A method for extracting nucleic acid based on magnetic beads

Abstract

The present invention provides a method for extracting nucleic acid based on a magnetic bead method, comprising the following steps: step one, surface modification of magnetic beads; step two, preparation of magnetic bead suspension; step three, sample lysis and incubation; step four, magnetic adsorption; step five, washing and removing impurities from magnetic beads. The present invention obtains phospholipid bilayer magnetic beads by surface modification of magnetic beads, which can reduce the interfacial tension between nucleic acid and magnetic beads, making it easier for nucleic acid to contact and bind with the surface of magnetic beads. The present invention uses a lysis solution of specific components and combines with ultraviolet light irradiation during sample lysis and incubation. By modifying the surface of magnetic beads and using a specific lysis solution for lysis and incubation, the magnetic beads of the present invention have a high degree of specific binding to nucleic acids, and only one washing is required, eliminating the elution step, and the obtained magnetic bead-nucleic acid complex can be directly used as a subsequent nucleic acid detection target, reducing the loss and dilution of nucleic acid caused during multiple washing and elution processes, and improving the concentration and purity of nucleic acids.

Description

Technical Field

[0001] The invention relates to the technical field of nucleic acid extraction, and in particular to a nucleic acid extraction method based on a magnetic bead method. Background Art

[0002] Extraction of nucleic acids is of vital importance in modern biological research. Nucleic acids include DNA and RNA, which are important genetic materials in life activities. They carry the genetic information of organisms, control the synthesis of proteins, and thus affect all physiological activities and characteristics of organisms. Extraction of nucleic acids allows us to directly access these genetic instructions, and then conduct in-depth research and analysis on them. Whether in gene sequencing, gene expression analysis, gene mutation screening, or in the early diagnosis and treatment of diseases, nucleic acid extraction is an indispensable step. Existing nucleic acid extraction is mainly divided into physical method, chemical method, and biological method. Among them, the physical method is further divided into boiling method, freeze-thaw method, ultrasonic method, grinding method, etc. Its principle is to use high temperature, high pressure, freezing, ultrasonic mechanical effect, thermal effect, cavitation, etc. to destroy the cell structure of the sample to be tested, release the cell contents, and nucleic acid molecules can be extracted. This operation method is simple and easy to operate, but the extraction efficiency is low and the purity of the extracted product is low. Among them, the chemical method includes alkaline lysis method, phenol-chloroform extraction method, salting out method, Trizol method, potassium iodide method, etc. Chemical methods can effectively remove impurities such as proteins, polysaccharides, and lipids, so the extracted product has a higher purity. However, the chemical extraction process is relatively cumbersome and time-consuming. The principle of the biological method is to use lysozyme, proteinase K, etc. to lyse cells, release cell contents, and extract nucleic acids. However, this method has the disadvantages of easy enzyme inactivation and high cost. Therefore, a new nucleic acid extraction method represented by solid phase extraction has been developed, mainly including silica matrix extraction method, centrifugal column extraction method, paper-based extraction method, magnetic ionic liquid extraction method and magnetic bead method. Among them, the magnetic bead method is a safe and environmentally friendly nucleic acid extraction method, which is flexible in operation, high in safety, and high in product purity. However, the current magnetic bead method has many steps and requires multiple washing and elution steps, so it may cause nucleic acid loss and dilution, affecting the concentration and purity of nucleic acids. Summary of the invention

[0003] In view of this, the present invention proposes a nucleic acid extraction method based on a magnetic bead method to solve the above problems.

[0004] The technical solution of the present invention is achieved as follows: a method for extracting nucleic acid based on a magnetic bead method, comprising the following steps:

[0005] Step 1, surface modification of magnetic beads: at 40-60°C, magnetic microspheres, phosphatidylinositol, and methanol are shaken and mixed at 80-100 r / min for 60-90 seconds, 3-morpholinepropanesulfonic acid and sodium chloride are slowly added to the mixture, and the pH is adjusted to 6.8-7.2, reacted for 25-35 minutes, and the magnetic microspheres are washed with sterile deionized water to obtain phospholipid bilayer magnetic beads;

[0006] Step 2, preparation of magnetic bead suspension: add Tween 20 and polyethylene glycol to physiological saline, stir at 100-200 r / min for 1-3 min, add phospholipid bilayer magnetic beads and shake at 20-40 r / min for 10-20 s to obtain a magnetic bead suspension;

[0007] Step 3: Sample lysis and incubation: Take the sample and add it to a 1.5 mL centrifuge tube. Add the magnetic bead suspension and cell lysis solution to the centrifuge tube, shake and mix at 20-40 r / min for 10-20 seconds, and incubate at 24-26°C under ultraviolet light for 4-6 minutes.

