Extraction method of nucleic acid

Abstract

The invention provides a nucleic acid extraction method, and relates to the technical field of nucleic acid extraction, the nucleic acid extraction method comprises the following steps: putting a to-be-detected sample, a lysis solution and magnetic beads into a reaction container, and splitting the to-be-detected sample by the lysis solution to release nucleic acid; performing ultrasonic treatment on the solution in the reaction container for preset time, so that the magnetic beads are dispersed in the solution and are combined with the nucleic acid; focusing the magnetic beads on a moving rod; transferring the magnetic beads from the reaction container to a subsequent solution through a moving rod, and carrying out ultrasonic treatment on the subsequent solution, so that the magnetic beads are re-dispersed in the subsequent solution; and removing the magnetic beads from the subsequent solution to obtain the nucleic acid. The magnetic beads are dispersed in a solution in a non-contact manner through sound streams generated by ultrasonic waves and flow in a vortex shape, the specific surface area is greatly increased, the magnetic beads and nucleic acid can be fully combined without contact with the solution in an ultrasonic manner, the pollution problem caused by existing direct stirring is avoided, the probability of contact pollution is reduced, and the production efficiency is improved. The subsequent nucleic acid extraction purity is effectively improved.

Description

Technical Field

[0001] The present application relates to the technical field of nucleic acid extraction, and in particular to a method for extracting nucleic acid. Background Art

[0002] Nucleic acids are an essential component of known life forms and are important markers in molecular diagnosis. With the increasing attention paid to the prevention and treatment of infectious diseases and the development of molecular biological detection methods, molecular diagnostic technology has received widespread attention and attention in the fields of clinical diagnosis, environmental testing, and food safety testing. Nucleic acid extraction is one of the key steps in molecular biology and molecular diagnosis. Molecular diagnostic technology detects diseases, pathogens, or genetic variations by analyzing the nucleic acid sequences of genomes, transcriptomes, or microorganisms. The accuracy and reliability of these technologies largely rely on high-quality nucleic acid samples. Therefore, nucleic acid extraction is of great significance in clinical diagnosis, scientific research, and biotechnology applications.

[0003] Among them, the magnetic bead method is widely used due to its high efficiency, easy automation, high purity and high yield, and wide applicability. This method mainly uses silica modified with ferroferric oxide or other materials that can specifically bind to nucleic acid molecules as magnetic beads, which reversibly bind to nucleic acid molecules under specific salt concentration and pH conditions, and realize solid-liquid separation and transfer by means of an external magnetic field. The magnetic beads need to be fully mixed in the mixed solution so that the nucleic acids can fully bind to the magnetic beads to obtain nucleic acids with higher concentrations in the subsequent process. At present, the mixing of magnetic beads is mainly carried out by stirring the mixed solution vertically, rotating, etc., and the agitator is immersed in the sample to combine the magnetic beads and nucleic acids. This method has problems such as sample contamination and low specific surface area of ​​contact between the magnetic beads and the sample, which is not conducive to the extraction of high-purity nucleic acids. Summary of the invention

[0004] The purpose of the embodiments of the present application is to provide a method for extracting nucleic acid, which can combine magnetic beads and nucleic acid by a non-contact method, thereby improving the extraction purity of nucleic acid.

[0005] In one aspect of an embodiment of the present application, a method for extracting nucleic acid is provided, comprising placing a sample to be tested, a lysis solution and magnetic beads into a reaction container, wherein the lysis solution lyses the sample to be tested to release nucleic acid;

[0006] Performing ultrasonic treatment on the solution in the reaction container for a preset time so that the magnetic beads are dispersed in the solution and combined with the nucleic acid;

[0007] focusing the magnetic beads on a moving rod;

[0008] Transferring the magnetic beads from the reaction container to a subsequent solution by means of the moving rod, and subjecting the subsequent solution to ultrasonic treatment so as to redisperse the magnetic beads in the subsequent solution;

[0009] The magnetic beads are removed from the subsequent solution to obtain the nucleic acid.

[0010] Optionally, the lysate comprises a mixture of sodium salt, a protein denaturant, a surfactant and alcohol, the volume concentration of the surfactant is 1% to 30%, and the volume concentration of the alcohol is 5% to 40%.

[0011] Optionally, the step of subjecting the solution in the reaction container to ultrasonic treatment for a preset time so that the magnetic beads are dispersed in the solution and bind to the nucleic acid comprises:

[0012] placing the reaction container on an ultrasonic transducer so that the reaction container is in contact with the ultrasonic transducer;

[0013] The solution in the reaction container is ultrasonically scanned with the resonant frequency of the ultrasonic transducer as the center and the range of ±0.2 kHz as the ultrasonic working frequency.

