Preparation method of magnetic beads for total nucleic acid extraction of trace cell sample, extraction reagent and extraction method

Abstract

The 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 a magnetic bead for total nucleic acid extraction of a trace cell sample. The preparation method comprises the following steps: 1, preparing magnetic fluid Fe4O3; 2, carrying out silicon-based coating on the surface of the prepared magnetic fluid Fe4O3; and 3, carrying out carboxyl modification on the surface of the magnetic bead obtained through silicon-based modification to obtain the magnetic bead for nucleic acid extraction, namely the carboxyl magnetic bead. The carboxyl magnetic beads obtained by the process provided by the invention are more favorable for obtaining high-concentration nucleic acid under the condition of low sample size, and can meet the requirements of third-generation sequencing or single-cell sequencing.

Description

Technical Field

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

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

[0003] There are some problems with the existing methods for extracting nucleic acids from samples: for example, traditional extraction methods using organic solvents such as chloroform are suitable for extracting large amounts of nucleic acids, but they result in large nucleic acid losses, low purity, complex operations, and long time, and can also cause a lot of environmental pollution; for centrifugal column chromatography, the nucleic acid extraction purity is high, but the price is relatively expensive, the operation is complex, and it takes a long time, and large-scale equipment such as ultracentrifugation is required; compared with traditional methods and centrifugal column methods, the extraction of nucleic acids by magnetic beads can solve the problem of large-scale automated nucleic acid extraction, but the extraction of nucleic acids based on the existing magnetic bead extraction reagents on the market has the problem of a large amount of magnetic beads used, an excessively large elution volume, and a too low nucleic acid concentration, which cannot meet the needs of scientific research and clinical practice for the complete and rapid extraction of whole nucleic acids from trace cell samples, and cannot meet the needs of subsequent third-generation direct sequencing. Summary of the invention

[0004] The present invention aims to provide a preparation method, extraction reagent and extraction method of magnetic beads for extracting whole nucleic acid from trace cell samples.

[0005] According to a first aspect of the present invention, the present invention provides a method for preparing magnetic beads for extracting whole nucleic acid from a trace cell sample, the preparation method comprising: 1. Preparation of magnetic fluid Fe 4 O 3 ; 2. In the prepared magnetic fluid Fe 4 O 3 The surface of the 3. Carboxyl modification is performed on the surface of the magnetic beads obtained by silicon-based modification to obtain the magnetic beads for extracting whole nucleic acid from trace cells, namely carboxyl magnetic beads.

[0006] Most of the magnetic beads used for nucleic acid extraction disclosed in the prior art are synthesized magnetic fluid Fe 4 O 3 Afterwards, the magnetic fluid Fe 4 O 3 The surface is coated with silicon hydroxyl groups to obtain silicon-based magnetic beads; for the above-mentioned silicon-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 the nucleic acids in the solution under high salt and low pH conditions through hydrophobic effects, hydrogen bonds and electrostatic effects, thereby achieving nucleic acid enrichment. The magnetic beads are mainly used for the study of a large number of samples. The amount of magnetic beads used is large, the elution volume is too large, and the resulting nucleic acid concentration is too low. It cannot meet the scientific research and clinical needs for the complete, rapid and high-concentration extraction of whole nucleic acids in trace cell samples (such as peripheral blood trace cell diagnosis, circulating tumor cell research, etc.), and cannot effectively meet the needs of subsequent third-generation direct sequencing. The existing public technology also has synthetic magnetic fluid Fe 4 O 3 Afterwards, the magnetic fluid Fe 4 O 3 The surface is directly coated with carboxyl groups to obtain carboxyl magnetic beads, but such carboxyl magnetic beads are often used to directly immobilize proteins (including but not limited to drug target proteins, streptavidin or polyantibody capture for immunoassay), small molecule ligands, nucleic acids, etc. on the magnetic beads for magnetic bioaffinity separation or targeted delivery. Such carboxyl magnetic beads have poor chemical stability, and there is no technology for using similar carboxyl magnetic beads for research on nucleic acid extraction or adsorption, let alone for using them for extraction and adsorption of whole nucleic acids in trace cell samples.

