Preparation method of magnetic beads for extracting total nucleic acid from trace cell samples, extraction reagent and extraction method

Abstract

The present invention belongs to the technical field of nucleic acid extraction and magnetic bead preparation, and relates to a preparation method, an extraction reagent and an extraction method of magnetic beads for extracting total nucleic acid from trace cell samples. The preparation method includes: First, preparing magnetic fluid Fe3O4; Second, performing silicon-based coating on the surface of the prepared magnetic fluid Fe3O4; Third, performing carboxyl modification on the surface of the magnetic beads obtained by silicon-based modification to obtain the magnetic beads for nucleic acid extraction, that is, carboxyl magnetic beads. The carboxyl magnetic beads obtained by the process provided by the present invention are more conducive to obtaining high-concentration nucleic acid under the condition of low sample volume, and can meet the requirements of third-generation sequencing or single-cell sequencing.

Description

Technical Field

[0001] The present invention belongs to the technical fields of total nucleic acid extraction and magnetic bead preparation, and relates to a preparation method, an extraction reagent and an extraction method of magnetic beads for extracting total nucleic acid from trace cell samples. Background Art

[0002] Nucleic acid extraction is crucial in processes such as medical-related scientific research, clinical tests, disease treatment, and disease course follow-up. Nucleic acid extraction methods include organic solvent extraction methods such as chloroform, centrifugal column chromatography methods, and magnetic bead methods. Among them, the organic solvent extraction method using chloroform and the like is to first lyse cells with strong detergents and the like to release nucleic acids, and then precipitate nucleic acids with organic solvents such as chloroform to remove impurities such as proteins and salts; the centrifugal column chromatography method is to specifically adsorb nucleic acids with a silicon matrix membrane by adjusting the salt concentration and pH value, and remove impurities such as proteins by high-speed centrifugation to purify nucleic acids; the magnetic bead method is: by adjusting the salt concentration and pH value, specifically adsorb and enrich nucleic acids using magnetic nanomaterials.

[0003] There are some problems with the existing publicly disclosed methods for extracting nucleic acids from samples: for example, the traditional organic solvent extraction method using chloroform and the like is suitable for extracting a large amount of nucleic acids, with large nucleic acid loss, low purity, complex operation, long time consumption, and at the same time, it will cause a large amount of environmental pollution; for the centrifugal column chromatography method, the nucleic acid extraction purity is high, but the price is relatively expensive, the operation is complex, the time consumption is long, and large-scale equipment such as ultracentrifugation is required; compared with traditional methods and centrifugal column methods, etc., extracting nucleic acids by the magnetic bead method can solve the problem of large-scale automated nucleic acid extraction. However, based on the existing magnetic bead method extraction reagents on the market for nucleic acid extraction, there are problems such as a large amount of magnetic beads used, too large an elution volume, and too low a nucleic acid concentration obtained, which cannot meet the requirements of scientific research and clinical for the complete and rapid extraction of total nucleic acid from trace cell samples, and cannot meet the subsequent requirements such as third-generation direct sequencing. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method, an extraction reagent and an extraction method of magnetic beads for extracting total nucleic acid from trace cell samples.

[0005] According to the first aspect of the present invention, the present invention provides a preparation method of magnetic beads for extracting total nucleic acid from trace cell samples, and the preparation method includes:

[0006] I. Prepare magnetic fluid Fe3O4;

[0007] II. Perform silicon-based coating on the surface of the prepared magnetic fluid Fe3O4;

[0008] III. Perform carboxyl modification on the surface of the magnetic beads obtained by silicon-based modification to obtain the magnetic beads for extracting total nucleic acid from trace cells, that is, carboxyl magnetic beads.

[0009] Most of the magnetic beads for nucleic acid extraction disclosed in the prior art are obtained by coating the surface of magnetic fluid Fe3O4 with silanol groups after synthesizing the magnetic fluid Fe3O4 to obtain silica-based magnetic beads. For the above silica-based magnetic beads, a large number of silanol groups (hydroxyl groups) are modified on the surface of the magnetic beads, which can specifically bind to nucleic acids in the solution through hydrophobic interaction, hydrogen bond interaction and electrostatic interaction under the conditions of high salt and low pH value, so as to enrich nucleic acids. This kind of magnetic beads is mainly used for the research of a large number of samples. There are problems such as a large amount of magnetic beads used, too large elution volume, and too low concentration of the obtained nucleic acids, which cannot meet the requirements of scientific research and clinical for the complete, rapid and high-concentration extraction of total nucleic acids in trace cell samples (such as peripheral blood trace cell diagnosis, circulating tumor cell research, etc.), and cannot effectively meet the subsequent requirements of third-generation direct sequencing and the like. There are also prior art disclosures of directly coating the surface of magnetic fluid Fe3O4 with carboxyl groups after synthesizing magnetic fluid Fe3O4 to obtain carboxyl magnetic beads. However, such carboxyl magnetic beads are often used for directly immobilizing proteins (including but not limited to drug target proteins, streptavidin or polyclonal antibodies for immunoassay), small molecule ligands, nucleic acids, etc. on the magnetic beads for magnetic affinity separation or targeted delivery. The chemical stability of this kind of carboxyl magnetic beads is poor. There is no technology that has been reported to use similar carboxyl magnetic beads for nucleic acid extraction or adsorption research, and there is no technology that has been reported to use them for the extraction and adsorption of total nucleic acids in trace cell samples.

