well of the multi-well plate (i.e. containing the 1st, 2nd and 3rd standard wells, corresponding to the first, second and third wells of the present application), heat the wax to form a liquid wax, drop it onto the surface of the neutralizing solution, and cool and solidify to form a wax layer, and pre-dispense 600 μl of lysis solution above the wax layer; pre-dispense 700 μl of deionized water + 50 μl of hydroxyl magnetic beads II (MB) into the 4th standard well (corresponding to the fourth well of the present application); pre-dispense 700 μl of 80% ethanol + 50 μl of carboxyl magnetic beads I into the 5th standard well (corresponding to the fifth well of the present application); and pre-dispense 100 μl of elution solution into the 6th standard well (corresponding to the sixth well of the present application).

[0145] 3. Specific running program (fully automatic operation): after placing the kit into the reagent placement position of the extraction device, align the magnetic bar and the magnetic bar sleeve with the wells:

[0146] (1) Mix the bacterial solution and the lysis solution uniformly in the 2nd standard well by moving the magnetic bar sleeve horizontally to make the cell lysis more thorough.

[0147] (2) Break the wax layer by moving the magnetic bar with the sleeve vertically through the wax to make the neutralizing solution contact the solution above the wax layer.

[0148] (3) Disperse the hydroxyl magnetic beads II in the 4th standard well to make the hydroxyl magnetic beads II disperse better.

[0149] (4) Disperse the carboxyl magnetic beads in the 5th standard well.

[0150] (5) Transfer the dispersed carboxyl magnetic beads I from the 5th standard well to the three-in-one well, disperse the carboxyl magnetic beads I, and make the impurities adsorb onto the carboxyl magnetic beads I.

[0151] (6) Transfer the hydroxyl magnetic beads II from the 4th standard well to the ethanol solution in the 5th standard well.

[0152] (7) Transfer the carboxyl magnetic beads II from the three-in-one well to the deionized water in the 4th standard well.

[0153] (8) Transfer the hydroxyl magnetic beads II from the 5th standard well to the three-in-one well to make the plasmid combine with the hydroxyl magnetic beads II better.

[0154] (9) Transfer the hydroxyl magnetic beads II with plasmid to the ethanol in the 5th standard hole for cleaning.

[0155] (10) Adsorb the ethanol cleaned hydroxyl magnetic beads II in the air, and evaporate the ethanol left on the magnetic beads.

[0156] (11) Transfer the cleaned hydroxyl magnetic beads II with plasmid to the 6th standard hole for plasmid elution, and dissolve the plasmid into the TE buffer.

[0157] (12) Take the hydroxyl magnetic beads II, and transfer the extracted hydroxyl magnetic beads II to the 5th standard hole.

[0158] Example 3 (negative control example)

[0159] 1. Reagent formula required for the experiment: lysing solution (200-500 mM strong base and 20-100 mM SDS), neutralizing solution (0.5-1 M potassium acetate / acetic acid buffer solution and 3-5 M guanidine hydrochloride), guanidine hydrochloride (concentration of 1-3 M, abbreviated as BR), 80% ethanol, hydroxyl magnetic beads II, carboxyl magnetic beads I, elution solution (5-15 mM Tris-HCl and 0.2-2 mM EDTA).

[0160] 2. Pre-dispensing of the reagent kit

[0161] Pre-dispense 950 μl of the neutralizing solution into the three-in-one hole of the multi-well plate (i.e. containing the 1st, 2nd and 3rd standard holes, corresponding to the first hole of the present application), heat the wax to form a liquid wax, drop it onto the surface of the neutralizing solution, and cool and solidify to form a wax layer, and pre-dispense 600 μl of the lysing solution above the wax layer; pre-dispense 700 μl of guanidine hydrochloride + 50 μl of carboxyl magnetic beads I into the 4th standard hole (corresponding to the second hole of the present application); pre-dispense 700 μl of 80% ethanol + 50 μl of hydroxyl magnetic beads II into the 5th standard hole (corresponding to the third hole of the present application); and pre-dispense 100 μl of the elution solution into the 6th standard hole (corresponding to the fourth hole of the present application).