[0008] Step 4: Magnetic adsorption: Place the above centrifuge tube in a magnetic rack and let it stand for 1-2 minutes to allow the magnetic beads to be completely adsorbed on the magnetic rack. Use a pipette to absorb and discard the supernatant;

[0009] Step 5. Wash and remove impurities from magnetic beads: Add washing solution to the above centrifuge tube, shake and mix at 20-40r / min for 20-30s, let stand for 15-25s to allow the magnetic beads to be completely adsorbed to the magnetic rack, use a pipette to absorb and discard the supernatant to obtain the magnetic bead-nucleic acid complex and complete the nucleic acid extraction.

[0010] Furthermore, the magnetic microspheres in step 1 are one of hydroxyl magnetic microspheres, carboxyl magnetic microspheres, epoxy magnetic microspheres, and ferroferric oxide magnetic microspheres, and the particle size of the magnetic microspheres is 500-1000 nm.

[0011] Furthermore, in step 1, the mass volume ratio of magnetic microspheres, phosphatidylinositol, methanol, 3-morpholinepropanesulfonic acid, and sodium chloride is (6-10):(3-5):(5-7):(1-3):(1-3).

[0012] Furthermore, in step 2, the volume ratio of Tween 20, polyethylene glycol, and physiological saline is (0.1-0.3):(1.5-2.5):(90-100), and the concentration of the phospholipid bilayer magnetic beads in the solution is 10-30 mg / mL.

[0013] Furthermore, in step three, the amount of sample added is 100-200 μL, the amount of magnetic bead suspension added is 15-25 μL, and the amount of cell lysate added is 100-200 μL.

[0014] Furthermore, the cell lysate is composed of quinine, proteinase K, sodium dodecyl sulfate, 4,4'-dihydroxyazobenzene, and tromethamine in a mass volume ratio of g / mL of (3-5):(0.5-1.0):(1-2):(0.4-0.6):(1-3).

[0015] Furthermore, when irradiating with ultraviolet light, the wavelength is set to 300-400nm and the light intensity is set to 800-1200Lux.

[0016] Furthermore, the amount of washing solution added is 400-600 μL.

[0017] Furthermore, the washing liquid is composed of cocamidopropyl betaine, urea and ethanol in a mass volume ratio of g / mL of (5-10):(5-10):(70-80).

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention obtains phospholipid bilayer magnetic beads by surface modification of magnetic beads, and the phospholipid bilayer has unique biocompatibility and biomimetic properties. When the surface of the magnetic beads is modified with a phospholipid bilayer, this biomimetic interface can reduce the interfacial tension between the nucleic acid and the magnetic beads, making it easier for the nucleic acid to contact and bind to the surface of the magnetic beads. And phospholipid molecules usually contain negatively charged phosphate groups and hydrophobic fatty acid chains. When the phospholipid bilayer is covered on the surface of the magnetic beads, these negative charges can electrostatically interact with the positively charged regions on the nucleic acid molecules, which helps to pull the nucleic acid molecules closer to the surface of the magnetic beads and promote their binding. The present invention uses a lysis solution with specific components and combines it with ultraviolet light irradiation during sample lysis and incubation, which not only improves the lysis efficiency, but also further improves the degree of lysis, and the extracted nucleic acid has a higher concentration and purity. By modifying the surface of the magnetic beads and using a specific lysis solution for lysis and incubation, the magnetic beads of the present invention have a high degree of specific binding to nucleic acids. After only one washing, the elution step is eliminated. The obtained magnetic bead-nucleic acid complex can be directly used as a target for subsequent nucleic acid detection, reducing the loss and dilution of nucleic acids caused by multiple washing and elution processes, and improving the concentration and purity of nucleic acids. DETAILED DESCRIPTION