[0010] Based on the urgent need and existing problems in the extraction of total nucleic acids from trace cell samples, the present invention provides a new method for preparing magnetic beads: First, the magnetic fluid Fe3O4 is synthesized in the present invention, and then the surface of the magnetic beads is coated with silicon hydroxyl groups to synthesize silicon-based magnetic beads. Then, a carboxyl group modifying substance (maleic anhydride) is added for surface carboxyl modification, that is, a large number of carboxyl functional groups are connected on the basis of the silicon-based magnetic beads, and carboxyl-modified magnetic beads are obtained. First, the carboxyl magnetic beads prepared by the preparation method provided by the present invention contain rich carboxyl groups and silanol groups (hydroxyl groups) on the surface at the same time. The carboxyl groups can form an ionic bridge with the negatively charged phosphate groups of nucleic acids through dissociated salt ions (such as Na+), so that DNA is non-specifically adsorbed on the surface of the carboxyl magnetic beads; at the same time, the silanol groups (hydroxyl groups) can also efficiently and non-specifically adsorb nucleic acids, thus achieving the effect of a small amount of magnetic beads adsorbing a large amount of nucleic acids; Second, through the magnetic bead preparation method provided by the present invention, the obtained magnetic beads have irregular shapes, which is beneficial to the adsorption of DNA or RNA with different fragment sizes, and is especially suitable for the extraction and adsorption requirements of total nucleic acids in trace cell samples; Third, the magnetic beads obtained by the magnetic bead preparation method provided by the present invention have small particle sizes. Therefore, under the condition of the same mass, the magnetic beads prepared by the preparation method of the present invention can achieve a much higher adsorption efficiency than the existing silicon-based magnetic beads on the market with a smaller usage amount, so that the elution volume during nucleic acid extraction can be made smaller, thereby realizing the high-concentration extraction of total nucleic acids from trace cells. The carboxyl magnetic beads obtained by the preparation method provided by the present invention are more conducive to obtaining high-concentration nucleic acids under the condition of trace samples, and can meet the requirements for direct detection of native DNA or total RNA by third-generation sequencing (the requirements for third-generation sequencing are that the DNA quality is not less than 1.5 μg, the volume is not more than 48 μl; the total RNA quality is not less than 1 μg, the volume is not more than 10 μl; the mRNA quality is not less than 100 ng, and the total volume is not more than 10 μl).

[0011] Specifically, the "total nucleic acid extraction" in the present invention mainly refers to the co-extraction of DNA and RNA from trace nucleic acid samples.

[0012] In some embodiments of the present invention, in step one, the preparation of magnetic fluid Fe3O4 includes: mixing a ferrous salt, a surfactant, and a ferric salt; adding an alkali solution precipitant and continuing to stir; after the stirring is completed, performing high-temperature aging treatment; after the high-temperature aging treatment, magnetically attracting to obtain magnetic fluid Fe3O4, and washing the obtained magnetic fluid Fe3O4 with a cleaning solution; the cleaning solution includes components with the following weight percentages: 0.1‰ - 0.5‰ sodium hypochlorite, 0.5‰ - 1‰ calcium chloride, 0.02‰ - 0.05‰ Tween 20. Compared with the cleaning methods used in the prior art, such as "repeatedly washing with 1% ammonia water, 5% NaCl, and deionized water by mass" or "repeatedly washing with deionized water", washing with the cleaning solution provided by the present invention can make the prepared magnetic beads have significantly better dispersion and suspension properties; at the same time, the prior art has also disclosed a solution of using a combination of sodium hypochlorite, calcium chloride, and a polymer (such as polyacrylamide, polyethylene glycol 2000, polyethylene glycol 4000, or polyethylene glycol 8000) as a cleaning solution. Compared with such cleaning solutions, the advantage of the cleaning solution used in the present invention is that the presence of the buffer solution can ensure that the pH value of the system does not change significantly with the change of the composition and content of the cleaning solution, avoiding the influence of the change of pH on the dispersion and suspension properties of the magnetic beads, thereby ensuring the specific surface area of the magnetic beads provided by the present invention and the total nucleic acid extraction effect of the magnetic bead microcell samples.

[0013] In some embodiments of the present invention, in the step of preparing magnetic fluid Fe3O4, the addition ratios of the ferrous salt, the surfactant, the ferric salt, and the alkali solution precipitant are: 0.6 - 3 g : 0.5 - 3 g : 0.01 - 0.5 g : 1 - 5 ml : 8 - 30 ml. By further adjusting the ratios of the ferrous salt, the surfactant, the ferric salt, and the alkali solution precipitant, the present invention prepares magnetic fluid Fe3O4 without introducing nitrogen or argon, simplifies the process while saving costs, and achieves better effects at the same time.

[0014] In some embodiments of the present invention, the divalent salt is selected from at least one of ferrous chloride tetrahydrate and ferrous sulfate.

[0015] In some embodiments of the present invention, the trivalent salt is selected from ferric chloride hexahydrate.

[0016] In some embodiments of the present invention, the surfactant is selected from at least one of polyethylene glycol 800, polyethylene glycol 1500, polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000, polyvinylpyrrolidone, and oleic acid.

[0017] In some embodiments of the present invention, the alkali solution precipitating agent is selected from at least one of ammonia water and sodium hydroxide.

[0018] In some embodiments of the present invention, in step two, the silicon-based coating on the surface of the prepared magnetic fluid Fe3O4 includes: successively adding an ethanol solution, a dispersant, an alkali solution, and a tetraethyl orthosilicate ethanol solution to the prepared magnetic fluid Fe3O4 for reaction. After the reaction is completed, the magnetic beads are separated by magnetic attraction, and the magnetic beads are washed with a cleaning solution to obtain silicon-based coated magnetic beads; in the step of washing the obtained magnetic beads with the cleaning solution, the cleaning solution includes components with the following weight ratios: 0.1‰ - 0.5‰ sodium hypochlorite, 0.5‰ - 1‰ calcium chloride, and 0.02‰ - 0.05‰ Tween20. Compared with the cleaning methods used in the prior art, such as "washing 3 times with absolute ethanol and then repeatedly washing with ultrapure water..." or "washing twice with absolute ethanol, then washing 2 times with 1 mol dilute hydrochloric acid, and then washing with deionized water until neutral...", washing with the cleaning solution provided by the present invention can make the prepared magnetic beads have significantly better suspension and other properties.