[0162] 3. Specific operation program (fully automatic operation): after placing the reagent kit into the extraction equipment, align the magnetic bar and the magnetic bar sleeve with the hole:

[0163] (1) Mix the bacterial solution and the lysing solution uniformly by moving the magnetic bar sleeve horizontally in the 2nd standard hole, so as to lyse the cells more thoroughly.

[0164] (2) Break the wax layer by moving the magnetic bar sleeve vertically through the wax, so as to make the acidic neutralizing solution contact the solution above the wax layer.

[0165] (3) Mix the neutralizing solution and the solution above the layer better by horizontal mixing, so as to neutralize the mixed solution in the three-in-one hole.

[0166] (4) In the No. 4 standard hole, the carboxyl magnetic beads I are scattered to disperse better, and the scattered carboxyl magnetic beads I are adsorbed to the magnetic rod.

[0167] (5) The adsorbed carboxyl magnetic beads I are transferred from the No. 4 standard hole to the three-in-one hole, the carboxyl magnetic beads I are scattered to disperse better, and the dispersed carboxyl magnetic beads I are better adsorbed to the solid impurities of the three-in-one hole.

[0168] (6) The carboxyl magnetic beads I adsorbed with impurities are transferred from the three-in-one hole to the BR of the No. 4 standard hole.

[0169] (7) In the No. 5 standard hole, the hydroxyl magnetic beads II are scattered and adsorbed to the magnetic rod, and then transferred to the three-in-one hole, the hydroxyl magnetic beads II are scattered to make the plasmid better combined with the hydroxyl magnetic beads II.

[0170] (8) The hydroxyl magnetic beads II carrying the plasmid are transferred from the three-in-one hole to the ethanol in the No. 5 standard hole.

[0171] (9) The hydroxyl magnetic beads II adsorbed with ethanol are washed, and the residual ethanol on the hydroxyl magnetic beads II is evaporated in the air.

[0172] (10) The hydroxyl magnetic beads II with plasmid after washing are subjected to plasmid elution in the No. 6 standard hole, and the plasmid is dissolved into the TE buffer.

[0173] (11) The hydroxyl magnetic beads II are sucked, and the extracted hydroxyl magnetic beads II are transferred to the No. 5 standard hole.

[0174] Example 4

[0175] The difference from Example 3 is only that the hydroxyl magnetic beads II in the No. 5 standard hole are not pre-existing in ethanol (i.e. the No. 5 standard hole only has ethanol before the kit is used), and the hydroxyl magnetic beads II are put into the No. 5 standard hole when the kit is used, and the other reagents and steps are the same as those of Example 3, which is used to compare the influence of the contact time of the hydroxyl magnetic beads II and ethanol on the plasmid extraction activity.

[0176] Result comparison

[0177] It should be noted that the kits in Examples 1-4 are all kits stored for 3 days.

[0178] 1. The related activities of the plasmids extracted in Examples 1-4 are compared, the centrifugal tubes are labeled, the corresponding plasmids are sucked, 12000g centrifugation for 5min, the supernatant is sucked, and the DNA concentration is measured using nanordrop, and the specific data are shown in Table 1 and Table 2.

[0179] Table 1: Characterization results of plasmid DNA concentration and purity

[0180] Table 2: Characterization results of plasmid DNA concentration and purity

[0181] In Table 1 and Table 2, the serial number represents different groups of parallel experiments for each embodiment, and the average concentration refers to the average concentration of the extracted plasmid of each embodiment.

[0182] Table 1 and Table 2 show the characterization results of the concentration and purity of the plasmid DNA extracted by each embodiment. As shown in Table 1, the 260 / 280 of embodiments 1 and 2 is basically around 1.8, indicating that the extracted plasmid has high purity. As shown in Table 2, the 260 / 280 of embodiments 3 and 4 is basically around 1.9, indicating that there are impurities such as RNA in the plasmid. 260 / 280 represents the absorption peak under ultraviolet light, 260 nm represents the nucleic acid absorption peak, and 280 nm represents the protein absorption peak. The concentration of the plasmid extracted by each embodiment can reach more than 50 ng / ul.