[0007] Based on the urgent need and existing problems for the extraction of whole nucleic acid from trace cell samples, the present invention provides a new method for preparing magnetic beads: the present invention first synthesizes a magnetic fluid Fe 4 O 3, then the surface of the magnetic beads is coated with silicon hydroxyl groups to synthesize silicon-based magnetic beads, and then a carboxyl-modified substance (maleic anhydride) is added to modify the surface carboxyl groups, that is, a large number of carboxyl functional groups are connected to the silicon-based magnetic beads to obtain carboxyl-modified magnetic beads. First, the carboxyl magnetic beads prepared by the preparation method provided by the present invention have rich carboxyl groups and silanol groups (hydroxyl groups) on the surface. The carboxyl group can form an ion bridge with the negatively charged phosphate group of the nucleic acid through the dissociated salt ions (such as Na+), so that the DNA is non-specifically adsorbed to the surface of the carboxyl magnetic beads; at the same time, the silanol group (hydroxyl group) can also efficiently and non-specifically adsorb nucleic acids, thereby achieving the effect of a small amount of magnetic beads adsorbing a large amount of nucleic acids; secondly, the magnetic beads obtained by the magnetic bead preparation method provided by the present invention have an irregular shape, which is conducive to the adsorption of DNA or RNA fragments of different sizes, and is particularly suitable for the extraction and adsorption of whole nucleic acids in trace cell samples; thirdly, the magnetic beads obtained by the magnetic bead preparation method provided by the present invention have a small particle size. Therefore, under the condition of equal quality, the magnetic beads prepared by the preparation method of the present invention can achieve an adsorption efficiency far higher than that of 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 smaller, thereby achieving high-concentration extraction of whole nucleic acids in 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 trace sample conditions, and can meet the requirements of third-generation sequencing for direct detection of natural DNA or total RNA (third-generation sequencing requires that the DNA mass is not less than 1.5ug and the volume is not more than 48ul; the total RNA mass is not less than 1ug and the volume is not more than 10ul; the mRNA mass is not less than 100ng and the total volume is not more than 10ul).

[0008] Specifically, the "total nucleic acid extraction" in the present invention mainly refers to the co-extraction of DNA and RNA in trace nucleic acid samples.

[0009] In some embodiments of the present invention, in step 1, the preparation of magnetic fluid Fe 4 O 3 The method comprises: mixing a divalent iron salt, a surfactant and a trivalent iron salt; adding an alkali solution precipitant and continuing stirring; performing a high temperature aging treatment after the stirring is completed; and obtaining a magnetic fluid Fe by magnetic absorption after the high temperature aging treatment. 4 O 3 , use cleaning liquid to clean the magnetic fluid Fe 4 O 3Cleaning is performed; the cleaning solution includes the following components in weight proportion: 0.1‰-0.5‰ sodium hypochlorite, 0.5‰-1‰ calcium chloride, and 0.02‰-0.05‰ Twen20. Compared with the cleaning method used in the prior art, such as "repeatedly washing with 1% ammonia water, 5% NaCl and deionized water by mass concentration..." 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 also discloses a solution of combining sodium hypochlorite, calcium chloride and a high molecular 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 will not change significantly with the change of the composition and content of the cleaning solution, thereby avoiding the influence of the dispersion and suspension properties of the magnetic beads due to the change of pH, thereby ensuring the specific surface area of ​​the magnetic beads provided by the present invention and the whole nucleic acid extraction effect of the magnetic beads trace cell sample.

[0010] In some embodiments of the present invention, the preparation of magnetic fluid Fe 4 O 3 In the step of preparing the magnetic fluid Fe 4 O 3 In the step, the addition ratio of the divalent iron salt, the surfactant, the trivalent iron salt and the alkali solution precipitant is: 0.6-3g: 0.5-3g: 0.01-0.5g: 1-5ml: 8-30ml. The present invention further adjusts the ratio of the divalent iron salt, the surfactant, the trivalent iron salt and the alkali solution precipitant to carry out the magnetic fluid Fe under the premise of not introducing nitrogen or argon. 4 O 3 The preparation method simplifies the process, saves costs, and achieves better results.

[0011] In some embodiments of the present invention, the divalent salt is selected from at least one of ferrous chloride tetrahydrate and ferrous sulfate.

[0012] In some embodiments of the present invention, the trivalent salt is selected from ferric chloride hexahydrate.

[0013] 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, polyvinyl pyrrolidone, and oleic acid.

[0014] In some embodiments of the present invention, the alkali solution precipitant is selected from at least one of ammonia water and sodium hydroxide.

[0015] In some embodiments of the present invention, in step 2, the prepared magnetic fluid Fe 4 O 3 The surface of the silicon-based coating includes: 4 O 3 An ethanol solution, a dispersant, an alkali solution, and an ethanol solution of tetraethyl orthosilicate are sequentially added to react. After the reaction is completed, the magnetic beads are separated by magnetic attraction, and the magnetic beads are cleaned with a cleaning solution to obtain silicon-coated magnetic beads; in the step of cleaning the obtained magnetic beads with a cleaning solution, the cleaning solution includes the following components in weight proportion: 0.1‰-0.5‰ sodium hypochlorite, 0.5‰-1‰ calcium chloride, and 0.02‰-0.05‰ Twen20. Compared with the cleaning method used in the prior art, such as "washing with anhydrous ethanol three times, and then repeatedly washing with ultrapure water several times..." or "washing with anhydrous ethanol twice, then washing with 1 mol of dilute hydrochloric acid twice, and then washing with deionized water to neutral...", washing with the cleaning solution provided by the present invention can make the prepared magnetic beads have significantly better suspension and other properties.

[0016] In some embodiments of the present invention, in step three, the carboxyl modification on the surface of the silicon-modified magnetic beads includes: dispersing the silicon-coated magnetic beads in anhydrous ethanol, adding a carboxyl-modified substance for carboxyl modification, and after the reaction is completed, washing the reactants with a washing solution, and after the washing is completed, obtaining carboxyl-modified carboxyl magnetic beads by magnetic attraction.