[0019] In some embodiments of the present invention, in step three, the carboxyl modification on the surface of the magnetic beads obtained by silicon-based modification includes: dispersing the silicon-based coated magnetic beads in absolute ethanol, adding a carboxyl modifying substance for carboxyl modification. After the reaction is completed, the reactants are washed with a cleaning solution. After the washing is completed, the carboxyl magnetic beads obtained by carboxyl modification are obtained by magnetic attraction.

[0020] In some embodiments of the present invention, among them, the conditions in the step of carboxyl modification on the surface of the magnetic beads obtained by silicon-based modification include: the carboxyl modifying substance is selected from at least one of maleic anhydride, acrylic acid, α-methylacrylic acid, and undecylenic acid. Using the above substances for carboxyl modification of the silicon-based coated magnetic beads can make the surface of the magnetic beads carry rich carboxyl and silicon hydroxyl functional groups, improving the effect of extracting total nucleic acid from trace cell samples.

[0021] In some embodiments of the present invention, the dispersing the silicon-based coated magnetic beads in absolute ethanol includes: dispersing the silicon-based coated magnetic beads in absolute ethanol and performing ultrasonic stirring; the conditions of the ultrasonic stirring are: under the condition of 200 - 300 r / min, ultrasonic stirring for 10 - 20 min.

[0022] In some embodiments of the present invention, the adding a carboxyl modifying substance for carboxyl modification includes: adding a carboxyl modifying substance, ultrasonic stirring for 3 - 10 min, then turning off the ultrasonic and continuing normal stirring for 15 - 25 h.

[0023] In some embodiments of the present invention, in the step of washing the obtained magnetic beads with the washing solution, the washing solution comprises components with the following weight ratios: 0.1‰ - 0.5‰ of sodium hypochlorite, 0.5‰ - 1‰ of calcium chloride, and 0.02‰ - 0.05‰ of Tween 20.

[0024] According to the second aspect of the present invention, the present invention also provides a carboxyl magnetic bead prepared by the preparation method described in any one of the first aspect of the present invention.

[0025] According to the third aspect of the present invention, the present invention also provides an application of the magnetic bead described in the second aspect of the present invention in the extraction of total nucleic acid from a trace cell sample.

[0026] The trace cell sample includes a trace cell sample with a quantity as low as 10 cells.

[0027] According to the fourth aspect of the present invention, the present invention also provides a reagent for extracting total nucleic acid from a trace cell sample, the extraction reagent comprising: 2 - 20 μL of lysis-binding solution, 50 - 150 μL of washing solution, 2 - 5 μL of elution solution, and 0.1 - 5 μg of magnetic beads; the magnetic beads are selected from the carboxyl magnetic beads described in the second aspect of the present invention or the carboxyl magnetic beads prepared by the preparation method described in the first aspect of the present invention.

[0028] In the extraction reagent provided by the present invention, precisely because the magnetic beads prepared by the preparation method provided by the present invention have excellent adsorption efficiency and extraction efficiency for nucleic acids in the sample, high-concentration total nucleic acids can be obtained even under the condition of trace cell samples. Therefore, when using the magnetic beads provided by the present invention to extract total nucleic acids in the sample, the corresponding sample treatment volume, lysis-binding solution volume, magnetic bead volume, washing solution volume, and elution solution volume are significantly less than the magnetic bead usage disclosed in the prior art. The concentration of the total nucleic acid solution obtained by extraction is high, the volume is small, and no further concentration is required. Therefore, the entire experimental process has simple steps, is easy to operate, and has a higher total nucleic acid extraction efficiency. For the total nucleic acid extraction kit provided by the present invention, the usage amount of the lysis-binding solution is 2 - 20 μL, which means that the minimum usage amount of the lysis-binding solution can be only 2 μL on the premise of ensuring excellent total nucleic acid extraction efficiency; the usage amount of the washing solution is 50 - 150 μL, which means that the minimum usage amount of the washing solution is only 50 μL; the usage amount of the elution solution is 2 - 5 μL, which means that the minimum usage amount of the elution solution can be up to 2 μL; the usage amount of the magnetic beads is 0.1 - 5 μg, which means that the minimum usage amount of the magnetic beads can be only 0.1 μg. The usage amounts of these reagents are far less than those of the magnetic bead nucleic acid extraction kits disclosed in the prior art: most of the lysis-binding solution requires at least 30 μL, the usage amount of the magnetic beads needs to be 500 μg or more, the washing solution needs to be 500 μL or more, and the elution solution needs to be 50 μL or more. In addition, all the existing publicly available magnetic bead nucleic acid extraction kits can only extract DNA or RNA, and there is no magnetic bead nucleic acid extraction kit that can extract both DNA and RNA simultaneously. Therefore, the magnetic beads provided by the present invention have more significant total nucleic acid extraction efficiency and concentration, and the obtained product can meet the requirements of third-generation direct sequencing.

[0029] In some embodiments of the present invention, the lysis-binding solution includes: 0.001 - 0.12 mg of proteinase K, DEPC water, 0.5 - 6 M guanidine hydrochloride or guanidine isothiocyanate, pH 3.5 - 9.5; the washing solution includes 0.1 - 5‰ DEPC water, 1 - 8% PEG - 8000, 20 - 80% ethanol, 0.5 - 3 M NaCl; the elution solution includes: 0.1 - 2 mol / L Tris - HCl, 0.5 - 1 mol / L EDTA.