[0183] 2. Extract 5 ul of the plasmid obtained in each of embodiments 1-4 respectively for agarose gel electrophoresis characterization to observe whether the plasmid band is normal (see Figure 1).

[0184] Generally, a good plasmid has three bands, the lowermost one is the supercoiled band, the middle one is the half-open circular plasmid, and the upper one is the open circular plasmid. The higher the proportion of supercoiled plasmid, the better the plasmid band and the higher the quality of the plasmid. As shown in Figure 1, the supercoiled band in the plasmid obtained in embodiments 1, 2 and 4 is more obvious than that in embodiment 3, indicating that the quality of the plasmid obtained in embodiments 1, 2 and 4 is better.

[0185] Therefore, it can be proved that (1) the long-term contact of hydroxyl magnetic beads with alcohol solvents such as ethanol will affect the activity of the hydroxyl magnetic beads, resulting in a decrease in the quality of the extracted plasmid; (2) in the reagent kit, the use of water (deionized water or distilled water, etc.) instead of ethanol to store the hydroxyl magnetic beads has little effect on the concentration of the extracted plasmid, improves the purity of the extracted plasmid, and significantly increases the proportion of supercoiled plasmid, thereby improving the quality of the extracted plasmid.

[0186] Embodiments 5 and 6 are comparative experiments of automatic plasmid DNA extraction using double magnetic beads and conventional single magnetic beads of the present application

[0187] Embodiment 5

[0188] Double magnetic bead extraction experiment of the present application

[0189] First, prepare various solutions in the following proportions:

[0190] The lysis solution comprises 200-500 mM strong alkali and 20-100 mM SDS, the neutralization solution comprises 0.5-1 M potassium acetate and 4-5 M guanidine hydrochloride, the washing solution 1 comprises 1-3 M guanidine hydrochloride, the washing solution 2 comprises 60%-90% ethanol, the elution solution comprises 5-15 mM Tris-HCl and 0.2-2 mM EDTA, the concentration of the magnetic bead solution I is 15-25% V / V, and the concentration of the magnetic bead solution II is 25-35% V / V.

[0191] Specifically, the lysis solution comprises 250 mM sodium hydroxide and 41.6 mM SDS, the neutralization solution comprises 0.75 M potassium acetate and 4.5 M guanidine hydrochloride, the washing solution 1 comprises 2.5 M guanidine hydrochloride, the washing solution 2 comprises 80% ethanol, the elution solution comprises 10 mM Tris-HCl and 1 mM EDTA, the concentration of the magnetic bead solution I is 21% V / V, and the concentration of the magnetic bead solution II is 30% V / V.

[0192] The magnetic bead solution I is prepared by using the magnetic beads I, and the magnetic bead solution II is prepared by using the magnetic beads II.

[0193] The following steps are used to respectively complete pre-dispensing of the double-magnetic-bead impurity removal kit:

[0194] 1 volume part of the neutralization solution is dispensed into the first hole of the sample plate, liquid wax is formed by heating the wax, and the liquid wax is dropped onto the surface of the neutralization solution to form a wax layer, and 0.6 volume parts of the lysis solution are dispensed above the wax layer; 0.7 volume parts of the washing solution 1 and 0.05 volume parts of the magnetic bead solution I are dispensed into the second hole; 0.7 volume parts of the washing solution 2 and 0.05 volume parts of the magnetic bead solution II are dispensed into the third hole; and 0.1 volume parts of the elution solution are dispensed into the fourth hole.

