CN115478072A - Magnetic bead-based rapid nucleic acid extraction method - Google Patents
Magnetic bead-based rapid nucleic acid extraction method Download PDFInfo
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- CN115478072A CN115478072A CN202211363369.XA CN202211363369A CN115478072A CN 115478072 A CN115478072 A CN 115478072A CN 202211363369 A CN202211363369 A CN 202211363369A CN 115478072 A CN115478072 A CN 115478072A
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Abstract
The invention discloses a magnetic bead-based nucleic acid rapid extraction method, which relates to the technical field of biology, and adopts a nucleic acid extraction kit, wherein the nucleic acid extraction kit comprises lysis solution, magnetic beads, washing solution, eluent, a nucleic acid protective agent, a deep hole plate and a magnetic rod sleeve, the deep hole plate comprises a box panel, a concave surface, a hole head, a threaded joint, a flexible tube, a torsion spring, a support plate, a bottom plate, a limiting groove, an extension plate and a magnetic strip, the concave surface is arranged on the surface of the box panel, and the limiting groove is also arranged on the surface of the bottom plate. This method of extracting nucleic acid fast based on magnetic bead, soft test tube have the compliance can realize folding, and the backup pad can rotate to the concave surface inside through the torsional spring, and the outer mouthful size of box panel and the interior mouthful size looks adaptation of limiting plate, and the box panel that consequently can make and the soft test tube after folding place between the limiting plate, and the volume of this deep hole board of greatly reduced from this to carry out large batch transportation.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a magnetic bead-based rapid nucleic acid extraction method.
Background
The principle of the magnetic bead method is that the surface of superparamagnetic nano particles is improved and surface modified by using a nanotechnology to prepare superparamagnetic silicon oxide nano magnetic beads, the magnetic beads can be specifically identified and efficiently combined with nucleic acid molecules on a microscopic interface, and DNA and RNA can be separated from samples such as blood, animal tissues, food, pathogenic microorganisms and the like under the action of Chaotropic salts (guanidine hydrochloride, guanidine isothiocyanate and the like) and an external magnetic field by utilizing the superparamagnetism of the silica-coated nano magnetic microspheres.
The deep-well plate used in the existing magnetic bead method for extracting nucleic acid is made of plastic, specifically, 96 tube positions are arranged on square hard plastic, the size of the deep-well plate is greatly increased due to the arrangement of the tube positions, the number of the deep-well plates which can be stored in a fixed storage space is limited, the deep-well plate cannot be transported in a large batch under the condition that the transportation cost is not increased, and how to transport the deep-well plate in a large batch while keeping the low transportation cost is also a great factor for improving the nucleic acid extraction speed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for quickly extracting nucleic acid based on magnetic beads, which solves the problems in the background technology.
In order to achieve the purpose, the invention is realized by the following technical scheme: the nucleic acid rapid extraction method based on the magnetic beads adopts a nucleic acid extraction kit, the nucleic acid extraction kit comprises lysate, magnetic beads, washing liquid, eluent, a nucleic acid protective agent, a deep hole plate and a magnetic rod sleeve, the deep hole plate comprises a box panel, a concave surface, a hole head, a threaded joint, a flexible tube, a torsion spring, a support plate, a bottom plate, a limiting groove, an extension plate and a magnetic stripe, the concave surface is formed in the surface of the box panel, the hole head is arranged at the bottom of the box panel, the threaded joint is connected to the outer wall of the hole head in a threaded mode, the flexible tube is fixed to the bottom of the threaded joint, the torsion springs are arranged on two sides of the box panel, the support plate is connected to the outer wall of the torsion spring, the bottom plate is arranged below the box panel in parallel, the limiting plate is arranged on two sides of the surface of the bottom plate, the limiting groove is formed in the surface of the bottom plate, the limiting groove is located between the limiting plates, the extension plate is connected to the support plate in a sliding mode, and the magnetic stripe is fixed to the end portion of the extension plate.