[0019] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0020] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0021] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources. Example 1

[0022] A method for extracting nucleic acid based on a magnetic bead method comprises the following steps:

[0023] Step 1, surface modification of magnetic beads: at 40°C, magnetic microspheres, phosphatidylinositol, and methanol were shaken and mixed at 80r / min for 90s, 3-morpholinepropanesulfonic acid and sodium chloride were slowly added to the mixture, and the pH was adjusted to 6.8, reacted for 35min, and the magnetic microspheres were washed with sterile deionized water to obtain phospholipid bilayer magnetic beads. The magnetic microspheres are hydroxy magnetic microspheres with a particle size of 500nm, and the mass volume ratio of magnetic microspheres, phosphatidylinositol, methanol, 3-morpholinepropanesulfonic acid, and sodium chloride is 6:3:5:1:1 g / ml.

[0024] Step 2, preparation of magnetic bead suspension: add Tween 20 and polyethylene glycol to normal saline, the volume ratio of Tween 20, polyethylene glycol and normal saline is 0.1:1.5:90, stir at 100 r / min for 3 min, add phospholipid bilayer magnetic beads to make the concentration of phospholipid bilayer magnetic beads in the solution is 10 mg / mL, shake and mix at 20 r / min for 20 s to obtain a magnetic bead suspension.

[0025] Step 3, sample lysis and incubation: take 100 μL of sample and add it to a 1.5 mL centrifuge test tube, add 15 μL of magnetic bead suspension, 100 μL of cell lysis solution composed of quinine, proteinase K, sodium dodecyl sulfate, 4,4'-dihydroxyazobenzene and tromethamine with a mass volume ratio of 3:0.5:1:0.4:1 g / mL to the above centrifuge test tube, shake and mix at 20 r / min for 20 s, incubate at 24 ° C under ultraviolet light with a wavelength of 300 nm and a light intensity of 800 Lux for 6 min.

[0026] Step 4: Magnetic adsorption: Place the above centrifuge tube in a magnetic rack and let it stand for 1 minute to allow the magnetic beads to be completely adsorbed to the magnetic rack. Use a pipette to absorb and discard the supernatant.

[0027] Step 5, washing and removing impurities from the magnetic beads: add 400 μL of a washing solution consisting of cocamidopropyl betaine, urea, and ethanol in a mass volume ratio of 5:5:70 g / mL to the above centrifuge tube, shake and mix at 20 r / min for 30 s, let stand for 15 s to allow the magnetic beads to be completely adsorbed to the magnetic rack, use a pipette to absorb and discard the supernatant to obtain a magnetic bead-nucleic acid complex, and complete the nucleic acid extraction. Example 2

[0028] A method for extracting nucleic acid based on a magnetic bead method comprises the following steps:

[0029] Step 1, surface modification of magnetic beads: at 40°C, magnetic microspheres, phosphatidylinositol, and methanol were shaken and mixed at 80r / min for 90s, 3-morpholinepropanesulfonic acid and sodium chloride were slowly added to the mixture, and the pH was adjusted to 6.8, reacted for 35min, and the magnetic microspheres were washed with sterile deionized water to obtain phospholipid bilayer magnetic beads. The magnetic microspheres are carboxyl magnetic microspheres with a particle size of 500nm, and the mass volume ratio of magnetic microspheres, phosphatidylinositol, methanol, 3-morpholinepropanesulfonic acid, and sodium chloride is 10:5:7:3:3 g / ml.

[0030] Step 2, preparation of magnetic bead suspension: add Tween 20 and polyethylene glycol to normal saline, the volume ratio of Tween 20, polyethylene glycol and normal saline is 0.3:2.5:100, stir at 100 r / min for 3 min, add phospholipid bilayer magnetic beads to make the concentration of phospholipid bilayer magnetic beads in the solution is 30 mg / mL, shake and mix at 20 r / min for 20 s to obtain a magnetic bead suspension.