[0195] The double-magnetic-bead impurity removal kit pre-dispensed above is used to extract plasmid DNA, and an instrument automatic extraction program is set to obtain a nucleic acid elution solution, and the specific steps are as follows:

[0196] (1) 1-2 volume parts of the bacterial solution are transferred into the bacterial solution cavity of the first hole;

[0197] (2) RNAse solution is selectively added into the first hole according to the need;

[0198] (3) The separation layer of the first hole is broken to uniformly mix the lysis solution, the neutralization solution and the bacterial solution, and even the RNAse;

[0199] (4) The magnetic beads I are transferred from the second hole to the first hole for adsorption and impurity removal, and then transferred to the second hole to be discarded by magnetic separation;

[0200] (5) The second magnetic beads are transferred from the third hole site to the first hole site for adsorbing nucleic acids, and then are transferred to the third hole site for washing the magnetic beads II with the washing solution II;

[0201] (6) The magnetic beads II adsorbed with nucleic acids are transferred from the third hole site to the fourth hole site for elution, and the magnetic beads II are discarded by magnetic separation in the fourth hole site, and the target nucleic acids are retained in the elution solution.

[0202] The plasmid DNA in the elution solution is characterized by agarose gel electrophoresis, as shown in FIG. 2.

[0203] The plasmid DNA in the elution solution is characterized by agarose gel electrophoresis, as shown in FIG. 2.

[0204] The protein synthesis is an IVTT reaction, and the final concentrations of the components are as follows: 9.78 mM Tris-HCl (pH 8.0), 80 mM potassium acetate, 5 mM magnesium acetate, 1.8 mM nucleotide triphosphate mixture (adenine nucleotide triphosphate, guanine nucleotide triphosphate, cytosine nucleotide triphosphate, and uracil nucleotide triphosphate, each at a concentration of 1.8 mM), 0.7 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine, each at a concentration of 0.1 mM), 15 mM glucose, 320 mM maltodextrin (molar concentration calculated based on glucose units, mass concentration of 52 mg / mL), 24 mM potassium phosphate, 2% (w / v) polyethylene glycol 8000, and finally 50% (by volume) of Kluyveromyces lactis cell extract containing endogenously expressed T7 RNA polymerase.

[0205] The Kluyveromyces lactis cell extract is prepared by the method described in CN109423496A. Briefly, the coding gene of T7 RNA polymerase is integrated into the genome of Kluyveromyces lactis, and the obtained genetically modified strain is cultured to obtain an appropriate amount of cells for preparing the cell extract.

[0206] Example 6

[0207] Conventional single-magnetic-bead adsorption experiment

[0208] The solutions are prepared according to the proportions in Example 5(1), and the following steps are performed to complete the pre-dispensing of the conventional single-magnetic-bead adsorption kit:

[0209] The 1 volume part of the neutralizing liquid is divided into the first hole of the sample plate, the wax is heated to form a liquid wax, which is dropped onto the surface of the neutralizing liquid, and the wax layer is formed by cooling and solidification. The 0.6 volume part of the lysis liquid is divided into the upper part of the wax layer; the 0.7 volume part of the washing liquid 1 is divided into the second hole; the 0.05 volume part of the magnetic bead liquid II and the 0.7 volume part of the washing liquid 2 are divided into the third hole; and the 0.1 volume part of the elution liquid is divided into the fourth hole.

[0210] The above-prepared conventional single-magnetic-bead adsorption kit is used, and an instrument automatic extraction program is set to obtain the plasmid DNA elution liquid. The specific steps are as follows:

[0211] (1) The 1-2 volume parts of the bacterial liquid are transferred to the bacterial liquid cavity of the first hole;

[0212] (2) The RNAse solution is selectively added to the first hole according to the need;

[0213] (3) The separation layer of the first hole is broken, so that the lysis liquid, the neutralizing liquid and the bacterial liquid are mixed uniformly;

[0214] (4) The magnetic beads II in the third hole are transferred to the first hole to adsorb nucleic acids, and then transferred to the second hole to wash the magnetic beads II with the washing liquid 1;

[0215] (5) The magnetic beads II adsorbing nucleic acids are transferred from the second hole to the third hole to wash the magnetic beads II with the washing liquid 2;

[0216] (6) The magnetic beads II adsorbing nucleic acids are transferred from the third hole to the fourth hole for elution. The magnetic beads II are discarded by magnetic separation in the fourth hole, and the target plasmid DNA is retained in the elution liquid.