[0014] Optionally, placing the reaction container on an ultrasonic transducer so that the reaction container contacts the ultrasonic transducer comprises:

[0015] The ultrasonic transducer is placed at the bottom of the reaction container; or, the ultrasonic transducer is placed around the reaction container.

[0016] Optionally, the operating power of the ultrasonic transducer is between 0 and 30 W, and the ultrasonic treatment time is between 10 s and 120 s.

[0017] Optionally, focusing the magnetic beads on the moving rod comprises:

[0018] A magnetic bar is used as the moving bar to non-contactly adsorb the magnetic beads in the reaction container, so that the magnetic beads are gathered on the magnetic bar.

[0019] Optionally, focusing the magnetic beads on the moving rod comprises:

[0020] The magnetic bar is moved into the magnetic bar sleeve, and the magnetic bar and the magnetic bar sleeve are integrated as the moving rod and extended into the solution in the reaction container to adsorb the magnetic beads, so that the magnetic beads are gathered on the magnetic bar sleeve.

[0021] Optionally, the transferring of the magnetic beads from the reaction container to a subsequent solution by the moving rod, and ultrasonic treatment of the subsequent solution to redisperse the magnetic beads in the subsequent solution, comprises:

[0022] Using the moving rod to move the magnetic beads from the reaction container to a cleaning solution, and dispersing the magnetic beads in the cleaning solution by ultrasonic treatment;

[0023] The moving rod is then used to move the magnetic beads from the cleaning solution to the elution solution, and the magnetic beads are dispersed in the elution solution by ultrasonic treatment, and the elution solution is used to separate the nucleic acid bound to the magnetic beads from the magnetic beads.

[0024] Optionally, after the moving rod is used to move the magnetic beads from the reaction container to a cleaning solution and the magnetic beads are dispersed in the cleaning solution by ultrasonic treatment, the method further comprises:

[0025] Using the moving rod to move the magnetic beads out of the cleaning solution;

[0026] The moving rod and the magnetic beads are dried.

[0027] Optionally, the ultrasonic transducer includes a piezoelectric ceramic transducer or a sandwich transducer.

[0028] The nucleic acid extraction method provided in the embodiment of the present application includes: placing the sample to be tested, lysate and magnetic beads into a reaction vessel, the lysate lyses the sample to be tested to release nucleic acid; subjecting the solution in the reaction vessel to ultrasonic treatment for a preset time so that the magnetic beads are dispersed in the solution and combined with the nucleic acid; focusing the magnetic beads on a moving rod; transferring the magnetic beads from the reaction vessel to a subsequent solution by the moving rod, subjecting the subsequent solution to ultrasonic treatment so that the magnetic beads are redispersed in the subsequent solution; removing the magnetic beads from the subsequent solution to obtain nucleic acid. The magnetic beads and nucleic acid are combined by ultrasonic means, and the acoustic flow generated by ultrasonic waves disperses the magnetic beads in the solution in a non-contact manner and flows in a vortex shape, greatly increasing the specific surface area. The ultrasonic method does not need to contact the solution to fully combine the magnetic beads and nucleic acid, avoiding the pollution problem caused by existing direct stirring, reducing the probability of contact pollution, and effectively improving the purity of subsequent nucleic acid extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 It is a flow chart of the method for extracting nucleic acid provided in this embodiment. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application.

[0032] In the description of this application, it should be noted that the terms "inside", "outside", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship in which the product of the application is usually placed when in use, which is only for the convenience of describing this application and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on this application. In addition, the terms "first", "second", etc. are only used to distinguish the description, and cannot be understood as indicating or implying relative importance.

[0033] It should also be noted that, unless otherwise clearly specified and limited, the terms "disposed" and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a direct connection, or an indirect connection through an intermediate medium, or it can be the internal communication of two elements. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0034] Please refer to Figure 1 The present invention provides a method for extracting nucleic acid, comprising:

[0035] S100: The sample to be tested, the lysis solution and the magnetic beads are placed in a reaction container, and the lysis solution lyses the sample to be tested to release nucleic acid.

[0036] First, add the sample to be tested and the lysis solution into the reaction container to fully lyse the sample to be tested and release the nucleic acid; then add magnetic beads into the reaction container and the nucleic acid is adsorbed on the magnetic beads.

[0037] Generally, the lysis solution includes a mixture of sodium salt, protein denaturant, surfactant and alcohol to lyse the sample to be tested to obtain nucleic acid; wherein the volume concentration of the surfactant is 1% to 30%, and the volume concentration of the alcohol is 5% to 40%.