[0031] Step 3, sample lysis and incubation: take 200 μL of sample and add it to a 1.5 mL centrifuge test tube, add 25 μL of magnetic bead suspension, 200 μL of cell lysis solution composed of quinine, proteinase K, sodium dodecyl sulfate, 4,4'-dihydroxyazobenzene and tromethamine with a mass volume ratio of 5:1.0:2:0.6:3 g / mL to the above centrifuge test tube, shake and mix at 20 r / min for 20 s, incubate at 24 ° C under ultraviolet light with a wavelength of 300 nm and a light intensity of 800 Lux for 6 min.

[0032] Step 4: Magnetic adsorption: Place the above centrifuge tube in a magnetic rack and let it stand for 2 minutes to allow the magnetic beads to be completely adsorbed on the magnetic rack. Use a pipette to absorb and discard the supernatant;

[0033] Step 5, washing and removing impurities from the magnetic beads: add 600 μL of a washing solution consisting of cocamidopropyl betaine, urea, and ethanol with a mass volume ratio of g / mL of 10:10:80 to the above centrifuge tube, shake and mix at 20 r / min for 30 s, let stand for 25 s to allow the magnetic beads to be completely adsorbed to the magnetic rack, use a pipette to absorb and discard the supernatant to obtain a magnetic bead-nucleic acid complex, and complete the nucleic acid extraction. Example 3

[0034] A method for extracting nucleic acid based on a magnetic bead method comprises the following steps:

[0035] Step 1, surface modification of magnetic beads: at 50°C, mix the magnetic microspheres, phosphatidylinositol, and methanol at 90 r / min for 75 seconds, slowly add 3-morpholinepropanesulfonic acid and sodium chloride to the mixture and adjust the pH to 7.0, react for 30 minutes, and wash the magnetic microspheres with sterile deionized water to obtain phospholipid bilayer magnetic beads; the magnetic microspheres are epoxy magnetic microspheres with a particle size of 750 nm, and the mass volume ratio of the magnetic microspheres, phosphatidylinositol, methanol, 3-morpholinepropanesulfonic acid, and sodium chloride is 8:4:6:2:2 g / ml.

[0036] Step 2, preparation of magnetic bead suspension: add Tween 20 and polyethylene glycol to normal saline, the volume ratio of Tween 20, polyethylene glycol and normal saline is 0.2:2.0:95, stir at 150r / min for 2min, add phospholipid bilayer magnetic beads to make the concentration of phospholipid bilayer magnetic beads in the solution be 20mg / mL, shake and mix at 30r / min for 15s to obtain a magnetic bead suspension;

[0037] Step 3, sample lysis and incubation: take 150 μL of sample and add it to a 1.5 mL centrifuge test tube, add 20 μL of magnetic bead suspension, 150 μL of cell lysis solution composed of quinine, proteinase K, sodium dodecyl sulfate, 4,4'-dihydroxyazobenzene and tromethamine with a mass volume ratio of g / mL of 4:0.8:1.5):0.5:2 to the above centrifuge test tube, shake and mix at 30 r / min for 15 s, incubate at 25 ° C under ultraviolet light with a wavelength of 3500 nm and an illumination intensity of 1000 Lux for 5 min.

[0038] Step 4: Magnetic adsorption: Place the above centrifuge tube in a magnetic rack and let it stand for 1.5 minutes to allow the magnetic beads to be completely adsorbed on the magnetic rack. Use a pipette to absorb and discard the supernatant;

[0039] Step 5, washing and removing impurities from the magnetic beads: add 500 μL of a washing solution consisting of cocamidopropyl betaine, urea, and ethanol in a mass volume ratio of 8:8:75 g / mL to the above centrifuge tube, shake and mix at 30 r / min for 25 s, let stand for 20 s to allow the magnetic beads to be completely adsorbed to the magnetic rack, use a pipette to absorb and discard the supernatant to obtain a magnetic bead-nucleic acid complex, and complete the nucleic acid extraction. Example 4

[0040] A method for extracting nucleic acid based on a magnetic bead method comprises the following steps:

[0041] Step 1, surface modification of magnetic beads: at 60°C, magnetic microspheres, phosphatidylinositol, and methanol were shaken and mixed at 100 r / min for 60 seconds, 3-morpholinepropanesulfonic acid and sodium chloride were slowly added to the mixture, and the pH was adjusted to 6.8, reacted for 25 minutes, and the magnetic microspheres were washed with sterile deionized water to obtain phospholipid bilayer magnetic beads. The magnetic microspheres are ferroferric oxide magnetic microspheres with a particle size of 1000 nm, and the mass volume ratio of magnetic microspheres, phosphatidylinositol, methanol, 3-morpholinepropanesulfonic acid, and sodium chloride is 6:3:5:1:1 g / ml.