[0217] The plasmid DNA in the elution liquid is characterized by agarose gel electrophoresis, as shown in FIG. 2.

[0218] The plasmid DNA in the elution liquid is characterized by agarose gel electrophoresis, as shown in FIG. 2.

[0219] Example 7. Automatic plasmid DNA extraction by conventional double-magnetic-bead adsorption

[0220] The solutions are prepared according to the proportions of Example 5(1), and the following steps are used to complete the pre-dispensing of the conventional double-magnetic-bead adsorption kit:

[0221] The 1 part of the neutralizing liquid is divided into the first hole of the sample plate, the wax is heated to form a liquid wax, which is dropped onto the surface of the neutralizing liquid, and the wax layer is formed by cooling and solidification. The 0.6 parts of the lysis liquid is divided into the upper part of the wax layer; the 0.7 parts of the washing liquid 1 and the 0.05 parts of the magnetic bead liquid II are divided into the second hole; the 0.7 parts of the washing liquid 2 and the 0.05 parts of the magnetic bead liquid II are divided into the third hole; and the 0.1 parts of the elution liquid is divided into the fourth hole.

[0222] The pre-dispensed sample plate is used, and an automatic extraction program of the instrument is set to obtain the nucleic acid elution liquid. The specific steps are as follows:

[0223] (1) 1-2 parts of the bacterial liquid is transferred to the bacterial liquid cavity of the first hole;

[0224] (2) The RNAse solution is selectively added to the first hole according to the need;

[0225] (3) The separation layer of the first hole is broken, so that the lysis liquid, the neutralizing liquid and the bacterial liquid are mixed uniformly;

[0226] (4) The magnetic beads II in the second hole are transferred to the first hole to adsorb the nucleic acid, and then transferred to the second hole to wash the magnetic beads II with the washing liquid 1;

[0227] (5) The magnetic beads II adsorbed with the nucleic acid in the second hole are transferred to the third hole to wash the magnetic beads II with the washing liquid 2;

[0228] (6) The magnetic beads II adsorbed with the nucleic acid in the third hole are transferred to the fourth hole for elution. The magnetic beads II are discarded by magnetic separation in the fourth hole, and the target plasmid DNA is retained in the elution liquid.

[0229] The plasmid DNA in the elution liquid is characterized by agarose gel electrophoresis, as shown in FIG. 2.

[0230] The plasmid DNA in the elution liquid is characterized by agarose gel electrophoresis, as shown in FIG. 2.

[0231] As shown in FIG. 2, compared with the conventional single-magnetic-bead adsorption extraction in Example 6 and the conventional double-magnetic-bead adsorption extraction in Example 7, the nucleic acid electrophoresis in Example 5 shows a brighter plasmid DNA band, which indicates that the quality of the plasmid DNA extracted by the double-magnetic-bead adsorption extraction in Example 5 is better.

[0232] As can be seen from FIG. 3, the protein synthesis of Example 5 has higher fluorescence value and synthesizes more protein than that of Example 6 (conventional single magnetic bead adsorption extraction) and Example 7 (conventional double magnetic bead adsorption extraction), which indicates that the plasmid DNA extracted by the double magnetic bead extraction of the application in Example 5 has better quality.

[0233] Based on the above ideal embodiments according to the application, through the above description, relevant personnel can make various changes and modifications without deviating from the technical idea of the application. The technical scope of the application is not limited to the content in the specification, and the technical scope must be determined according to the scope of claims.

Claims

1. A magnetic bead extraction reagent, characterized by, The kit comprises magnetic bead solution I and magnetic bead solution II; the magnetic bead solution I comprises magnetic beads I; the magnetic bead solution II comprises magnetic beads II; the magnetic beads I are used for removing impurities from a biological sample containing nucleic acid to be extracted; and the magnetic beads II are used for adsorbing the nucleic acid.

2. The extraction reagent according to claim 1, characterized in that: The magnetic bead solution II comprises magnetic beads II and does not comprise an alcohol solvent.