Further, soft test tube and spacing groove are a one-to-one and correspond the setting, and the bottom of soft test tube is located the spacing inslot portion.
Furthermore, the supporting plate is elastically connected with the box panel through a torsion spring, and the side face of the supporting plate is attached to the inner side face of the limiting plate.
Furthermore, the size of the outer opening of the box panel is matched with the size of the inner opening of the limiting plate, and the box panel forms a concave structure through a concave surface.
Further, the components of the lysis solution comprise the following components in mass-volume ratio:
0.1177% Tris,1.753% NaCl,0.1862% EDTA.2Na, 5% PEG6000, 41.36% guanidinium isothiocyanate and 5% TritonX-100 by volume, pH 8.5, wherein Tris is named as Tris (hydroxymethyl) aminomethane and has the molecular formula of (HOCH) 2 )3CNH 2 NaCl is named as sodium chloride in Chinese, EDTA-2 Na is named as disodium ethylenediamine tetraacetate in Chinese, and PEG6000 is named as polyethylene glycol 6000 in Chinese, and the molecular formula is HO (CH) 2 CH 2 O) nH, wherein n represents the average number of oxyethylene groups, tritonX-100, which is a nonionic surfactant, is known as polyoxyethylene octylphenyl ether.
Further, the washing solution is ethanol with the concentration of 80%.
Further, the eluent is ultrapure water treated by DEPC.
Further, the nucleic acid protective agent selects tris (2-carboxyethyl) phosphine hydrochloride with the mass volume ratio of 14.34%.
Furthermore, the magnetic rod is sleeved and inserted into the soft test tube.
Further, the method for rapidly extracting the nucleic acid comprises the following specific steps:
the method comprises the following steps: adding lysis solution into a soft test tube, then adding a throat swab sample into the soft test tube, wherein if scientific research grade RNA needs to be obtained, adding nucleic acid protective solution;
step two: inserting a magnetic rod sleeve into the soft test tube, sucking magnetic beads by using the magnetic rod, enabling the magnetic beads to penetrate through the magnetic rod sleeve, adding the magnetic beads into the soft test tube, uniformly mixing reagents in the soft test tube at a temperature, and releasing RNA from a sample and adsorbing the RNA on the surfaces of the magnetic beads;
step three: and taking out the magnetic beads adsorbed with the RNA by using the magnetic rod again, removing redundant impurities from the magnetic beads through a washing solution, putting the magnetic beads into an eluent, and dissolving the RNA in the eluent at the temperature so as to finish the extraction of the RNA.
The invention provides a magnetic bead-based rapid nucleic acid extraction method, which has the following beneficial effects:
1. according to the rapid extraction method of nucleic acid based on magnetic beads, a lysis solution enables cells to be lysed, nucleic acid is released, meanwhile, nuclease is inhibited under the assistance of a nucleic acid protective agent, and the nucleic acid is protected from being hydrolyzed by the nuclease; under the environment of high salt and low PH, silica gel on the surface of the magnetic beads efficiently adsorbs released nucleic acid; washing the magnetic particles with a washing solution to remove impurities such as protein, salt and the like; under the environment of low salt and high PH, the elution liquid is used to make the silica gel release the adsorbed nucleic acid, thereby separating and purifying the nucleic acid quickly, and being beneficial to improving the extraction speed of the nucleic acid.
2. This method of extracting nucleic acid fast based on magnetic bead, soft test tube have the compliance can realize folding, and the backup pad can rotate to the concave surface inside through the torsional spring, and the outer mouthful size of box panel and the interior mouthful size looks adaptation of limiting plate, and the box panel that consequently can make and the soft test tube after folding place between the limiting plate, and the volume of this deep hole board of greatly reduced from this to carry out large batch transportation.
3. This method for extracting nucleic acid fast based on magnetic bead, the inside embolism that has inserted of hole head is inside in order to seal soft test tube, can pull out extension board from the backup pad when the backup pad passes through the torsional spring can rotate to the concave surface inside, and two extension boards are close to each other and adsorb each other through the magnetic stripe this moment, and this setting is favorable to strengthening the sealed effect of hole head.