[0042] Step 2, preparation of magnetic bead suspension: add Tween 20 and polyethylene glycol to normal saline, the volume ratio of Tween 20, polyethylene glycol and normal saline is 0.1:1.5:90, stir at 200r / min for 13min, add phospholipid bilayer magnetic beads to make the concentration of phospholipid bilayer magnetic beads in the solution is 10mg / mL, shake and mix at 40r / min for 10s to obtain a magnetic bead suspension.

[0043] Step 3, sample lysis and incubation: take 100 μL of sample and add it to a 1.5 mL centrifuge test tube, add 15 μL of magnetic bead suspension, 100 μL of cell lysis solution composed of quinine, proteinase K, sodium dodecyl sulfate, 4,4'-dihydroxyazobenzene and tromethamine with a mass volume ratio of 3:0.5:1:0.4:1 g / mL to the above centrifuge test tube, shake and mix at 40 r / min for 10 s, incubate at 26 ° C under ultraviolet light with a wavelength of 400 nm and an illumination intensity of 1200 Lux for 4 min.

[0044] Step 4: Magnetic adsorption: Place the above centrifuge tube in a magnetic rack and let it stand for 1 minute to allow the magnetic beads to be completely adsorbed on the magnetic rack. Use a pipette to absorb and discard the supernatant;

[0045] Step 5, washing and removing impurities from the magnetic beads: add 400 μL of a washing solution consisting of cocamidopropyl betaine, urea, and ethanol in a mass volume ratio of 5:5:70 g / mL to the above centrifuge tube, shake and mix at 40 r / min for 20 s, let stand for 15 s to allow the magnetic beads to be completely adsorbed to the magnetic rack, use a pipette to absorb and discard the supernatant to obtain a magnetic bead-nucleic acid complex, and complete the nucleic acid extraction. Example 5

[0046] A method for extracting nucleic acid based on a magnetic bead method comprises the following steps:

[0047] Step 1, surface modification of magnetic beads: at 60°C, mix the magnetic microspheres, phosphatidylinositol, and methanol at 100 r / min for 60 seconds, slowly add 3-morpholinepropanesulfonic acid and sodium chloride to the mixture and adjust the pH to 7.2, react for 25 minutes, and wash the magnetic microspheres with sterile deionized water to obtain phospholipid bilayer magnetic beads; the magnetic microspheres are epoxy magnetic microspheres with a particle size of 1000 nm, and the mass volume ratio of the magnetic microspheres, phosphatidylinositol, methanol, 3-morpholinepropanesulfonic acid, and sodium chloride is 10:5:7:3:3 g / ml.

[0048] Step 2, preparation of magnetic bead suspension: add Tween 20 and polyethylene glycol to normal saline, the volume ratio of Tween 20, polyethylene glycol and normal saline is 0.3:2.5:100, stir at 200 r / min for 1 min, add phospholipid bilayer magnetic beads to make the concentration of phospholipid bilayer magnetic beads in the solution to be 30 mg / mL, shake and mix at 40 r / min for 10 s to obtain a magnetic bead suspension;

[0049] Step 3, sample lysis and incubation: take 200 μL of sample and add it to a 1.5 mL centrifuge test tube, add 25 μL of magnetic bead suspension, 200 μL of cell lysis solution composed of quinine, proteinase K, sodium dodecyl sulfate, 4,4'-dihydroxyazobenzene and tromethamine with a mass volume ratio of 5:1.0:2:0.6:3 g / mL to the above centrifuge test tube, shake and mix at 40 r / min for 10 s, incubate at 26 ° C under ultraviolet light with a wavelength of 400 nm and an illumination intensity of 1200 Lux for 4 min.