[0017] In some embodiments of the present invention, the conditions in the step of performing carboxyl modification on the surface of the silicon-modified magnetic beads include: the carboxyl-modified substance is selected from at least one of maleic anhydride, acrylic acid, α-methylacrylic acid, and undecylenic acid. Carboxyl modification of silicon-coated magnetic beads using the above substances can make the surface of the magnetic beads have abundant carboxyl and silanol functional groups, thereby improving the effect of the magnetic beads on the extraction of whole nucleic acid from trace cell samples.

[0018] In some embodiments of the present invention, dispersing the silicon-coated magnetic beads in anhydrous ethanol includes: dispersing the silicon-coated magnetic beads in anhydrous ethanol and performing ultrasonic stirring; the conditions of the ultrasonic stirring are: ultrasonic stirring for 10-20 min at 200-300 r / min.

[0019] In some embodiments of the present invention, the adding of carboxyl-modifying substances for carboxyl modification comprises: adding carboxyl-modifying substances, ultrasonically stirring for 3-10 minutes, then turning off the ultrasound, and continuing conventional stirring for 15-25 hours.

[0020] In some embodiments of the present invention, in the step of washing the obtained magnetic beads with a washing solution, the washing solution comprises the following components in weight proportions: 0.1‰-0.5‰ sodium hypochlorite, 0.5‰-1‰ calcium chloride, and 0.02‰-0.05‰ Twen20.

[0021] According to the second aspect of the present invention, the present invention also provides carboxyl magnetic beads prepared by any preparation method as described in the first aspect of the present invention.

[0022] According to the third aspect of the present invention, the present invention also provides a use of the magnetic beads as described in the second aspect of the present invention in extracting whole nucleic acid from trace cell samples.

[0023] The micro-cell sample includes a micro-cell sample with a number as low as 10.

[0024] According to the fourth aspect of the present invention, the present invention also provides an extraction reagent for total nucleic acid of a trace cell sample, the extraction reagent comprising: 2-20 μL of lysis 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 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.

[0025] In the extraction reagent provided by the present invention, 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, so even under the condition of trace cell samples, a higher concentration of whole nucleic acid can be obtained. Therefore, when the magnetic beads provided by the present invention are used to extract whole nucleic acid in the sample, the corresponding sample processing volume, lysis binding liquid volume, magnetic bead volume, cleaning liquid volume and elution liquid volume are significantly less than the amount of magnetic beads disclosed in the prior art, and the whole nucleic acid solution obtained by extraction has a high concentration and a small volume, and does not need to be concentrated again. Therefore, the entire experimental process is simple and easy to operate, and the whole nucleic acid extraction efficiency is higher. For the whole nucleic acid extraction kit provided by the present invention, the dosage of 2-20 μL of lysis and binding solution means that under the premise of ensuring an excellent whole nucleic acid extraction efficiency, the dosage of lysis and binding solution can be as low as 2 μL; the dosage of 50-150 μL of cleaning solution means that the minimum dosage of cleaning solution is only 50 μL; the dosage of 2-5 μL of eluent means that the minimum dosage of eluent can be as low as 2 μL; the dosage of 0.1-5 μg of magnetic beads means that the minimum dosage of magnetic beads can be as low as 0.1 μg. The dosage of these reagents is much smaller than the dosage of the magnetic bead nucleic acid extraction kit disclosed in the prior art: most of the lysis and binding solutions require a minimum of 30 μL, the dosage of magnetic beads requires 500 μg and above, the cleaning solution requires 500 μL and above, and the eluent requires 50 μL and above. In addition, the existing public 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 DNA and RNA at the same time. Therefore, the magnetic beads provided by the present invention have more significant total nucleic acid extraction efficiency and concentration, and the obtained products can meet the requirements of third-generation direct sequencing.

[0026] In some embodiments of the present invention, the lysis and binding solution includes: 0.001-0.12 mg proteinase K, DEPC water, 0.5-6 M guanidine hydrochloride or guanidine isothiocyanate, pH 3.5-9.5; the cleaning solution includes 0.1-5‰ DEPC water, 1-8% PEG-8000, 20-80% ethanol, 0.5-3M NaCl; the elution solution includes: 0.1-2mol / L Tris-HCl, 0.5-1mol / LEDTA.

[0027] In some embodiments of the present invention, the micro-cell sample comprises: a micro-cell sample with a quantity as low as 1-10.

[0028] According to a fifth aspect of the present invention, the present invention further provides a method for extracting total nucleic acid from a trace cell sample, the extraction method comprising: extracting with any extraction reagent described in the fourth aspect of the present invention; the extraction method comprising: (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.

[0029] In some embodiments of the present invention, in step (2), the treatment conditions include: binding at room temperature for 3-5 minutes, magnetic separation, and discarding the filtrate.

[0030] In some embodiments of the present invention, in step (3), the washing conditions include: washing at room temperature for 1-3 minutes, vortex mixing, magnetic separation, suction and discarding of waste liquid, and drying.

[0031] In some embodiments of the present invention, in step (4), the elution conditions include: 50-60°C, elution 2-5 min.