[0038] S110: The solution in the reaction container is subjected to ultrasonic treatment for a preset time so that the magnetic beads are dispersed in the solution and combined with the nucleic acid.

[0039] Ultrasonic treatment can accelerate the binding of nucleic acid and magnetic beads in step S100. After the magnetic beads dispersed in the solution are fully bound to the nucleic acid in the solution, a higher concentration of nucleic acid can be extracted subsequently.

[0040] Furthermore, in order to further increase the binding concentration of nucleic acid and magnetic beads, the ultrasonic treatment time is maintained within a preset time, for example, the ultrasonic treatment time can be 10s to 120s.

[0041] Specifically, the reaction container is placed on the ultrasonic transducer so that the reaction container is in contact with the ultrasonic transducer, so that the solution in the reaction container is ultrasonically treated by the ultrasonic transducer; wherein the ultrasonic transducer can be located at the bottom of the reaction container or around the reaction container, as long as the two are in contact.

[0042] Then, the solution in the reaction container is ultrasonically scanned with the resonant frequency of the ultrasonic transducer as the center and the range of ±0.2 kHz as the ultrasonic working frequency.

[0043] During ultrasonic treatment, the power of the ultrasonic transducer is between 0 and 30 W. This is conducive to sufficient oscillation, so that the magnetic beads and nucleic acids are fully combined.

[0044] In addition, the ultrasonic transducer may be a piezoelectric ceramic transducer or a sandwich transducer.

[0045] The magnetic beads and nucleic acids are combined by ultrasound. The acoustic flow generated by ultrasound disperses the magnetic beads in the solution in a non-contact manner and makes them flow in a vortex, greatly increasing the specific surface area. The ultrasonic method can fully combine the magnetic beads and nucleic acids without contacting the solution, avoiding the pollution problems caused by existing direct stirring, reducing the chance of contact contamination, and effectively improving the purity of subsequent nucleic acid extraction.

[0046] S120: Focus the magnetic beads on the moving rod.

[0047] After the magnetic beads and nucleic acids are fully combined, the nucleic acids are firmly adsorbed on the magnetic beads. After the magnetic beads are aggregated, it is convenient for the subsequent extraction of nucleic acids.

[0048] The moving rod may be a magnetic rod, or may be a whole formed by a magnetic rod and a magnetic rod sleeve.

[0049] Specifically, when the moving rod is a whole formed by a magnetic rod and a magnetic rod sleeve, the magnetic rod is moved into the magnetic rod sleeve and the whole is extended into the solution in the reaction container to adsorb the magnetic beads. The magnetic beads are attached to the outer bottom wall of the magnetic rod sleeve and gathered on the inner bottom wall of the reaction container. In this way, when the magnetic rod sleeve and the magnetic rod are removed from the solution as a whole, the magnetic beads and the nucleic acids adsorbed on the magnetic beads can be taken out.

[0050] Of course, this process must ensure that the magnetic rod and the magnetic rod cover are clean to avoid contaminating the solution in the reaction container.

[0051] In other embodiments, in order to further improve the purity of nucleic acid extraction, a non-contact magnetic bead aggregation method can also be used. A magnetic bar is used as a moving rod, and the magnetic bar is used to non-contactly adsorb the magnetic beads in the reaction container. The magnetic attraction of the magnetic bar can be used to suck the magnetic beads out of the reaction container and adsorb and aggregate them on the magnetic bar to transfer the magnetic beads and the nucleic acids adsorbed on the magnetic beads. When this method aggregates and transfers magnetic beads, the magnetic bar does not need to extend into the reaction container, which can ensure that the solution in the reaction container is not contaminated.

[0052] S130: transferring the magnetic beads from the reaction container to a subsequent solution by moving the rod, and subjecting the subsequent solution to ultrasonic treatment to redisperse the magnetic beads in the subsequent solution.

[0053] After the magnetic beads and nucleic acids are transferred from the reaction vessel using a moving rod, the magnetic beads are first moved from the reaction vessel to the cleaning solution, and the magnetic beads are dispersed in the cleaning solution through ultrasonic treatment. The purpose of this is to use the cleaning solution to wash away non-nucleic acid substances on the surface of the magnetic beads. Ultrasonic treatment can ensure sufficient cleaning.

[0054] The magnetic beads are then moved from the cleaning solution to the elution solution using a moving rod, and the magnetic beads are dispersed in the elution solution through ultrasonic treatment, and the elution solution is used to separate the nucleic acids bound to the magnetic beads from the magnetic beads.