[0030] In some embodiments of the present invention, the trace cell sample includes: a trace cell sample with a quantity as low as 1 - 10 cells.

[0031] According to the fifth aspect of the present invention, the present invention also provides a method for extracting total nucleic acids from a trace cell sample, and the extraction method includes: extracting with the extraction reagent according to any one of the fourth aspects of the present invention; the extraction method includes:

[0032] (1) Take a trace cell sample from which nucleic acids are to be extracted;

[0033] (2) Add magnetic beads and lysis binding solution to the sample for treatment;

[0034] (3) Wash the magnetic beads obtained after lysis binding treatment with a washing solution;

[0035] (4) After adding an elution solution to elute the washed magnetic beads, perform magnetic separation to obtain a supernatant, which is the extracted total nucleic acid sample.

[0036] In some embodiments of the present invention, in step (2), the conditions for the treatment include: binding at room temperature for 3 - 5 min, magnetic separation, and aspirating the filtrate.

[0037] In some embodiments of the present invention, in step (3), the conditions for the washing include: washing at room temperature for 1 - 3 min, vortex mixing, magnetic separation, aspirating the waste liquid, and air drying.

[0038] In some embodiments of the present invention, in step (4), the conditions for the elution include: 50 - 60 °C, eluting for 2 - 5 min.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) The present invention provides a method for preparing magnetic beads for extracting total nucleic acid from trace cell samples. First, magnetic fluid Fe3O4 is synthesized in the present invention, and then the surface is coated with silicon hydroxyl groups to synthesize silicon-based magnetic beads. Then, a carboxyl group-modifying substance is added for surface carboxyl modification, that is, a large number of carboxyl functional groups are connected on the basis of the silicon-based magnetic beads, and the obtained carboxyl magnetic beads are obtained. The carboxyl magnetic beads prepared by the preparation method provided by the present invention have rich carboxyl groups on the surface. The carboxyl group can form an ionic bridge with the negatively charged phosphate group of nucleic acid through dissociated salt ions (such as Na+), so that DNA is non-specifically adsorbed on the surface of the carboxyl magnetic beads; at the same time, the magnetic beads of this patent also have silanol groups (hydroxyl groups), which can efficiently non-specifically adsorb nucleic acid. Therefore, under the condition of the same mass, the adsorption efficiency of the nucleic acid by the magnetic beads is much higher than that of the silicon-based magnetic beads used in the prior art or the carboxyl magnetic beads directly coated with carboxyl groups on the surface of magnetic fluid Fe3O4. The carboxyl magnetic beads obtained by the process provided by the present invention are more conducive to obtaining high-concentration nucleic acid under the condition of trace cell samples.

[0041] (2) Based on the method for preparing magnetic beads for total nucleic acid extraction from trace cell samples provided by the present invention, a reagent for total nucleic acid extraction from trace cell samples and a method for total nucleic acid extraction are further provided. Because the magnetic beads provided by the present invention have excellent adsorption efficiency and extraction efficiency for total nucleic acid in trace cell samples, during nucleic acid extraction, the corresponding sample treatment volume, lysis and binding solution volume, magnetic bead volume, washing solution volume, and elution solution volume are significantly less than the amounts of various reagents in the magnetic bead-based nucleic acid extraction kits disclosed in the prior art. At the same time, the nucleic acid extraction efficiency is also higher. The present invention solves the problems existing in the magnetic bead-based nucleic acid extraction kits on the existing market, such as the relatively large amount of magnetic beads used, the too large elution volume, the too low nucleic acid concentration obtained, the inability to meet the requirements for the complete, rapid, and high-concentration extraction of RNA and DNA information in trace cell samples, and the inability to meet the subsequent requirements for direct sequencing of the third-generation sequencing. Description of the Drawings

[0042] Figure 1 It is a scanning electron microscope display diagram of the magnetic bead product prepared by the method of Example 1 of the present invention;

[0043] Figure 2 It is a microscope display diagram of the magnetic bead product prepared by the method of Example 1 of the present invention;

[0044] Figure 3 It is a macroscopic observation display diagram of the magnetic bead product prepared by the method of Example 1 of the present invention placed in a centrifuge tube;

[0045] Figure 4 It is the hysteresis loop of the magnetic bead product prepared by the method of Example 1 of the present invention; It can be seen from Figure 4 that the saturation magnetization intensity of the magnetic bead product prepared by the method of Example 1 is 40.03 emu / g;

[0046] Figure 5 It is the analysis result of the nucleic acid fragment distribution of the nucleic acid extraction product extracted from the Hela cell sample with 5 cells in Example 2 of the present invention;

[0047] Figure 6 It is an overlay diagram of the analysis results of the nucleic acid extraction products extracted from the Hela cell samples with 5, 10, 20, and 50 cells respectively in Example 2 of the present invention;

[0048] Figure 7The comparison results of the semi-settling of magnetic beads prepared by different preparation processes in Comparative Example 1 of the present invention; among them, "1" is the semi-settling result of the magnetic beads prepared in Group 1, "2" is the semi-settling result of the magnetic beads prepared in Group 2, "3" is the semi-settling result of the magnetic beads prepared in Group 3, "4" is the semi-settling result of the magnetic beads prepared in Group 4 (Example 1), "A" is the comparison diagram of the settling effect corresponding to a settling time of 15 minutes, "B" is the comparison diagram of the settling effect corresponding to a settling time of 30 minutes, "C" is the comparison diagram of the settling effect corresponding to a settling time of 60 minutes, and "D" is the comparison diagram of the settling effect corresponding to a settling time of 120 minutes;