[0032] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a method for preparing magnetic beads for extracting whole nucleic acid from trace cell samples. The present invention first synthesizes a magnetic fluid Fe 4 O 3 , and then the surface is coated with silicon hydroxyl groups to synthesize silicon-based magnetic beads, and then a carboxyl-modified substance is added to modify the surface carboxyl groups, that is, a large number of carboxyl functional groups are connected to the silicon-based magnetic beads to obtain carboxyl magnetic beads. The carboxyl magnetic beads prepared by the preparation method provided by the present invention have abundant carboxyl groups on the surface. The carboxyl groups can form ion bridges with the negatively charged phosphate groups of nucleic acids through dissociated salt ions (such as Na+), so that DNA is non-specifically adsorbed to the surface of the carboxyl magnetic beads; at the same time, the magnetic beads of this patent also carry silanol groups (hydroxyl groups), which can efficiently and non-specifically adsorb nucleic acids. Therefore, under the condition of equal quality, the adsorption efficiency of nucleic acids by the magnetic beads is much higher than that of the silicon-based magnetic beads used in the prior art or directly in the magnetic fluid Fe 4 O 3 Carboxyl magnetic beads coated with carboxyl groups on the surface. The carboxyl magnetic beads obtained by the process provided by the present invention are more conducive to obtaining high-concentration nucleic acids under the condition of trace cell samples.

[0033] (2) Based on the preparation method of magnetic beads for extracting whole nucleic acid from trace cell samples provided by the present invention, a reagent for extracting whole nucleic acid from trace cell samples and a method for extracting whole nucleic acid are further provided. Because the magnetic beads provided by the present invention have excellent adsorption efficiency and extraction efficiency for whole nucleic acid in trace cell samples, the corresponding sample processing volume, lysis binding liquid volume, magnetic bead volume, cleaning liquid volume and elution liquid volume are significantly less than the amount of various reagents in the magnetic bead method nucleic acid extraction kit disclosed in the prior art. At the same time, the nucleic acid extraction efficiency is also higher. The present invention solves the problems of the existing magnetic bead method nucleic acid extraction kits on the market, such as large amount of magnetic beads, large elution volume, low nucleic acid concentration, inability to meet the requirements of complete, rapid and high-concentration extraction of RNA and DNA information in trace cell samples, and inability to meet the requirements of subsequent third-generation sequencing direct measurement. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a scanning electron microscope image of the magnetic bead product prepared by the method of Example 1 of the present invention; Figure 2 This is a microscope display of the magnetic bead product prepared by the method of Example 1 of the present invention; Figure 3 This is a macroscopic observation diagram showing a magnetic bead product prepared by the method of Example 1 of the present invention placed in a centrifuge tube; Figure 4 is the hysteresis loop of the magnetic bead product prepared by the method of Example 1 of the present invention; Figure 4 It can be seen that the saturation magnetization intensity of the magnetic beads product prepared by the method of Example 1 is 40.03 emu / g; Figure 5 The nucleic acid fragment distribution analysis result of the nucleic acid extraction product extracted from the Hela cell sample with 5 cells in Example 2 of the present invention; Figure 6 It is an overlay of analysis results of nucleic acid extraction products extracted from Hela cell samples with 5, 10, 20 and 50 cells, respectively, in Example 2 of the present invention; Figure 7It is a comparison result of the semi-sedimentation of magnetic beads prepared by different preparation processes in Comparative Example 1 of the present invention; wherein, "1" is the semi-sedimentation result of magnetic beads prepared by group one, "2" is the semi-sedimentation result of magnetic beads prepared by group two, "3" is the semi-sedimentation result of magnetic beads prepared by group three, "4" is the semi-sedimentation result of magnetic beads prepared by group four (Example 1), "A" is a comparison chart of the sedimentation effect corresponding to a sedimentation time of 15 min, "B" is a comparison chart of the sedimentation effect corresponding to a sedimentation time of 30 min, "C" is a comparison chart of the sedimentation effect corresponding to a sedimentation time of 60 min, and "D" is a comparison chart of the sedimentation effect corresponding to a sedimentation time of 120 min; Figure 8 These are the semi-sedimentation comparison results of magnetic beads prepared by different cleaning methods in Comparative Example 2 of the present invention; wherein, "1" is the semi-sedimentation result of magnetic beads prepared in Group 1, "2" is the semi-sedimentation result of magnetic beads prepared in Group 2, "3" is the semi-sedimentation result of magnetic beads prepared in Group 3 (Example 1), "A" is a sedimentation effect comparison chart corresponding to a sedimentation time of 15 min, "B" is a sedimentation effect comparison chart corresponding to a sedimentation time of 30 min, "C" is a sedimentation effect comparison chart corresponding to a sedimentation time of 60 min, and "D" is a sedimentation effect comparison chart corresponding to a sedimentation time of 120 min. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below by specific embodiments, which do not limit 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.

[0036] As used in the present invention, including as used in the examples and unless otherwise expressly provided, all numbers may be read as if beginning with the wording "substantially", "approximately" or "about", even if the term does not explicitly appear. The phrase "approximately" or "about" may be used when describing the magnitude and / or position to indicate that the described value and / or position is within a reasonable expected value and / or position range. For example, the value may be ±0.1% of the value (or value range), ±1% of the value (or value range), ±2% of the value (or value range), ±5% of the value (or value range), ±10% of the value (or value range), ±15% of the value (or value range), ±20% of the value (or value range), etc. Any numerical range described in the present invention is intended to include all subranges or intermediate values ​​contained therein.