[0050] Step 4: Magnetic adsorption: Place the above centrifuge tube in a magnetic rack and let it stand for 2 minutes to allow the magnetic beads to be completely adsorbed on the magnetic rack. Use a pipette to absorb and discard the supernatant;

[0051] Step 5, washing and removing impurities from the magnetic beads: add 600 μL of a washing solution consisting of cocamidopropyl betaine, urea, and ethanol with a mass volume ratio of g / mL of 10:10:80 to the above centrifuge tube, shake and mix at 40 r / min for 20 s, let stand for 15 s to allow the magnetic beads to be completely adsorbed to the magnetic rack, use a pipette to absorb and discard the supernatant to obtain a magnetic bead-nucleic acid complex, and complete the nucleic acid extraction. Comparative Example 1

[0052] The difference between this comparative example and Example 3 is that the surface of the magnetic beads is not modified. Comparative Example 2

[0053] The difference between this comparative example and Example 3 is that the lysate in step 3 consists of proteinase K, sodium dodecyl sulfate and tromethamine in a mass volume ratio of g / mL of 0.5:3:3. Comparative Example 3

[0054] The difference between this comparative example and Example 3 is that no ultraviolet light is used for irradiation during the sample lysis and incubation in step 3. Comparative Example 4

[0055] The difference between this comparative example and Example 3 is that the washing liquid in step 5 is an ethanol solution with a concentration of 70%wt.

[0056] Nucleic acid concentration and purity detection

[0057] Taking the extraction of Escherichia coli DNA as an example, 10 samples cultivated from the same Escherichia coli colony were cultured, and nucleic acid was extracted from the Escherichia coli samples according to the extraction methods of Examples 1-5 and Comparative Examples 1-5, respectively, to obtain a magnetic bead-nucleic acid complex. PCR amplification was performed according to the following method: the PCR amplification system was 20 μL, including 12.0 μL 2x PCR buffer, 1.0 μL upstream primer, 1.0 μL downstream primer, 0.2 μL DNA polymerase, 0.5 μL fluorescent dye, 2 μL magnetic bead-nucleic acid complex, and 3.3 μL sterile enzyme-free water. The PCR amplification program was set as follows: pre-denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 55°C for 30 s. During the cycles, the absorbance was measured at 55°C at wavelengths of 230 nm, 260 nm, and 280 nm. The absorbance value A260 and the absorbance ratios A260 / A280 and A260 / A230 were calculated. The results are shown in Table 1.

[0058] Table 1

[0059] A260 A260 / A280 A260 / A230 Example 1 20.09 1.83 2.38 Example 2 20.14 1.78 2.40 Example 3 21.37 1.81 2.45 Example 4 19.98 1.82 2.39 Example 5 20.36 1.77 2.37 Comparative Example 1 11.35 1.39 1.61 Comparative Example 2 14.02 1.47 1.76 Comparative Example 3 16.59 1.53 1.85 Comparative Example 4 17.53 1.65 1.92

[0060] The higher the A260 value, the higher the nucleic acid concentration. The closer the A260 / A280 ratio is to 1.8, the less protein in the nucleic acid and the higher the nucleic acid purity. The closer the A260 / A280 ratio is to 2.5, the less salts and sugars in the nucleic acid and the higher the nucleic acid purity. It can be seen from Table 1 that in Examples 1-5, the A260 value is high, the A260 / A280 ratio is close to 1.8, and the A260 / A230 ratio is close to 2.5. It can be seen that the nucleic acid extraction method based on magnetic beads of the present invention has a high nucleic acid concentration and high purity. From Table 1, it can be seen that Example 3 has the best effect.

[0061] By comparing Example 3 with Comparative Example 1, when modifying the phospholipid bilayer on the surface of the magnetic microsphere, the phosphatidylinositol in the reaction solution has a unique molecular structure, and its head group contains phosphoinositide, and this special structure can enhance the binding stability between the phospholipid bilayer and the magnetic microsphere, so that the prepared phospholipid bilayer magnetic beads are more uniform and stable. In addition, phosphatidylinositol is an important component of the cell membrane and participates in a variety of signal transduction pathways. The lipid bilayer magnetic beads prepared using phosphatidylinositol may retain or simulate these biological activities, thereby having higher biocompatibility and functionality. Due to the special structure of phosphatidylinositol, better self-assembly characteristics are exhibited during the preparation process, making the preparation of lipid bilayer magnetic beads more simple and efficient. In addition, the nitrogen atom and hydroxyl of 3-morpholinepropanesulfonic acid may also form hydrogen bonds with the phosphoric acid or hydroxyl of phosphatidylinositol, thereby stabilizing the arrangement of phospholipid molecules on the surface of the magnetic beads. The specific three-dimensional structure of 3-morpholinepropanesulfonic acid may match the spatial configuration of phosphatidylinositol, and this structural compatibility contributes to the close combination between the two. When 3-morpholinepropanesulfonic acid molecules come into contact with phosphatidylinositol, they may attract each other through weak interactions such as van der Waals forces, prompting the phospholipid molecules to form an orderly arrangement on the surface of the magnetic beads.