3. The extraction reagent according to claim 1 or 2, characterized in that: The magnetic microsphere body of the magnetic beads I has at least one branched polymer with a functional group on the outer surface of the magnetic microsphere body; and / or the nucleic acid is DNA or RNA, and further, the DNA is genomic DNA or plasmid DNA. Preferably, one end of the branched polymer is covalently coupled to the outer surface of the magnetic microsphere body, and the other part is free on the outer surface of the magnetic microsphere body; further, the polymer is a linear polymer; and further, the main chain of the polymer is a polyolefin main chain.

4. The extraction reagent of claim 3, wherein: The branched polymer is obtained by polymerization of one or a combination of acrylic acid, an acrylic acid salt, an acrylic ester, methacrylic acid, a methacrylic acid salt, a methacrylic ester, and other acrylic monomers. Further, the main chain of the polymer is formed without a crosslinking agent.

5. The extraction reagent according to claim 2 or 3, characterized in that: The functional group is one or a combination of a carboxyl group, a hydroxyl group, an amino group, and a mercapto group.

6. The magnetic bead extraction reagent according to any one of claims 2-5, wherein the magnetic beads are coated with a polymer. The preparation method of the magnetic beads I is as follows: (1) chemically modifying the magnetic microsphere body to introduce an amino group to the outer surface of the magnetic microsphere body to form magnetic microspheres A; Further, the magnetic microsphere body is chemically modified by using a silane coupling agent; (2) covalently coupling acrylic acid molecules to the outer surface of the magnetic microspheres A by covalent reaction between a carboxyl group and an amino group to introduce a carbon-carbon double bond, thereby forming magnetic microspheres B; (3) under the condition of no crosslinking agent, polymerizing acrylic monomers by using the polymerization reaction of the carbon-carbon double bond, covalently coupling the obtained polymer to the outer surface of the magnetic microspheres B, performing solid-liquid separation, removing the liquid phase, and obtaining the magnetic beads I; or The preparation method of the magnetic beads I is as follows: (Ⅰ) chemically modifying the magnetic microsphere body by using a trimethoxysilane-modified acrylic molecule to introduce a carbon-carbon double bond to the outer surface of the magnetic microsphere body, thereby forming magnetic microspheres B; Further, the trimethoxysilane-modified acrylic molecule is preferably γ-methacryloyloxypropyl trimethoxysilane; (Ⅱ) under the condition of no crosslinking agent, polymerizing acrylic monomers by using the polymerization reaction of the carbon-carbon double bond, covalently coupling the obtained polymer to the outer surface of the magnetic microspheres B, performing solid-liquid separation, removing the liquid phase, and obtaining the magnetic beads I.

7. The magnetic bead extraction reagent according to any one of claims 1-6, wherein the magnetic beads are coated with a polymer. The magnetic beads II have the same structure as the magnetic beads I, or the outer surface of the magnetic microsphere body of the magnetic beads II is directly modified with a silicon hydroxyl group or a silicon carboxyl group.

8. The magnetic bead extraction reagent according to any one of claims 1-7, wherein, The reagent further comprises any one or more components of a lysis solution, a neutralization solution, a washing solution, and an elution solution. Further, the reagent further comprises a suspension component; and / or The storage of each component in the extraction reagent comprises any one of the following modes: (1) each component in the extraction reagent is stored independently; (2) the magnetic bead solution I is stored in the neutralization solution and / or the lysis solution, and the rest of the components are stored independently; (3) the magnetic bead solution I is stored in the washing solution, and the rest of the components are stored independently. (4) the magnetic bead solution II is stored in the washing solution 2, and the rest of the components are stored independently.

9. The magnetic bead extraction reagent according to any one of claims 1-8, wherein, The alcohol solvent includes any one or more of an ethanol solvent, a propanol solvent or an isopropanol solvent; and / or the magnetic bead solution II comprises magnetic beads II and water; preferably, the water is selected from deionized water or distilled water.