[0037] The disclosure of the numerical values ​​and numerical ranges of specific parameters (such as temperature, weight percentage, weight fraction, etc.) does not exclude other numerical values ​​and numerical ranges useful to the present invention. It is conceivable that two or more specific example numerical values ​​of a given parameter can determine the endpoints of the numerical range that the parameter can require. For example, if parameter X is exemplified herein as having numerical value A and also exemplified as having numerical value Z, it is expected that parameter X can have a numerical range from about A to about Z. Similarly, it is expected that two or more numerical ranges of disclosed parameters (whether these ranges are nested, overlapped or distinct) include all possible combinations of numerical ranges that can be required using the endpoints of the disclosed ranges. For example, if parameter X is exemplified herein as having a value in the range of 1-10, it also describes a sub-range of parameter X, including only as an example, 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. Ranges include their endpoints and values ​​within the endpoints, for example, the range 0-5 includes 0, >0, 1, 2, 3, 4, <5, and 5.

[0038] Hereinafter, the present invention will be described in more detail by using specific examples and comparative examples.

[0039] Example 1 This embodiment provides a method for preparing magnetic beads for nucleic acid extraction, and the specific steps are as follows: 1. Magnetic fluid Fe 4 O 3 Preparation 1.1. Add the divalent iron salt ferrous chloride tetrahydrate (FeCl 2 ·4H 2 O), surfactant polyethylene glycol-2000 (PEG-2000) and ferric iron salt ferric chloride hexahydrate (FeCl 3 6H 2 O) are transferred into a reaction container in a certain order and an appropriate amount of ultrapure water is added as a solvent, and stirred at room temperature for 15 minutes at a stirring speed of 260r / min; wherein, the addition amount of ferrous chloride tetrahydrate is 3g, the addition amount of polyethylene glycol-2000 is 0.5g, the addition amount of ferric chloride hexahydrate is 2.5g, and the addition amount of ultrapure water as a solvent is 30ml, and the divalent iron salt, the surfactant and the trivalent iron salt are dissolved in three groups of solvents respectively, and then the three groups of dissolved reactants are transferred into a reaction container in the order of the divalent iron salt, the surfactant and the trivalent iron salt and the remaining solvent is added; 1.2. After stirring, add alkali liquid precipitant ammonia water dropwise into the reaction vessel, and with the addition of the alkali liquid precipitant, adjust the stirring speed to 560r / min accordingly. After the alkali liquid precipitant is added dropwise, stir at a stirring speed of 560r / min for 6h. The above operation needs to be carried out under the condition that the reaction temperature is controlled below 35°C; wherein, the amount of alkali liquid precipitant added is 5ml, and the dropping speed of the alkali liquid precipitant is 30ml / h; 1.3. After the stirring is completed, the stirred reaction system is heated and reacted at 85°C for 3 hours for high temperature aging treatment; 1.4. After the high temperature aging treatment is completed, pour out the reaction solution, let it settle naturally for 6 hours, remove the supernatant by magnetic suction, and use a cleaning solution to clean the precipitated magnetic fluid Fe 4 O 3 Clean the magnetic fluid Fe 4 O 3 The magnetic fluid Fe 3 O 4 ; 2. Preparation of magnetic beads 2.1. Silicon-based coating: Take the magnetic fluid Fe prepared in step 1 3 O 4 320g, after stirring evenly, transfer to a reaction container, and add a certain proportion (the volume ratio of ethanol solution to magnetic beads is 6:1) of ethanol solution to a fixed concentration, and ultrasonically stir at 300r / min for 3h at room temperature; after the ultrasonic stirring is completed, add dispersant trisodium citrate to the system, the addition amount of dispersant trisodium citrate is 10g, and stir at 300r / min for 15min; after the stirring is completed, add 80ml of ammonia water and continue ultrasonic stirring for 15min; after the ultrasonic stirring is completed, add 120ml of tetraethyl orthosilicate ethanol solution by a microinjection pump, and control the dropping speed of tetraethyl orthosilicate ethanol solution to 150ml / h. After the dropwise addition is completed, the reaction solution is ultrasonically stirred for 10min, the ultrasound is turned off, and then ordinary stirring is performed for 0.5h. After the ordinary stirring is completed, it is allowed to stand for about 16h; after the standing is completed, the supernatant is removed by magnetic suction, and the obtained magnetic beads are cleaned with a cleaning solution to obtain intermediate magnetic beads, i.e., silica-coated magnetic beads; 2.2. Carboxyl modification: 350 g of the washed intermediate magnetic beads were dispersed in anhydrous ethanol, wherein the volume ratio of anhydrous ethanol to magnetic beads was 4:1, and stirred evenly with a glass rod, and ultrasonically stirred for 15 min at 240 r / min; after sedimentation, stirred evenly with a glass rod, replaced in a 20 L reaction container, and ultrasonically stirred for 10 min at 240 r / min, and then 20 g of dissolved maleic anhydride (the solvent used for dissolution was pure water, and the amount of solvent was 100 ml) was added, and ultrasonically stirred for 5 min, and then the ultrasound was turned off and continued to stir for 20 h; after the reaction was completed, the reactant was poured out and washed with a washing solution; after washing, irregular-shaped magnetic beads, i.e., carboxyl magnetic beads, were obtained by magnetic attraction, and the obtained carboxyl magnetic beads were diluted to 10 mg / ml with ultrapure water to obtain magnetic beads for nucleic acid extraction; Among them, in the above steps, the cleaning solution used is prepared from 0.1‰ sodium hypochlorite, 0.5‰ calcium chloride and 0.05‰ Twen20. The preparation method of the cleaning solution is: weigh 0.1g sodium hypochlorite, 0.5g calcium chloride, dissolve in 999.95ml pure water, and then add 0.05ml Twen20 and stir well.