[0062] When the surface of the magnetic beads is modified with a phospholipid bilayer, this biomimetic interface can reduce the interfacial tension between the nucleic acid and the magnetic beads, making it easier for the nucleic acid to contact and bind with the surface of the magnetic beads. And phospholipid molecules generally contain negatively charged phosphate groups and hydrophobic fatty acid chains. When the phospholipid bilayer is covered on the surface of the magnetic beads, these negative charges can electrostatically interact with the positively charged regions on the nucleic acid molecules, which helps to pull the nucleic acid molecules closer to the surface of the magnetic beads and promote their binding. Hydrogen bonds may be formed between the polar heads of the phospholipid molecules and the nucleic acid molecules, and this relatively weak chemical bonding helps to enhance the binding stability of nucleic acids and magnetic beads. In addition, the phospholipid bilayer has certain flexibility and fluidity, which enables it to better adapt to nucleic acid molecules of different shapes and sizes. When nucleic acid molecules are close to the phospholipid layer, the phospholipid molecules can adjust their own arrangement to adapt to and wrap the nucleic acid molecules, thereby enhancing the binding ability. Due to the presence of the phospholipid layer, it is helpful to reduce the non-specific binding with impurity molecules, thereby improving the selective binding efficiency of the target nucleic acid, so the nucleic acid concentration and purity of Example 3 are higher than those of Comparative Example 1.

[0063] By comparing Example 3 with Comparative Example 2, quinine helps to separate nucleic acids from cells more effectively by interfering with the stability of the cell membrane, thereby improving the extraction efficiency. Under the irradiation of ultraviolet light, 4,4'-dihydroxyazobenzene can undergo a photochemical reaction to produce an active substance that can assist in the lysis of cells. This active substance can penetrate into the cell membrane and react with the lipids in the cell membrane, thereby accelerating the cell lysis process. In addition, proteinase K can degrade proteins bound to nucleic acids, allowing nucleic acids to be extracted more purely. Sodium dodecyl sulfate can reduce surface tension, which helps in the lysis of cells and the release of nucleic acids. Tromethamine is used to adjust the pH value of the lysate and provide a suitable chemical environment to promote the lysis process. Therefore, compared with Comparative Example 2, Example 3 is more fully lysed, and the concentration and purity of the extracted nucleic acids are also higher.

[0064] By comparing Example 3 with Comparative Example 3, when 4,4'-dihydroxyazobenzene is irradiated with ultraviolet light, its molecular structure changes from a stable trans structure to a cis structure. This structural change can lead to charge redistribution and polarity changes within the molecule. This change not only enhances the interaction between 4,4'-dihydroxyazobenzene and cell membrane lipids, but may also produce a local high-energy state, which helps to destroy the stability of the cell membrane. 4,4'-dihydroxyazobenzene under ultraviolet light irradiation may undergo peroxidation with lipids on the cell membrane to generate reactive oxygen species, such as hydrogen peroxide or superoxide anions. These reactive oxygen species are highly oxidizing and can destroy cell membranes, thereby promoting cell lysis. In addition, 4,4'-dihydroxyazobenzene may also produce a transient, local high temperature effect under ultraviolet light excitation, which can instantly destroy the structure of the cell membrane, making it more fragile and brittle. This local high temperature effect works together with reactive oxygen species to greatly accelerate the cell lysis process. Therefore, compared with Comparative Example 3, Example 3 not only improves the lysis efficiency, but also further improves the lysis degree, and the extracted nucleic acid has higher concentration and purity.