10. The magnetic bead extraction reagent according to any one of claims 7-9, characterized in that, The lysis solution comprises: 200-500 mM, 200-250 mM, 250-300 mM, 300-350 mM, 350-400 mM, 400-450 mM or 450-500 mM of a strong base and 20-100 mM, 20-30 mM, 30-40 mM, 40-50 mM, 50-60 mM, 60-70 mM, 70-80 mM, 80-90 mM or 90-100 mM of SDS; The neutralization solution comprises 0.5-1 M, 0.5-0.8 M, 0.6-1.0 M, 0.7-1.0 M, 0.8-0.9 M or 0.9-1 M of potassium acetate / acetic acid buffer solution and 3-5 M, 3-4 M, 3.5-4.5 M or 4-5 M of guanidine hydrochloride; The washing solution comprises washing solution 2, and the washing solution 2 comprises 60%-90%, 60%-80%, 70%-90%, 75%-90% or 65%-85% of an alcohol solvent, preferably the alcohol solvent is ethanol; The elution solution comprises 5-15 mM, 5-10 mM, 7-12 mM, 9-11 mM, 11-13 mM or 10-15 mM of Tris-HCl and 0.2-2 mM, 0.3-0.5 mM, 0.5-0.8 mM, 0.8-1.0 mM, 1.0-1.2 mM, 1.2-1.5 mM, 1.5-1.8 mM or 1.2-2.0 mM of EDTA; The concentration of the magnetic bead solution I is 5-25% V / V, 5-15% V / V, 5-10% V / V, 5-20% V / V, 10-20% V / V or 25-25% V / V, and the concentration of the magnetic bead solution II is 5-35% V / V, 5-15% V / V, 10-20% V / V, 15-25% V / V, 20-25% V / V, 25-30% V / V or 30-35% V / V; Preferably, the volume of the lysis solution is 1-10 portions, 5-10 portions or 3-8 portions; The volume of the neutralization solution is 5-10 portions, 5-8 portions, 6-8 portions or 10 portions; The volume of the washing solution 2 is 1-10 portions, 5-10 portions, 3-8 portions or 6-8 portions; The volume of the magnetic bead solution I is 0.1-2 portions, 0.1-1 portion, 0.2-2 portions, 0.3-1 portion, 0.3-0.5 portion, 0.5-0.8 portion or 0.8-1 portion; The volume of the magnetic bead solution II is 0.1-2 portions, 0.1-1 portion, 0.2-2 portions, 0.3-1 portion, 0.3-0.5 portion, 0.5-0.8 portion or 0.8-1 portion; The volume of the eluent is 0.5-2 portions, 0.5-1 portion, 0.5-1.5 portions, 0.8-1.5 portions, 1.2-2 portions or 1.5-2 portions; More preferably, the washing solution further comprises washing solution 1, and the washing solution 1 comprises 1-3M, 1-2M, 2-3M, 1.5-2.5M or 2.5-3M guanidine hydrochloride; further, the volume of the washing solution 1 is 5-10 portions or 6-8 portions.

11. A magnetic bead extraction kit comprising the magnetic bead extraction reagent of any one of claims 1-10, preferably, the storage of each component in the extraction reagent comprises any one of the following modes: (1) each component in the extraction reagent is stored independently; (2) the magnetic bead solution I is stored in the neutralizing solution and / or the lysis solution, and the remaining components are stored independently; (3) the magnetic bead solution I is stored in the washing solution, and the remaining components are stored independently. (4) the magnetic bead solution II is stored in the washing solution 2, and the remaining components are stored independently.