[0040] The scanning electron microscopy image of the magnetic beads prepared by the above method is as follows Figure 1 As shown, the microscopic image of the magnetic beads is Figure 2 As shown, the macroscopic appearance of magnetic beads in the centrifuge tube is shown in Figure 3 As shown, the hysteresis loop of the magnetic bead is as follows Figure 4 As shown. Figure 1-4 The results shown show that the magnetic beads are irregular in shape, with a magnetic saturation intensity of about 40.03emu / g, an average particle size of about 410.5nm, and a polydispersity index of 0.010 obtained from the particle size test distribution curve of the magnetic beads using a laser particle size analyzer. The beads are in a monodisperse state, have a larger specific surface area, and correspondingly have better adsorption performance.

[0041] Example 2 Based on the magnetic beads prepared in Example 1, this example provides a method for extracting nucleic acids from trace samples. The nucleic acid extraction samples used in this example are samples with a very small amount of nucleic acids, and the samples are human HeLa cells with 5, 10, 20, and 50 cells, respectively. The specific steps are as follows: (1) Place the four gradient cell quantity samples taken above into 200 μL centrifuge tubes respectively; (2) Lysis and binding: Add 0.02 mg of proteinase K, 4 μL of lysis solution binding buffer (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; (3) Cleaning: Use 100 μL of cleaning solution (DEPC water, 80% ethanol, 0.5-3M NaCl) to clean the magnetic beads obtained in step (2) at room temperature for 1 min, vortex to mix; magnetic separation, aspirate and discard the waste liquid, and air dry for 2 min; (4) Elution: Add 2 μL of elution buffer to the magnetic beads obtained after drying in step (3), and elute at 56°C for 3 min. After elution, perform magnetic separation and aspirate the supernatant, which is the extracted complete nucleic acid sample (and DNA).

[0042] The nucleic acid samples in the trace amount of human HeLa cell samples extracted by the above method were run on Agilent's pre-made high-sensitivity RNA gel, and then the results were analyzed using the Agilent 4200 TapeStation system. The results are as follows: Figure 5 , Figure 6 As shown, Figure 5 The analysis results of the nucleic acid extraction product obtained from a sample of 5 human HeLa cells (Hela cells); Figure 6 This is a superposition of the analysis results of nucleic acid extraction products extracted from human HeLa cell (Hela cell) samples with 5, 10, 20, and 50 cells, respectively.

[0043] like Figure 5 The results shown, Figure 5 In the figure, 18S and 28S correspond to the ribosomal RNA bands, which are used to determine whether the RNA has been extracted and whether it has been degraded. The band on the far right, ">10000", corresponds to the DNA band, which can be used to further determine whether the DNA has been extracted and whether it has been degraded. "824", "1404" and "1893" represent other atypical nucleic acid fragments. Figure 5 The results shown show that the magnetic beads provided by the present invention can achieve the co-extraction of DNA and RNA in samples with extremely small amounts of nucleic acid (5 cells); Figure 6 The results shown in the figure show 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 content of nucleic acid with the same molecular weight decreases, and the two trends are consistent, and the positions of the peaks are the same and the bands are consistent, further indicating that the magnetic beads provided by the present invention can be used to extract the whole nucleic acid from human HeLa cell (Hela cell) samples with 5, 10, 20, and 50 cells, respectively, and according to Figure 6 The results shown further indicate that the results of extracting nucleic acids from corresponding samples using the magnetic beads provided by the present invention are relatively stable, further indicating the stability of the magnetic bead products and extraction methods provided by the present invention.

[0044] Comparative Example 1 Based on Example 1, this comparative example further explores the effects of preparing magnetic beads based on different preparation processes on the performance of the magnetic beads finally prepared, as follows: Group 1: magnetic beads prepared according to the corresponding patent document (CN103908945A); Group 2: magnetic beads prepared according to the corresponding patent document (CN111330558A); Group 3: Based on Example 1, the process parameters in the carboxyl modification step 2.2 were changed. The specific method is as follows: Take 350 g of the washed intermediate magnetic beads, disperse them in anhydrous ethanol, stir them evenly with a glass rod, and stir them for 15 minutes at 240 r / min; after sedimentation, stir them evenly with a glass rod, replace them in a 20 L reaction container, continue stirring for 10 minutes at 240 r / min, then add 20 g of dissolved maleic anhydride and stir for 20 hours; other conditions remain unchanged as in Example 1, and carboxyl-modified magnetic beads are prepared.