[0065] By comparing Example 3 with Comparative Example 4, when cocamidopropyl betaine is combined with ethanol, ethanol can enhance the solubility and dispersibility of cocamidopropyl betaine, so that it can act on the surface of magnetic beads more effectively, and remove attached impurities and pollutants. In addition, the volatility of ethanol helps to quickly remove the residual washing liquid, thereby reducing the interference with subsequent experimental steps. The addition of ethanol can also reduce the surface tension of the washing liquid, making it easier to penetrate into the tiny gaps inside the magnetic beads. And urea, through its denaturation, enhances the ability to remove deep pollutants. The three work together so that the washing liquid can clean the magnetic beads more comprehensively and reduce residual pollutants. When cocamidopropyl betaine, urea and ethanol are used in combination, their respective advantages are amplified, and the synergistic effect between each other produces a kind of "1+1+1>3" effect, and this comprehensive effect greatly improves the efficiency and purity of nucleic acid extraction.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting nucleic acid based on magnetic beads, characterized in that: The following steps are involved: Step 1, surface modification of magnetic beads: at 40-60°C, mix magnetic microspheres, phosphatidylinositol, and methanol at 80-100 r / min for 60-90s, slowly add 3-morpholinepropanesulfonic acid and sodium chloride to the mixture and adjust the pH to 6.8-7.2, react for 25-35min, and wash the magnetic microspheres with sterile deionized water to obtain phospholipid bilayer magnetic beads; the mass volume ratio of the magnetic microspheres, phosphatidylinositol, methanol, 3-morpholinepropanesulfonic acid, and sodium chloride is (6-10):(3-5):(5-7):(1-3):(1-3); Step 2, preparation of magnetic bead suspension: add Tween 20 and polyethylene glycol to normal saline, stir at 100-200 r / min for 1-3 min, add phospholipid bilayer magnetic beads and shake and mix at 20-40 r / min for 10-20 s to obtain a magnetic bead suspension; the volume ratio of Tween 20, polyethylene glycol and normal saline is (0.1-0.3):(1.5-2.5):(90-100), and the concentration of the phospholipid bilayer magnetic beads in the solution is 10-30 mg / mL; Step 3, sample lysis and incubation: take a sample and add it to a 1.5mL centrifuge test tube, add a magnetic bead suspension and a cell lysis solution to the centrifuge test tube, shake and mix at 20-40r / min for 10-20s, and incubate at 24-26°C under ultraviolet light for 4-6min; the sample addition amount is 100-200μL, the magnetic bead suspension addition amount is 15-25μL, and the cell lysis solution addition amount is 100-200μL; the cell lysis solution is composed of quinine, proteinase K, sodium dodecyl sulfate, 4,4'-dihydroxyazobenzene, and tromethamine in a mass volume ratio of g / mL of (3-5):(0.5-1.0):(1-2):(0.4-0.6):(1-3); Step 4: Magnetic adsorption: Place the above centrifuge tube in a magnetic rack and let it stand for 1-2 minutes to allow the magnetic beads to be completely adsorbed on the magnetic rack. Use a pipette to absorb and discard the supernatant; Step 5, washing and removing impurities from the magnetic beads: adding washing solution to the above centrifuge tube, shaking and mixing at 20-40r / min for 20-30s, standing for 15-25s, so that the magnetic beads are completely adsorbed to the magnetic rack, using a pipette to absorb and discard the supernatant to obtain a magnetic bead-nucleic acid complex, and completing nucleic acid extraction; the amount of the washing solution added is 400-600μL; the washing solution is composed of cocamidopropyl betaine, urea, and ethanol with a mass volume ratio of g / mL of (5-10):(5-10):(70-80).

2. A method for extracting nucleic acid based on magnetic beads as claimed in claim 1, characterized in that: The magnetic microspheres described in step 1 are one of hydroxyl magnetic microspheres, carboxyl magnetic microspheres, epoxy magnetic microspheres, and ferroferric oxide magnetic microspheres, and the particle size of the magnetic microspheres is 500-1000 nm.

3. A method for extracting nucleic acid based on magnetic beads as claimed in claim 1, characterized in that: When the ultraviolet light is irradiated, the wavelength is set to 300-400nm and the light intensity is set to 800-1200Lux.