4. According to the method for rapidly extracting the nucleic acid based on the magnetic beads, the supporting plate is manually rotated to enable the bottom side face of the supporting plate to enter the limiting plate, the supporting plate is stably arranged on the bottom plate based on the elastic rotation action of the torsion spring, the soft test tube is reset to be straight, the bottom of the soft test tube is located in the limiting groove to improve the straight degree, and the soft test tube is prevented from being bent due to the flexibility of the soft test tube.
5. This nucleic acid rapid extraction method based on magnetic bead, soft tube pass through screwed joint and hole head threaded connection, and this sets up the later stage and can replace alone the soft tube that appears the damage to prevent that single test tube damage from just abandoning the condition production of whole deep hole board.
Drawings
FIG. 1 is a schematic diagram of a front view of a soft test tube of the deep hole plate of the present invention;
FIG. 2 is a schematic top view of a bottom plate of the deep hole plate according to the present invention;
fig. 3 is a schematic top view of the panel of the deep hole plate according to the present invention.
In the figure: 1. a box panel; 2. a concave surface; 3. a hole head; 4. a threaded joint; 5. a soft test tube; 6. a torsion spring; 7. a support plate; 8. a base plate; 9. a limiting plate; 10. a limiting groove; 11. an extension plate; 12. and (4) a magnetic strip.
Detailed Description
Embodiments of the present invention will be described in further detail with reference to the drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
As shown in fig. 1-3, the present invention provides a technical solution: a nucleic acid rapid extraction method based on magnetic beads adopts a nucleic acid extraction kit, the nucleic acid extraction kit comprises lysis solution, magnetic beads, washing solution, eluent, a nucleic acid protective agent, a deep hole plate and a magnetic rod sleeve, the deep hole plate comprises a box panel 1, a concave surface 2, a hole head 3, a threaded joint 4, a flexible test tube 5, a torsion spring 6, a support plate 7, a bottom plate 8, a limit plate 9, a limit groove 10, an extension plate 11 and a magnetic strip 12, the concave surface 2 is arranged on the surface of the box panel 1, the hole head 3 is arranged on the bottom array of the box panel 1, the threaded joint 4 is connected to the outer wall of the hole head 3 in a threaded manner, the flexible test tube 5 is fixed at the bottom of the threaded joint 4, the torsion springs 6 are arranged on two sides of the box panel 1, the outer wall of the torsion spring 6 is connected with a supporting plate 7, a bottom plate 8 is arranged below the box panel 1 in parallel, limiting plates 9 are arranged on two sides of the surface of the bottom plate 8, limiting grooves 10 are further formed in the surface of the bottom plate 8, the limiting grooves 10 are located between the limiting plates 9, an extending plate 11 is connected to the inside of the supporting plate 7 in a sliding mode, magnetic stripes 12 are fixed to the end portions of the extending plate 11, the flexible test tubes 5 and the limiting grooves 10 are correspondingly arranged in a one-to-one mode, the bottom ends of the flexible test tubes 5 are located inside the limiting grooves 10, the supporting plate 7 is elastically connected with the box panel 1 through the torsion spring 6, the side face of the supporting plate 7 is attached to the inner side face of the limiting plate 9, the size of the outer opening of the box panel 1 is matched with the size of the inner opening of the limiting plate 9, and the box panel 1 forms a concave-shaped structure through the concave face 2;