[0045] Group 4: Example 1.

[0046] First, the suspension performance of the magnetic beads prepared by the above group one, group two, group three and group four (Example 1) was tested, that is, the prepared magnetic beads were subjected to natural sedimentation for different time periods (15min, 30min, 60min, 120min), and the final semi-sedimentation effect was compared. The results are as follows: Figure 7 As shown. Figure 7 The results shown show that the half-sedimentation time of the magnetic beads prepared by different preparation processes is significantly different, indicating that the suspension performance is quite different. The half-sedimentation time of the magnetic beads prepared by Example 1 is greater than 2h, and the suspension performance is significantly better than that of Group 1, Group 2, and Group 3.

[0047] Secondly, the relevant indicators and performances of the magnetic beads prepared by the preparation processes of Group 1, Group 2, Group 3 and Group 4 (Example 1) are further compared, and the comparison results are shown in Table 1: Table 1 Comparison of related indicators and performance of various magnetic beads

[0048] According to the results shown in Table 1, the magnetic beads prepared by the method provided by the present invention have a small particle size and a large specific surface area, and the amount of nucleic acid adsorption per microgram of magnetic beads is greater. In addition, from the comparison results of the physical and chemical indicators of the magnetic beads shown in Table 1, it can be seen that the half-sedimentation time of the magnetic beads prepared by group one, group two, group three, and group four (Example 1) is significantly different, indicating that the suspension performance is quite different. The half-sedimentation time of the magnetic beads in group one and the magnetic beads in group two is about 10 minutes, and the half-sedimentation time of the magnetic beads in group three is 75 minutes. The half-sedimentation time of the magnetic beads prepared by group four (Example 1) is greater than 2 hours, and the suspension performance is significantly better than that of the other three groups.

[0049] Comparative Example 2 This comparative example is based on Example 1, and further explores the effects 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: Group 1: A cleaning method similar to that in patent document CN103908945A is used: that is, based on Example 1, in step 1.4 of step 1, "remove the supernatant by magnetic suction, and use a cleaning solution to clean the precipitated magnetic fluid Fe 3 O 4 "cleaning" is replaced by "separating the magnetic fluid Fe by magnetic attraction 3 O 4 , the precipitate was washed repeatedly three times with 1% ammonia water, 5% NaCl and deionized water in sequence until the washing solution was neutral"; and in step 2.1 of step 2, "after standing, the supernatant was removed by magnetic suction, and the obtained magnetic beads were washed with a washing solution" was replaced with "after standing, the beads were washed three times with anhydrous ethanol, and then washed repeatedly with ultrapure water several times until the pH value of the washing solution was 7" to prepare the corresponding carboxyl magnetic beads; Group 2: Using the cleaning method in patent document CN111330558A: That is, based on Example 1, in step 1.4 of step 1, "remove the supernatant by magnetic suction, and use the cleaning liquid to clean the precipitated magnetic fluid Fe 3 O 4 Wash with deionized water repeatedly until neutral”; and in step 2.1 of step 2, replace “after standing, remove the supernatant by magnetic suction, and wash the obtained magnetic beads with a cleaning solution” with “wash twice with anhydrous ethanol, then wash twice with 1 mol dilute hydrochloric acid, and then wash with deionized water until neutral” to prepare the corresponding carboxyl magnetic beads; Group 3: Example 1.

[0050] The suspension performance of the magnetic beads prepared by the above group 1, group 2, and group 3 was tested respectively, that is, the prepared magnetic beads were subjected to natural sedimentation for different time periods (15min, 30min, 60min, 120min), and the final semi-sedimentation effect was compared. The results are as follows Figure 8 As shown. Figure 8 The results shown show that the half-sedimentation time of the magnetic beads prepared based on different cleaning methods is significantly different, and the suspension performance is quite different. The half-sedimentation time of the magnetic beads prepared by group 3 (Example 1) is greater than 2h, and the suspension performance is significantly better than that of group 1 and group 2. That is, the suspension performance of the magnetic beads after washing with the cleaning solution provided by the present invention is good, and the 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 impurities, 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 better disperse and suspend, thereby improving the suspension performance of the magnetic beads; the buffer in the cleaning solution can adjust the pH value of the cleaning, so that the pH change will not be caused by the change in the composition and content of the cleaning solution, so that the extraction efficiency of the whole nucleic acid of the trace cell sample is higher.

[0051] Comparative Example 3 This comparative example further compares the extraction reagent buffer systems used by different magnetic beads, as follows: The extraction reagent buffer systems used for extracting nucleic acids from samples in Examples 2-3 of the present invention are compared with those in the prior art documents CN111330558A and CN103908945A. The comparison results are shown in Table 2: Table 2 Comparison of extraction reagent buffer systems

[0052] According to the comparison results presented in Table 2, based on the magnetic beads provided in the embodiment of the present invention, the corresponding sample processing volume, lysis binding liquid volume, magnetic bead volume, cleaning liquid volume and elution liquid volume are obviously less than the added amounts in the existing documents CN111330558A and CN103908945A, and the nucleic acid extraction efficiency is higher.