[0049] Figure 8 The comparison results of the semi-settling of magnetic beads prepared by different cleaning methods in Comparative Example 2 of the present invention; among them, "1" is the semi-settling result of the magnetic beads prepared in Group 1, "2" is the semi-settling result of the magnetic beads prepared in Group 2, "3" is the semi-settling result of the magnetic beads prepared in Group 3 (Example 1), "A" is the comparison diagram of the settling effect corresponding to a settling time of 15 minutes, "B" is the comparison diagram of the settling effect corresponding to a settling time of 30 minutes, "C" is the comparison diagram of the settling effect corresponding to a settling time of 60 minutes, and "D" is the comparison diagram of the settling effect corresponding to a settling time of 120 minutes. Detailed implementation manners

[0050] The technical solutions of the present invention are further described below through specific embodiments. The specific embodiments do not represent a limitation on the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0051] As used in the present invention, including as used in the embodiments and unless otherwise clearly specified, all numbers can be regarded as if they start with the words "substantially", "about" or "approximately", even if the term does not explicitly appear. When describing the amplitude and / or position to indicate that the described numerical value and / or position are within a reasonable expected value and / or position range, the phrase "about" or "approximately" can be used. For example, the numerical value can be ±0.1% of the described numerical value (or numerical range), ±1% of the described numerical value (or numerical range), ±2% of the described numerical value (or numerical range), ±5% of the described numerical value (or numerical range), ±10% of the described numerical value (or numerical range), ±15% of the described numerical value (or numerical range), ±20% of the described numerical value (or numerical range), etc. Any numerical range described in the present invention is intended to include all sub-ranges or intermediate values contained therein.

[0052] The disclosure of numerical values and numerical ranges of specific parameters (such as temperature, weight percentage, parts by weight, etc.) does not exclude other numerical values and numerical ranges useful for the present invention. It is contemplated that two or more specific example numerical values of a given parameter can define the endpoints of the numerical range that the parameter can claim. For example, if parameter X is exemplified herein as having a numerical value A and is also exemplified as having a numerical value Z, it can be expected that parameter X can have a numerical range from approximately A to approximately Z. Similarly, it is contemplated that the disclosure of two or more numerical ranges of a parameter (whether these ranges are nested, overlapping, or distinct) encompasses all possible combinations of numerical ranges that can be claimed using the endpoints of the disclosed ranges. For example, if parameter X is exemplified herein as having values in the range of 1 - 10, it also describes sub - ranges of parameter X, including by way of example only, such as: 1 - 9, 1 - 8, 1 - 7, 2 - 9, 2 - 8, 2 - 7, 3 - 9, 3 - 8, 3 - 7, 2 - 8, 3 - 7, 4 - 6 or 7 - 10, 8 - 10 or 9 - 10. A range includes its endpoints as well as the values within the endpoints, for example, the range 0 - 5 includes 0, >0, 1, 2, 3, 4, <5 and 5.

[0053] Hereinafter, the embodiments of the present invention will be described in more detail through specific examples and comparative examples.

[0054] Example 1

[0055] This example provides a method for preparing magnetic beads for nucleic acid extraction, and the specific steps are as follows:

[0056] I. Preparation of magnetic fluid Fe3O4

[0057] 1.1. Transfer ferrous chloride tetrahydrate (FeCl2·4H2O), a surfactant polyethylene glycol - 2000 (PEG - 2000), and ferric chloride hexahydrate (FeCl3·6H2O) into a reaction vessel in a certain order and add an appropriate amount of solvent ultrapure water. Stir at room temperature for 15 min under a stirring speed of 260 r / min; among them, the addition amount of ferrous chloride tetrahydrate is 3 g, the addition amount of polyethylene glycol - 2000 is 0.5 g, the addition amount of ferric chloride hexahydrate is 2.5 g, and the addition amount of solvent ultrapure water is 30 ml. Dissolve the ferrous salt, surfactant, and ferric salt in three groups of solvents respectively, and then transfer the three groups of dissolved reactants into the reaction vessel in the order of ferrous salt, surfactant, and ferric salt and add the remaining solvent;

[0058] 1.2 After stirring, add the alkaline solution precipitant ammonia water dropwise into the reaction vessel. Along with the addition of the alkaline solution precipitant, adjust the stirring speed to 560 r / min accordingly. After the addition of the alkaline solution precipitant is completed, stir for 6 h at a stirring speed of 560 r / min. The above operations need to be carried out under the condition that the reaction temperature is controlled below 35 °C. Among them, the dropping amount of the alkaline solution precipitant is 5 ml, and the dropping speed of the alkaline solution precipitant is 30 ml / h;

[0059] 1.3 After the stirring is completed, heat the stirred reaction system and react for 3 h at 85 °C for high-temperature aging treatment;

[0060] 1.4 After the high-temperature aging treatment is completed, pour out the reaction solution. After natural sedimentation for 6 h, remove the supernatant by magnetic attraction, wash the precipitated magnetic fluid Fe3O4 with the cleaning solution, and make up the volume of the washed magnetic fluid Fe3O4 in pure water to obtain the magnetic fluid Fe3O4;

[0061] II. Preparation of magnetic beads

[0062] 2.1 Silicon-based coating: Take 320 g of the magnetic fluid Fe3O4 prepared in Step 1, transfer it to the reaction vessel after stirring evenly, and add an ethanol solution with a certain ratio (the volume ratio of the ethanol solution to the magnetic beads is 6:1) to fix the concentration. Stir ultrasonically at 300 r / min at room temperature for 3 h. After the ultrasonic stirring is completed, add the dispersant trisodium citrate to the system. The addition amount of the dispersant trisodium citrate is 10 g, and stir at 300 r / min for 15 min. After the stirring is completed, add 80 ml of ammonia water and continue to stir ultrasonically for 15 min. After the ultrasonic stirring is completed, add 120 ml of tetraethyl orthosilicate ethanol solution using a micro-injection pump, control the dropping speed of the tetraethyl orthosilicate ethanol solution to be 150 ml / h. After the dropping is completed, stir the reaction solution ultrasonically for 10 min, then turn off the ultrasonic wave, and then carry out ordinary stirring for 0.5 h. After the ordinary stirring is completed, let it stand for about 16 h. After standing is completed, remove the supernatant by magnetic attraction, and wash the obtained magnetic beads with the cleaning solution to obtain the intermediate magnetic beads, that is, silicon-based coated magnetic beads;