12. The magnetic bead extraction kit of claim 11, wherein, The kit is provided with a sample adding plate comprising at least one set of first, second, third and fourth holes, wherein, First scheme: the first hole comprises at least two reaction liquid cavities completely separated by a separation layer: a neutralizing solution cavity and a lysis solution cavity, respectively used to contain the neutralizing solution and the lysis solution; the separation layer is easily broken; the second hole contains the magnetic bead solution II; the third hole contains the magnetic bead solution I, and further, the third hole also contains the washing solution; the fourth hole contains the eluent; or Second scheme: the first hole comprises at least two reaction liquid cavities completely separated by a separation layer: a magnetic bead solution I cavity and a lysis solution cavity, respectively used to contain the magnetic bead solution I and the lysis solution, and further, the magnetic bead solution I cavity also contains the neutralizing solution; the second hole contains the magnetic bead solution II; the third hole contains the washing solution; the fourth hole contains the eluent; or Third scheme: the first hole comprises two reaction cavities completely separated by a separation layer: a lysis solution cavity and a neutralizing solution cavity, respectively used to contain the lysis solution and the neutralizing solution; the separation layer is easily broken; the second hole contains the magnetic bead solution I, and further, the second hole also contains the washing solution 1; the third hole contains the magnetic bead solution II, and further, the third hole also contains the washing solution 2; the fourth hole contains the eluent; Preferably, the length of the first hole is at least 2 times, more preferably 3 times, and further preferably, the length of the other holes is a standard hole; and / or the sample adding plate is provided with two sets of the first, second, third and fourth holes.

13. A method for extracting nucleic acid using the magnetic bead extraction kit of claim 12, comprising the following steps: (1) bacterial solution lysis: adding the bacterial solution to be extracted to the lysis solution to release nucleic acid to obtain a reaction solution; ​ Further, RNase can be added after the lysis solution is added, After mixing, a reaction solution in which the released nucleic acid is dissolved is obtained; Further, a neutralizing solution can be further added to the reaction solution; The process of step (1) is performed in the first hole site; In the first aspect of claim 12, a cleaning solution is also stored in the third hole site, and the subsequent steps further include: (2.1) impurity removal treatment: the magnetic beads I in the third hole site are transferred to the first hole site for adsorption and impurity removal, the magnetic beads II in the second hole site are transferred to the third hole site for temporary storage, and the magnetic beads I after the impurity removal treatment are transferred to the second hole site, and the magnetic beads I are discarded by magnetic separation; (3.1) adsorption treatment: the magnetic beads II temporarily stored in the third hole site are transferred to the first hole site for adsorption of nucleic acid, and then transferred to the third hole site and cleaned with the cleaning solution to clean the magnetic beads II adsorbing the nucleic acid; (4.1) elution: the magnetic beads II adsorbing the nucleic acid are transferred from the third hole site to the fourth hole site for elution, and the target nucleic acid is preserved in the elution solution; In the second aspect of claim 12, the subsequent steps further include: (2.2) impurity removal treatment: the magnetic beads I are adsorbed in the first hole site for impurity removal, the magnetic beads II in the second hole site are transferred to the third hole site for temporary storage, and the magnetic beads I after the impurity removal treatment are transferred to the second hole site, and the magnetic beads I are discarded by magnetic separation; (3.2) adsorption treatment: the magnetic beads II temporarily stored in the third hole site are transferred to the first hole site for adsorption of nucleic acid, and then transferred to the third hole site and cleaned with the cleaning solution to clean the magnetic beads II adsorbing the nucleic acid; (4.2) elution: the magnetic beads II adsorbing the nucleic acid are transferred from the third hole site to the fourth hole site for elution, and the target nucleic acid is preserved in the elution solution; In the third aspect of claim 12, the second hole site also stores cleaning solution 1, and the third hole site also stores cleaning solution 2; the subsequent steps further include: (2.3) the magnetic beads I in the second hole site are transferred to the first hole site for adsorption and impurity removal, and then transferred to the second hole site for magnetic separation to discard the magnetic beads I; (3.3) the magnetic beads II in the third hole site are transferred to the first hole site for adsorption of nucleic acid, and then transferred to the third hole site to clean the magnetic beads II with the cleaning solution 2; (4.3) the magnetic beads II adsorbing the nucleic acid are transferred from the third hole site to the fourth hole site for elution; the magnetic beads II are discarded by magnetic separation in the fourth hole site, and the target nucleic acid is preserved in the elution solution; Preferably, the method does not require centrifugation; Further, the method is fully automatic.

Images