[0055] S140: removing the magnetic beads from the subsequent solution to obtain the nucleic acid.

[0056] After the magnetic beads carry the nucleic acid into the elution solution, the nucleic acid attached to the magnetic beads detaches from the magnetic beads and enters the elution solution, so that the magnetic beads and the nucleic acid are separated. The magnetic beads are adsorbed by a moving rod for recovery. After the magnetic beads are moved out of the elution solution, the nucleic acid to be extracted can be obtained.

[0057] Similarly, ultrasonic treatment allows the magnetic beads to be fully dispersed in the elution solution, so that the nucleic acid on the magnetic beads can be fully eluted to obtain nucleic acid with higher concentration and purity.

[0058] In addition, after the magnetic beads are transferred to the cleaning solution and before they are transferred to the elution solution, the magnetic beads can be dried to avoid bringing the cleaning solution into the elution solution and affecting the elution of the nucleic acid.

[0059] Specifically, the magnetic beads are moved out of the cleaning solution using a moving rod, and then the moving rod and the magnetic beads are dried.

[0060] When the magnetic beads are transferred from one solution to another, they always move together with the nucleic acid adsorbed thereon. The purpose is to achieve the transfer of nucleic acid between different solutions so as to wash, elute and extract the nucleic acid.

[0061] In summary, the method for extracting nucleic acid provided in the embodiments of the present application adopts a non-contact method such as ultrasound to effectively and fully combine the magnetic beads and nucleic acid. The magnetic beads are dispersed in the solution in a non-contact manner and flow in a vortex shape through the acoustic flow generated by ultrasound, which greatly increases the specific surface area and reduces the problem of contact contamination, thereby improving the extraction purity of the nucleic acid.

[0062] The following is explained by different embodiments. In the first embodiment, 1 ml of lysate containing 2.5%, 5%, 7.5%, 10% and 15% of surfactant is added to a reaction container, and then 25 μl of magnetic bead suspension is added. The solution in the reaction container is ultrasonically treated for 1 minute at room temperature, and the magnetic beads are suspended and distributed in the solution under the action of ultrasound. The ultrasonic transducer is placed at the bottom of the reaction container and is in close contact with the container.

[0063] The magnetic beads in the container were gathered with a magnetic bar. After the magnetic beads gathered on the magnetic bar and the inner bottom wall of the reaction container, the reaction container was ultrasonicated to disperse and mix the magnetic beads in the solution. The mixing time of the magnetic beads was statistically analyzed. The lysis solution containing 2.5% surfactant mixed the magnetic beads fastest under the action of ultrasound.

[0064] In the second embodiment, 1 ml of lysate containing 10% surfactant and 0%, 10%, 20%, and 30% isopropanol was added to a reaction container, and then 25 μl of magnetic bead suspension was added. The solution in the reaction container was ultrasonically treated for 1 minute at room temperature, and the magnetic beads were suspended and distributed in the solution under the action of ultrasound. The ultrasonic transducer was placed at the bottom of the reaction container and was in close contact with the container.

[0065] The magnetic beads in the container were gathered with a magnetic bar. After the magnetic beads were gathered on the magnetic bar and the inner bottom wall of the reaction container, the reaction container was ultrasonicated for 1 min to disperse and mix the magnetic beads in the solution. The mixing time of the magnetic beads was statistically analyzed. The lysis solution containing 30% isopropanol could mix the magnetic beads fastest under the action of ultrasound.

[0066] The above two embodiments do not add the sample to be tested, which can be used as a comparison with the following examples of adding the sample to be tested. It can be seen that the magnetic beads can be quickly mixed by ultrasound.

[0067] In the third embodiment, 500 μl of a lysate containing 20% ​​surfactant and 30% isopropanol, 500 μl of a bovine serum sample, and 25 μl of a magnetic bead suspension were added to a reaction container for lysis. Ultrasonic treatment was applied to the bottom of the reaction container for 20 seconds at room temperature, and the magnetic beads were fully dispersed in the solution in the reaction container under the action of ultrasonic waves.

[0068] Maintain the ultrasonic state and allow the magnetic beads to be distributed in the solution for 5 minutes. After the magnetic beads are fully combined with the nucleic acid, use a magnetic bar to aggregate the magnetic beads and transfer them to the cleaning solution or elution solution, and then perform ultrasonic treatment for 30 seconds. The magnetic beads are redispersed in the cleaning solution or elution solution within 15 seconds under the action of ultrasound.