[0063] 2.2. Carboxyl modification: Take 350 g of the washed intermediate magnetic beads and disperse them in absolute ethanol. The volume ratio of absolute ethanol to magnetic beads is 4:1. Stir evenly with a glass rod, and under the condition of 240 r / min, perform ultrasonic stirring for 15 min; after sedimentation, stir evenly with a glass rod, transfer to a 20 L reaction vessel, and under the condition of 240 r / min, continue ultrasonic stirring for 10 min. Then add 20 g of dissolved maleic anhydride (the solvent used for dissolution is pure water, and the dosage of the solvent is 100 ml), and perform ultrasonic stirring for another 5 min. Then turn off the ultrasonic and continue stirring for 20 h; at the end of the reaction, pour out the reactants and wash them with the cleaning solution; after washing, obtain irregular-shaped magnetic beads by magnetic attraction, that is, carboxyl magnetic beads. Dilute the obtained carboxyl magnetic beads to 10 mg / ml with ultrapure water to prepare magnetic beads for nucleic acid extraction;

[0064] Among them, in the above steps, the cleaning solution used is prepared from 0.1‰ sodium hypochlorite, 0.5‰ calcium chloride, and 0.05‰ Tween 20. The preparation method of the cleaning solution is: weigh 0.1 g of sodium hypochlorite and 0.5 g of calcium chloride, dissolve them in 999.95 ml of pure water, and then add 0.05 ml of Tween 20 and stir evenly.

[0065] The scanning electron microscope image of the magnetic beads prepared by the above method is as shown in Figure 1 shown, the microscope image of the magnetic beads is as shown in Figure 2 shown, the macroscopic appearance image of the magnetic beads in the centrifuge tube is as shown in Figure 3 shown, the hysteresis loop of the magnetic beads is as shown in Figure 4 shown. According to the results shown in Figures 1-4 it shows that the magnetic beads are irregular in shape, the magnetic saturation intensity is about 40.03 emu / g, the average particle size is about 410.5 nm, the polydispersity index obtained from the particle size test distribution curve of the magnetic beads by the laser particle size analyzer is 0.010, showing a monodisperse state, with a larger specific surface area and better corresponding adsorption performance.

[0066] Example 2

[0067] Based on the magnetic beads prepared in Example 1, this example provides a method for extracting nucleic acid from a trace sample. The nucleic acid extraction sample used in this example is a sample with an extremely small number of nucleic acids. The samples are human HeLa cells with cell numbers of 5, 10, 20, and 50 respectively. The specific steps are as follows:

[0068] (1) Place the above four gradient cell number samples taken into 200 μL centrifuge tubes respectively;

[0069] (2) Lysis and binding: Add 0.02 mg of proteinase K, 4 μL of lysis-binding solution (DEPC water, 0.8 - 6 M guanidine hydrochloride or guanidine isothiocyanate, pH 3.5 - 8.8), and 5 μg of magnetic beads to each centrifuge tube, and bind at room temperature for 4 min; perform magnetic separation and discard the waste liquid;

[0070] (3) Washing: Wash the magnetic beads obtained in step (2) with 100 μL of washing solution (DEPC water, 80% ethanol, 0.5 - 3 M NaCl) at room temperature for 1 min, and vortex to mix evenly; perform magnetic separation, discard the waste liquid, and air-dry for 2 min;

[0071] (4) Elution: Add 2 μL of elution solution to the air-dried magnetic beads obtained in step (3), elute at 56 °C for 3 min. After the elution is completed, perform magnetic separation, and aspirate the supernatant, which is the total nucleic acid sample (and DNA) extracted.

[0072] The nucleic acid samples in the trace human HeLa cell samples extracted by the above method were respectively run on an Agilent precast high-sensitivity RNA gel, and then the results were analyzed using an Agilent 4200 TapeStation system. The results obtained are as Figure 5 、 Figure 6 shown, Figure 5 is the analysis result of the nucleic acid extraction product extracted from a human HeLa cell sample with 5 cells; Figure 6 is the superposition of the analysis results of the nucleic acid extraction products extracted from human HeLa cell samples with 5, 10, 20, and 50 cells respectively.

[0073] As Figure 5 shown in the results, Figure 5 in, the bands corresponding to 18S and 28S are the bands of ribosomal RNA, which are used to determine whether RNA is extracted and whether there is degradation. The band of ">10000" on the far right corresponds to the band of DNA, and whether DNA is extracted and whether there is degradation can be further determined through this band; "824", "1404", and "1893" represent other non-typical nucleic acid fragments. As can be seen from Figure 5 the results shown, through the magnetic beads provided by the present invention, co-extraction of DNA and RNA in samples with extremely low nucleic acid amounts (5 cells) can be achieved; According to Figure 6The results shown indicate that the concentration / density of the corresponding nucleic acid fragments extracted from different samples is positively correlated with the number of cells in the samples. That is, as the number of cells decreases, the nucleic acid content of the same molecular weight decreases, and the two trends are consistent. Moreover, the positions where the peaks appear are the same and the bands are consistent, further indicating that the magnetic beads provided by the present invention can be used to extract total nucleic acids from samples of human Hela cells with 5, 10, 20, and 50 cells respectively, and according to Figure 6 The results shown further indicate that when the magnetic beads provided by the present invention are used to extract nucleic acids from the corresponding samples, the results are relatively stable, further indicating the stability of the magnetic bead product and the extraction method provided by the present invention.