the operation is as follows, when in idle or transportation state, the plug is inserted into the hole head 3 to seal the interior of the soft test tube 5, the support plate 7 rotates to the interior of the concave surface 2 based on the elastic rotation action of the torsion spring 6, at the moment, the extension plates 11 are pulled out manually, at the moment, the two extension plates 11 are close to each other and are adsorbed by the magnetic strip 12, the arrangement is favorable for enhancing the sealing effect of the hole head 3 to prevent the plug in the hole head 3 from loosening, the soft test tube 5 is of a flexible structure and can be folded, after the soft test tube 5 is folded, as the outer opening size of the box panel 1 is matched with the inner opening size of the limit plate 9, the box panel 1 and the folded soft test tube 5 are placed between the limit plates 9, at the moment, the volume of a single deep hole plate can be greatly reduced to increase the storable amount of the fixed storage space, so as to carry out large batch transportation or store, can avoid having the automatic appearance of drawing of nucleic acid because of lacking the sample to carry out nucleic acid extraction and be in idle state through the deep hole board of mass transportation, thereby it shortens the required time of whole nucleic acid extraction operation to become mutually, and when needing to carry out nucleic acid extraction, separate magnetic stripe 12 earlier and push into backup pad 7 inside with extension board 11, then artifical rotatory backup pad 7 makes its bottom get into inside limiting plate 9, make backup pad 7 spacing inside limiting plate 9 based on the elasticity gyration effect of torsional spring 6 this moment, so that box panel 1 is settled steadily on bottom plate 8, soft tube 5 resets simultaneously and is straight form, and soft tube 5 bottom is located inside improving straight degree in spacing groove 10, prevent that soft tube 5 from taking place the bending because of its compliance, then can carry out the nucleic acid extraction operation.
The components of the lysate comprise the following components in mass volume ratio:
0.1177% Tris,1.753% NaCl,0.1862% EDTA.2Na, 5% PEG6000, 41.36% guanidinium isothiocyanate and 5% TritonX-100 by volume, pH 8.5, wherein Tris is named as Tris (hydroxymethyl) aminomethane and has the molecular formula of (HOCH) 2 )3CNH 2 NaCl is named as sodium chloride in Chinese, EDTA-2 Na is named as disodium ethylenediamine tetraacetate in Chinese, and PEG6000 is named as polyethylene glycol 6000 in Chinese, and the molecular formula is HO (CH) 2 CH 2 O) nH, wherein n represents the average number of oxyethylene groups, tritonX-100 is named as polyethylene glycol octyl phenyl ether which is a nonionic surfactant, ethanol with the concentration of 80% is selected as a washing solution, ultrapure water treated by DEPC is selected as an eluent, tris (2-carboxyethyl) phosphine hydrochloride with the mass-volume ratio of 14.34% is selected as a nucleic acid protective agent, and a magnetic rod is sleeved in the soft test tube 5;
the method for quickly extracting the nucleic acid comprises the following specific steps:
the method comprises the following steps: adding 400ul of lysis solution into the soft test tube 5, then adding 200ul of throat swab sample into the soft test tube 5, wherein if scientific research grade RNA needs to be obtained, 10ul of nucleic acid protective solution needs to be added;
step two: inserting a magnetic rod sleeve into the soft test tube 5, sucking magnetic beads by using the magnetic rod, enabling the magnetic beads to penetrate through the magnetic rod sleeve, adding the magnetic beads into the soft test tube 5, uniformly mixing reagents in the soft test tube 5 at 65 ℃, and releasing RNA from a sample and adsorbing the RNA on the surface of the magnetic beads;
step three: and taking out the magnetic beads adsorbed with the RNA by using the magnetic rod again, removing redundant impurities from the magnetic beads through a washing solution, putting the magnetic beads into an eluent, and dissolving the RNA in the eluent at 70 ℃ so as to finish the extraction of the RNA.