[0053] It is to be understood that the present invention is described by some embodiments, and it is known to those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present invention. In addition, under the teachings of the present invention, these features and embodiments may be modified to adapt to specific circumstances 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 are within the scope of protection of 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 Fe4O3; 2. Silicon-based coating is performed on the surface of the prepared magnetic fluid Fe4O3; 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, namely carboxyl magnetic beads.

2. The preparation method according to claim 1, characterized in that: In step one, the preparation of magnetic fluid Fe4O3 includes: mixing divalent iron salt, surfactant, and trivalent iron salt; adding alkaline solution precipitant and continuing stirring; after the stirring is completed, performing high-temperature aging treatment; after the high-temperature aging treatment, magnetically absorbing the magnetic fluid Fe4O3, and cleaning the obtained magnetic fluid Fe4O3 with a cleaning liquid; the cleaning liquid includes the following components in weight proportion: 0.1‰-0.5‰ sodium hypochlorite, 0.5‰-1‰ calcium chloride, and 0.02‰-0.05‰ Twen20.

3. The preparation method according to claim 2, characterized in that: In the step of preparing the magnetic fluid Fe4O3, the addition ratio of the divalent iron salt, the surfactant, the trivalent iron salt and the alkali solution precipitant is: 0.6-3g: 0.5-3g: 0.01-0.5g: 1-5ml: 8-30ml; and / or, The divalent salt is selected from at least one of ferrous chloride tetrahydrate and ferrous sulfate; and / or, The trivalent salt is selected from ferric chloride hexahydrate; and / or, 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; and / or, The alkaline solution precipitant is selected from at least one of ammonia water and sodium hydroxide.

4. The preparation method according to claim 1, characterized in that: In step 2, the silicon-based coating on the surface of the prepared magnetic fluid Fe4O3 includes: adding ethanol solution, dispersant, alkali solution, and tetraethyl orthosilicate ethanol solution to the prepared magnetic fluid Fe4O3 in sequence 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-coated magnetic beads; in the step of washing the obtained magnetic beads with a cleaning solution, the cleaning solution includes the following components in weight proportion: 0.1‰-0.5‰ sodium hypochlorite, 0.5‰-1‰ calcium chloride, and 0.02‰-0.05‰ Twen20.

5. The preparation method according to claim 1, characterized in that: In step 3, the carboxyl modification of the surface of the magnetic beads obtained by silicon-based modification comprises: The silica-coated magnetic beads are dispersed in anhydrous ethanol, and a carboxyl-modified substance is added to perform carboxyl modification. After the reaction is completed, the reactant is washed with a washing solution. After the washing is completed, the carboxyl-modified carboxyl magnetic beads are obtained by magnetic absorption; Wherein, the conditions in the step of performing carboxyl modification on the surface of the silicon-modified magnetic beads include: The carboxyl-modified substance is selected from at least one of maleic anhydride, acrylic acid, α-methacrylic acid, and undecylenic acid; and / or, The dispersing of the silicon-coated magnetic beads in anhydrous ethanol comprises: dispersing the silicon-coated magnetic beads in anhydrous ethanol and performing ultrasonic stirring; the ultrasonic stirring condition is: ultrasonic stirring for 10-20 minutes at 200-300 r / min; and / or, The adding of carboxyl-modified substances to perform carboxyl modification comprises: adding the carboxyl-modified substances, stirring with ultrasound for 3-10 minutes, then turning off ultrasound, and continuing conventional stirring for 15-25 hours; and / or, In the step of washing the obtained magnetic beads with a washing solution, the washing solution comprises the following components in proportion by weight: 0.1‰-0.5‰ sodium hypochlorite, 0.5‰-1‰ calcium chloride, and 0.02‰-0.05‰ Twen20.

6. Carboxyl magnetic beads prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the magnetic beads as claimed in claim 5 in extracting whole nucleic acid from trace cell samples.

8. 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 6 / or the magnetic beads prepared by the preparation method described in any one of claims 1-5.

9. The extraction reagent according to claim 8, characterized in that The lysis and binding solution includes: 0.001-0.12 mg proteinase K, DEPC water, 0.5-6 M guanidine hydrochloride or guanidine isothiocyanate, pH 3.5-9.5; the cleaning solution includes 0.1-5‰ DEPC water, 1-8% PEG-8000, 20-80% ethanol, 0.5-3M NaCl; the elution solution includes: 0.1-2mol / L Tris-HCl, 0.5-1mol / L EDTA.

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 described in any one of claims 8-9; 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) Adding an elution solution to elute the washed magnetic beads, followed by magnetic separation to obtain a supernatant, which is the extracted whole nucleic acid sample; 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; and / or, In step (3), the washing conditions include: washing at room temperature for 1-3 minutes, vortex mixing, magnetic separation, suction and discarding of waste liquid, and drying; and / or, In step (4), the elution conditions include: 50-60°C, elution time 2-5 min.

Images