[0074] Comparative Example 1

[0075] Based on Example 1, this comparative example further explored the influence of different preparation processes on the performance of the magnetic bead products prepared, as follows:

[0076] Group 1: Magnetic beads prepared according to the corresponding patent document (CN103908945A);

[0077] Group 2: Magnetic beads prepared according to the corresponding patent document (CN111330558A);

[0078] Group 3: Based on Example 1, the process parameters in Step 2.2 of the carboxyl modification were changed. The specific method was as follows:

[0079] Take 350 g of the washed intermediate magnetic beads, disperse them in anhydrous ethanol, stir evenly with a glass rod, stir at 240 r / min for 15 min; after sedimentation, stir evenly with a glass rod, transfer to a 20 L reaction vessel, continue to stir at 240 r / min for 10 min, then add 20 g of dissolved maleic anhydride, and stir for 20 h; other conditions are the same as in Example 1 to prepare carboxyl-modified magnetic beads.

[0080] Group 4: Example 1.

[0081] First, the suspension performance of the magnetic beads prepared by the above Group 1, Group 2, Group 3, and Group 4 (Example 1) was tested respectively. That is, the prepared magnetic beads were subjected to natural sedimentation for different times (15 min, 30 min, 60 min, 120 min), and the final semi-sedimentation effect was compared. The results are as Figure 7 shown. According to Figure 7 the results shown, the semi-sedimentation times of the magnetic beads prepared by different preparation processes are significantly different, indicating that the suspension performances are quite different. The semi-sedimentation time of the magnetic beads prepared by Example 1 is more than 2 h, and the suspension performance is significantly better than that of Group 1, Group 2, and Group 3.

[0082] Secondly, the relevant indicators and performance of the magnetic beads prepared by the preparation processes of the above-mentioned Group 1, Group 2, Group 3, and Group 4 (Example 1) were further compared. The comparison results are shown in Table 1:

[0083] Table 1 Comparison of relevant indicators and performance of each magnetic bead

[0084]

[0085] According to the results shown in Table 1, by the method provided by the present invention, the prepared magnetic beads have small particle size, large specific surface area, and larger nucleic acid adsorption amount per microgram of magnetic beads. In addition, from the comparison results of the physical and chemical indexes of each magnetic bead shown in Table 1, it can be seen that the half-sedimentation times of the magnetic beads prepared by Group 1, Group 2, Group 3, and Group 4 (Example 1) are significantly different, indicating that there are large differences in suspension performance. The half-sedimentation times of the magnetic beads in Group 1 and Group 2 are about 10 minutes, the half-sedimentation time of the magnetic beads in Group 3 is 75 minutes, while the half-sedimentation time of the magnetic beads prepared by the method of Group 4 (Example 1) is greater than 2 hours, and the suspension performance is significantly better than the other three groups.

[0086] Comparative Example 2

[0087] Based on Example 1, this comparative example further explored the influence of different cleaning methods on the performance of magnetic bead products. The difference from Example 1 is only that the cleaning methods are different in each step, as follows:

[0088] Group 1: Adopt the cleaning method similar to that in the patent document CN103908945A: That is, based on Example 1, in step 1.4 of step one, replace "removing the supernatant by magnetic attraction and washing the precipitated magnetic fluid Fe3O4 with the cleaning solution" with "separating out the magnetic fluid Fe3O4 by magnetic attraction and repeatedly washing the precipitate 3 times with ammonia water with a mass concentration of 1%, 5% NaCl, and deionized water until the washing solution is neutral"; and in step 2.1 of step two, replace "after standing, removing the supernatant by magnetic attraction and washing the obtained magnetic beads with the cleaning solution" with "after standing, washing 3 times with absolute ethanol and repeatedly washing with ultrapure water several times until the pH value of the washing solution is 7" to prepare the corresponding carboxyl magnetic beads;

[0089] Group 2: The cleaning method in patent document CN111330558A is adopted: that is, based on Example 1, in step 1.4 of step one, "removing the supernatant by magnetic attraction and cleaning the precipitated magnetic fluid Fe3O4 with a cleaning solution" is replaced with "washing repeatedly with deionized water until neutral"; and in step 2.1 of step two, "after standing, removing the supernatant by magnetic attraction and cleaning the obtained magnetic beads with a cleaning solution" is replaced with "cleaning twice with absolute ethanol, then cleaning 2 times with 1 mol of dilute hydrochloric acid, and then washing with deionized water until neutral", and the corresponding carboxyl magnetic beads are prepared;

[0090] Group 3: Example 1.

[0091] The suspension performance of the magnetic beads prepared through the above Group 1, Group 2, and Group 3 is tested respectively, that is, the prepared magnetic beads are naturally settled for different times (15 min, 30 min, 60 min, 120 min), and the final semi-settling effect is compared. The results are as Figure 8 shown. According to Figure 8 the results shown, for the magnetic beads prepared based on different cleaning methods, the semi-settling time is significantly different, and the suspension performance varies greatly. The semi-settling time of the magnetic beads prepared through Group 3 (Example 1) is more than 2 h, and the suspension performance is significantly better than that of Group 1 and Group 2. That is, the suspension performance of the magnetic beads cleaned with the cleaning solution provided by the present invention is better. Sodium hypochlorite in the cleaning solution provided by the present invention can further remove the uncoated iron and free impurity ions on the magnetic beads, making the magnetic beads themselves cleaner, without attached debris, and increasing the number of effective functional groups of the magnetic beads; in addition, the divalent cations in the cleaning solution make it difficult for the magnetic bead particles to agglomerate through electrostatic repulsion, and thus better disperse and suspend, improving the suspension performance of the magnetic beads; the buffer solution in the cleaning solution can adjust the pH value of the cleaning, so as not to cause a change in pH due to changes in the composition and content of the cleaning solution, making the extraction efficiency of total nucleic acid from trace cell samples higher.