In summary, as shown in fig. 1-3, in the method for rapidly extracting nucleic acid based on magnetic beads, when in use, firstly, in an idle or transportation state, the inner part of the well head 3 is inserted into the plug to seal the inner part of the flexible test tube 5, and the support plate 7 rotates to the inner part of the concave surface 2 based on the elastic rotation action of the torsion spring 6, at this time, the extension plates 11 are manually pulled out, at this time, the two extension plates 11 approach each other and are adsorbed to each other through the magnetic strip 12, which is beneficial to enhancing the sealing effect of the well head 3 to prevent the plug in the well head 3 from loosening;
the flexible test tubes 5 are flexible structures and can be folded, after the flexible test tubes 5 are folded, the size of the outer opening of the box panel 1 is matched with the size of the inner opening of the limiting plates 9, so that the box panel 1 and the folded flexible test tubes 5 can be placed between the limiting plates 9, and at the moment, the volume of a single deep hole plate can be greatly reduced, so that the storage capacity of a fixed storage space is increased in a variable manner, and the large-batch transportation or storage is facilitated;
when nucleic acid extraction is needed, the magnetic stripe 12 is separated firstly, the extension plate 11 is pushed into the supporting plate 7, then the supporting plate 7 is rotated manually, the bottom of the supporting plate 7 enters the limiting plate 9, the supporting plate 7 is limited in the limiting plate 9 based on the elastic rotation effect of the torsion spring 6, the box panel 1 is stably arranged on the bottom plate 8, meanwhile, the soft test tube 5 is reset to be straight, the bottom of the soft test tube 5 is located in the limiting groove 10 to improve the straight degree, the soft test tube 5 is prevented from being bent due to the flexibility, and then nucleic acid extraction operation can be carried out;
the method for quickly extracting the nucleic acid comprises the following specific steps:
the method comprises the following steps: adding 400ul of lysis solution into the soft test tube 5, then adding 200ul of throat swab sample into the soft test tube 5, wherein if scientific research grade RNA needs to be obtained, 10ul of nucleic acid protective solution needs to be added;
step two: inserting a magnetic rod sleeve into the soft test tube 5, sucking magnetic beads by using the magnetic rod, enabling the magnetic beads to penetrate through the magnetic rod sleeve, adding the magnetic beads into the soft test tube 5, uniformly mixing reagents in the soft test tube 5 at 65 ℃, and releasing RNA from a sample and adsorbing the RNA on the surface of the magnetic beads;
step three: and taking out the magnetic beads adsorbed with the RNA by using the magnetic rod again, removing redundant impurities from the magnetic beads through a washing solution, putting the magnetic beads into an eluent, and dissolving the RNA in the eluent at 70 ℃ so as to finish the extraction of the RNA.
The embodiments of the present invention have been presented for purposes of illustration and description, and are not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to practitioners skilled in this art. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.
Claims (10)
1. A nucleic acid rapid extraction method based on magnetic beads is characterized in that: the method adopts a nucleic acid extraction kit, the nucleic acid extraction kit comprises lysis solution, magnetic beads, washing solution, eluent, a nucleic acid protective agent, a deep hole plate and a magnetic rod sleeve, the deep hole plate comprises a box panel (1), a concave surface (2), a hole head (3), a threaded joint (4), a soft test tube (5), a torsion spring (6), a supporting plate (7), a bottom plate (8), a limiting plate (9), a limiting groove (10), an extension plate (11) and a magnetic strip (12), the surface of the box panel (1) is provided with a concave surface (2), and the bottom of the box panel (1) is provided with hole heads (3) in an array manner, the outer wall of each hole head (3) is connected with a screwed joint (4) in a threaded manner, and the bottom of the screwed joint (4) is fixed with a soft test tube (5), torsion springs (6) are arranged on two sides of the box panel (1), the outer wall of the torsion spring (6) is connected with a supporting plate (7), a bottom plate (8) is arranged below the box panel (1) in parallel, and the two sides of the surface of the bottom plate (8) are provided with limit plates (9), the surface of the bottom plate (8) is also provided with a limit groove (10), and the limiting groove (10) is positioned between the limiting plates (9), the inner part of the supporting plate (7) is connected with an extending plate (11) in a sliding manner, and the end part of the extending plate (11) is fixed with a magnetic strip (12).
2. The method for rapidly extracting nucleic acid based on magnetic beads according to claim 1, wherein the method comprises the following steps: soft test tube (5) are the one-to-one with spacing groove (10) and correspond the setting, and the bottom of soft test tube (5) is located inside spacing groove (10).