[0069] In the fourth embodiment, 500 μl of a lysate containing 20% ​​surfactant and 30% isopropanol, 500 μl of a porcine serum sample, and 25 μl of a magnetic bead suspension were added to a reaction container for lysis. Ultrasonic treatment was applied to the bottom of the reaction container for 20 seconds at room temperature, and the magnetic beads were fully dispersed in the solution in the reaction container under the action of ultrasonic waves.

[0070] Maintain the ultrasonic state and distribute the magnetic beads in the solution for 5 minutes to allow the magnetic beads to fully bind to the nucleic acid. Then use a magnetic bar to aggregate the magnetic beads and transfer them to the cleaning solution or elution solution. Then perform ultrasonic treatment for 30 seconds. The magnetic beads will be redispersed in the cleaning solution or elution solution within 15 seconds under the action of ultrasound.

[0071] The above description is only an embodiment of the present application and is not intended to limit the protection scope of the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for extracting nucleic acid, characterized in that: include: Putting the sample to be tested, lysis solution and magnetic beads into a reaction container, wherein the lysis solution lyses the sample to be tested to release nucleic acid; Performing ultrasonic treatment on the solution in the reaction container for a preset time so that the magnetic beads are dispersed in the solution and combined with the nucleic acid; focusing the magnetic beads on a moving rod; Transferring the magnetic beads from the reaction container to a subsequent solution by means of the moving rod, and subjecting the subsequent solution to ultrasonic treatment so as to redisperse the magnetic beads in the subsequent solution; The magnetic beads are removed from the subsequent solution to obtain the nucleic acid.

2. The method for extracting nucleic acid according to claim 1, characterized in that: The lysate comprises a mixture of sodium salt, protein denaturant, surfactant and alcohol, wherein the volume concentration of the surfactant is 1% to 30%, and the volume concentration of the alcohol is 5% to 40%.

3. The method for extracting nucleic acid according to claim 1, characterized in that: The step of subjecting the solution in the reaction container to ultrasonic treatment for a preset time so that the magnetic beads are dispersed in the solution and combined with the nucleic acid comprises: placing the reaction container on an ultrasonic transducer so that the reaction container is in contact with the ultrasonic transducer; The solution in the reaction container is ultrasonically scanned with the resonant frequency of the ultrasonic transducer as the center and the range of ±0.2 kHz as the ultrasonic working frequency.

4. The method for extracting nucleic acid according to claim 3, characterized in that: Placing the reaction container on the ultrasonic transducer so that the reaction container contacts the ultrasonic transducer comprises: The ultrasonic transducer is placed at the bottom of the reaction container; or, the ultrasonic transducer is placed around the reaction container.

5. The method for extracting nucleic acid according to claim 3 or 4, characterized in that: The use power of the ultrasonic transducer is between 0 and 30 W, and the ultrasonic treatment time is between 10 s and 120 s.

6. The method for extracting nucleic acid according to claim 1, characterized in that: The step of focusing the magnetic beads on the moving rod comprises: A magnetic bar is used as the moving bar to non-contactly adsorb the magnetic beads in the reaction container, so that the magnetic beads are gathered on the magnetic bar.

7. The method for extracting nucleic acid according to claim 1, characterized in that: The step of focusing the magnetic beads on the moving rod comprises: The magnetic bar is moved into the magnetic bar sleeve, and the magnetic bar and the magnetic bar sleeve are integrated as the moving rod and extended into the solution in the reaction container to adsorb the magnetic beads, so that the magnetic beads are gathered on the magnetic bar sleeve.

8. The method for extracting nucleic acid according to claim 1, characterized in that: The step of transferring the magnetic beads from the reaction container to a subsequent solution by the moving rod and subjecting the subsequent solution to ultrasonic treatment so as to redisperse the magnetic beads in the subsequent solution comprises: Using the moving rod to move the magnetic beads from the reaction container to a cleaning solution, and dispersing the magnetic beads in the cleaning solution by ultrasonic treatment; The moving rod is then used to move the magnetic beads from the cleaning solution to the elution solution, and the magnetic beads are dispersed in the elution solution by ultrasonic treatment, and the elution solution is used to separate the nucleic acid bound to the magnetic beads from the magnetic beads.

9. The method for extracting nucleic acid according to claim 8, characterized in that: After the moving rod is used to move the magnetic beads from the reaction container to the cleaning solution and the magnetic beads are dispersed in the cleaning solution by ultrasonic treatment, the method further comprises: Using the moving rod to move the magnetic beads out of the cleaning solution; The moving rod and the magnetic beads are dried.

10. The method for extracting nucleic acid according to claim 3, characterized in that: The ultrasonic transducer includes a piezoelectric ceramic transducer or a sandwich transducer.

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