[0092] Comparative Example 3

[0093] This comparative example further compares the extraction reagent buffer systems used for different magnetic beads, as follows:

[0094] The extraction reagent buffer systems used for extracting nucleic acid from samples in Examples 2-3 of the present invention and existing documents CN111330558A and CN103908945A are compared respectively. The comparison results are shown in Table 2:

[0095] Table 2 Comparison of extraction reagent buffer systems

[0096]

[0097] According to the comparison results presented in Table 2, when extracting nucleic acid samples based on the magnetic beads provided in the embodiments of the present invention, the corresponding sample processing volume, lysis binding solution volume, magnetic bead volume, washing solution volume, and elution solution volume are significantly less than the addition amounts in the existing literatures CN111330558A and CN103908945A, and the nucleic acid extraction efficiency is higher.

[0098] It can be understood that the present invention is described through some embodiments. Those skilled in the art know that without departing from the spirit and scope of the present invention, various changes or equivalent replacements can be made to these features and embodiments. Additionally, under the teaching of the present invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of the present invention belong to the scope protected by the present invention.

Claims

1. A method for preparing magnetic beads for extracting whole nucleic acid from trace cell samples, characterized in that: The preparation method comprises:

1. Preparation of magnetic fluid Fe3O4: Mixing divalent iron salt, surfactant and trivalent iron salt; adding alkali solution precipitant and continuing stirring; after stirring, performing high temperature aging treatment; after high temperature aging treatment, magnetic fluid Fe3O4 is obtained by magnetic absorption, and the obtained magnetic fluid Fe3O4 is cleaned with washing liquid; 2. Silicon-based coating is performed on the surface of the prepared magnetic fluid Fe3O4: ethanol solution, dispersant, alkali solution, and tetraethyl orthosilicate ethanol solution are sequentially added to the prepared magnetic fluid Fe3O4 to react. After the reaction is completed, the magnetic beads are separated by magnetic attraction, and the magnetic beads are washed with a washing solution to obtain silicon-based coated magnetic beads; 3. Carboxyl modification is performed on the surface of the magnetic beads obtained by silica coating to obtain the magnetic beads for nucleic acid extraction, i.e., carboxyl magnetic beads: the magnetic beads after silica coating are dispersed in anhydrous ethanol and ultrasonically stirred; the ultrasonic stirring conditions are: ultrasonic stirring for 10-20 minutes under 200-300r / min; carboxyl modified substances are added, ultrasonic stirring is performed for 3-10 minutes, then the ultrasound is turned off, and conventional stirring is continued for 15-25 hours; after the reaction is completed, the reactants are washed with a washing solution, and after washing, the carboxyl modified carboxyl magnetic beads are obtained by magnetic absorption; Among them, in step 1, step 2 and step 3, the washing solution used includes the following components in weight proportion: 0.1‰-0.5‰ sodium hypochlorite, 0.5‰-1‰ calcium chloride, and 0.02‰-0.05‰ Tween20.

2. The preparation method according to claim 1, characterized in that: The divalent iron salt is selected from at least one of ferrous chloride tetrahydrate and ferrous sulfate.

3. The preparation method according to claim 1, characterized in that: The ferric iron salt is selected from ferric chloride hexahydrate.

4. The preparation method according to claim 1, characterized in that: The surfactant is selected from at least one of polyethylene glycol 800, polyethylene glycol 1500, polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000, polyvinyl pyrrolidone and oleic acid.

5. The preparation method according to claim 1, characterized in that: The alkaline solution precipitant is selected from at least one of ammonia water and sodium hydroxide solution.

6. The preparation method according to claim 1, characterized in that: The carboxyl-modified substance is selected from at least one of maleic anhydride, acrylic acid, α-methacrylic acid, and undecylenic acid.

7. Carboxyl magnetic beads prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the magnetic beads as claimed in claim 7 in extracting whole nucleic acid from trace cell samples.

9. A reagent for extracting whole nucleic acid from a trace cell sample, characterized in that: The extraction reagent includes: 2-20 μL of lysis and binding solution, 50-150 μL of cleaning solution, 2-5 μL of elution solution, and 0.1-5 μg of magnetic beads; the magnetic beads are selected from the magnetic beads described in claim 7 / or the magnetic beads prepared by the preparation method described in any one of claims 1-6.

10. A method for extracting whole nucleic acid from a trace cell sample, characterized in that: The extraction method comprises: extracting with the extraction reagent according to claim 9.

11. The extraction method according to claim 10, characterized in that The extraction method comprises: (1) Take a small amount of cell sample from which nucleic acid is to be extracted; (2) adding magnetic beads and lysis binding solution to the sample for treatment; (3) Using a cleaning solution to clean the magnetic beads obtained after the lysis and binding treatment; (4) After adding elution solution to elute the washed magnetic beads, magnetic separation is performed to obtain the supernatant, which is the extracted whole nucleic acid sample.

12. The extraction method according to claim 11, characterized in that The conditions in the extraction method include: in step (2), the treatment conditions include: combining at room temperature for 3-5 minutes, magnetic separation, and discarding the filtrate.

13. The extraction method according to claim 11, characterized in that In step (3), the cleaning conditions include: cleaning at room temperature for 1-3 minutes, vortex mixing, magnetic separation, suction and discarding of waste liquid, and drying.

14. The extraction method according to claim 11, characterized in that In step (4), the elution conditions include: 50-60°C, elution time 2-5 min.

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