3. The method for rapidly extracting nucleic acid based on magnetic beads according to claim 1, wherein the method comprises the following steps: the supporting plate (7) is elastically connected with the box panel (1) through the torsion spring (6), and the side face of the supporting plate (7) is attached to the inner side face of the limiting plate (9).
4. The method for rapidly extracting nucleic acid based on magnetic beads according to claim 1, wherein the method comprises the following steps: the size of the outer opening of the box panel (1) is matched with the size of the inner opening of the limiting plate (9), and the box panel (1) forms a concave structure through the concave surface (2).
5. The method for rapidly extracting nucleic acid based on magnetic beads according to claim 1, wherein the method comprises the following steps: the lysis solution comprises the following components in percentage by mass and volume:
0.1177% Tris,1.753% NaCl,0.1862% EDTA.2Na, 5% PEG6000, 41.36% guanidinium isothiocyanate and 5% Triton X-100 by volume, pH 8.5, wherein Tris is named Tris with molecular formula (HOCH) in Chinese 2 )3CNH 2 NaCl is named as sodium chloride in Chinese, EDTA-2 Na is named as disodium ethylenediamine tetraacetate in Chinese, and PEG6000 is named as polyethylene glycol 6000 in Chinese, and the molecular formula is HO (CH) 2 CH 2 O) nH, wherein n represents the average number of oxyethylene groups, triton x-100, chinese name polyethylene glycol octyl phenyl ether, which is a nonionic surfactant.
6. The method for rapidly extracting nucleic acid based on magnetic beads according to claim 1, wherein the method comprises the following steps: the washing liquid is ethanol with the concentration of 80%.
7. The method for rapidly extracting nucleic acid based on magnetic beads according to claim 1, wherein the method comprises the following steps: and the eluent is ultrapure water treated by DEPC.
8. A method for rapidly extracting nucleic acid based on magnetic beads according to claim 1, wherein the method comprises the following steps: the nucleic acid protective agent is tris (2-carboxyethyl) phosphine hydrochloride with the mass volume ratio of 14.34%.
9. The method for rapidly extracting nucleic acid based on magnetic beads according to claim 1, wherein the method comprises the following steps: the magnetic rod sleeve is used for being inserted into the soft test tube (5).
10. A method for rapid extraction of nucleic acid based on magnetic beads according to any of claims 1 to 9, wherein: the method for quickly extracting the nucleic acid comprises the following specific steps:
the method comprises the following steps: adding 400ul of lysis solution into the soft test tube (5), then adding 200ul of throat swab sample into the soft test tube (5), wherein if scientific research grade RNA needs to be obtained, 10ul of nucleic acid protective solution needs to be added;
step two: the magnetic rod sleeve is inserted into the soft test tube (5), then the magnetic rod is used for absorbing magnetic beads, the magnetic beads penetrate through the magnetic rod sleeve and are added into the soft test tube (5), then reagents in the soft test tube (5) are uniformly mixed under the condition of 65 ℃, and at the moment, RNA can be released from a sample and adsorbed on the surface of the magnetic beads;
step three: and taking out the magnetic beads adsorbed with the RNA by using the magnetic rod again, removing redundant impurities from the magnetic beads through a washing solution, putting the magnetic beads into an eluent, and dissolving the RNA in the eluent at 70 ℃ so as to finish the extraction of the RNA.
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| CN114655548A (en) * | 2022-02-08 | 2022-06-24 | 河南泰宇高分医疗器械有限公司 | DNA extraction kit based on paramagnetic particle method |
| CN217324042U (en) * | 2022-05-11 | 2022-08-30 | 江苏昱安生物科技有限公司 | Nucleic acid extraction kit capable of efficiently splitting virus |
| CN217409805U (en) * | 2022-06-13 | 2022-09-13 | 中国农业科学院油料作物研究所 | Portable foldable purification column extraction element |
| CN115140362A (en) * | 2022-09-05 | 2022-10-04 | 融越医疗科技(江苏)有限公司 | Nucleic acid detection test tube packaging device and using method thereof |
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