CN114657054A - Nucleic acid extraction centrifugal column tube, nucleic acid extraction device and nucleic acid extraction method - Google Patents
Nucleic acid extraction centrifugal column tube, nucleic acid extraction device and nucleic acid extraction method Download PDFInfo
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- CN114657054A CN114657054A CN202210295109.7A CN202210295109A CN114657054A CN 114657054 A CN114657054 A CN 114657054A CN 202210295109 A CN202210295109 A CN 202210295109A CN 114657054 A CN114657054 A CN 114657054A
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- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 135
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 135
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 135
- 238000000605 extraction Methods 0.000 title claims abstract description 64
- 238000007789 sealing Methods 0.000 claims abstract description 224
- 239000007788 liquid Substances 0.000 claims abstract description 193
- 238000005336 cracking Methods 0.000 claims abstract description 50
- 239000003480 eluent Substances 0.000 claims abstract description 46
- 230000009089 cytolysis Effects 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 28
- 239000002699 waste material Substances 0.000 claims abstract description 25
- 239000012528 membrane Substances 0.000 claims description 77
- 239000011148 porous material Substances 0.000 claims description 48
- 239000006166 lysate Substances 0.000 claims description 40
- 238000001471 micro-filtration Methods 0.000 claims description 22
- 238000001179 sorption measurement Methods 0.000 claims description 22
- 239000003153 chemical reaction reagent Substances 0.000 claims description 17
- 238000005119 centrifugation Methods 0.000 claims description 13
- 239000012535 impurity Substances 0.000 claims description 8
- 238000011045 prefiltration Methods 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 abstract description 34
- 239000012487 rinsing solution Substances 0.000 abstract description 8
- 238000010828 elution Methods 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 239000004743 Polypropylene Substances 0.000 description 2
- -1 Polytetrafluoroethylene Polymers 0.000 description 2
- 239000004809 Teflon Substances 0.000 description 2
- 229920006362 Teflon® Polymers 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 2
- 239000004810 polytetrafluoroethylene Substances 0.000 description 2
- 238000005096 rolling process Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 229920012266 Poly(ether sulfone) PES Polymers 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000036316 preload Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
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Abstract
The invention discloses a nucleic acid extraction centrifugal column tube, a nucleic acid extraction device and a nucleic acid extraction method, wherein the centrifugal column tube comprises: centrifuging the tube; the bottom of the cracking tube is provided with a spike structure; the pre-sealing liquid assembly comprises a plurality of pre-sealing liquid bags; a centrifugal column; the self-switching channel block is internally provided with a self-switching channel, and the self-switching channel is provided with a first outlet and a second outlet which can be switched on/off; and a nucleic acid sample bottle detachably connected with the first outlet and a waste liquid bottle detachably connected with the second outlet. According to the centrifugal column tube for extracting nucleic acid and the method for extracting nucleic acid based on the same, provided by the invention, the lysis solution, the binding solution, the rinsing solution and the eluent which are required to be used in the nucleic acid extraction process are pre-packaged in the centrifugal column tube, the addition of the lysis solution is realized by the linkage of the uncovering work during sample addition, the binding solution, the rinsing solution and the eluent can be sequentially and correspondingly released in each operation process by controlling the centrifugal force, and the rapid extraction of nucleic acid can be realized.
Description
Technical Field
The invention relates to the field of nucleic acid detection, in particular to a nucleic acid extraction centrifugal column tube, a nucleic acid extraction device and a nucleic acid extraction method.
Background
A centrifugal column method for extracting nucleic acid is one of the mainstream nucleic acid extraction methods at present, and the basic principle is that a lysate is utilized to promote cell disruption, so that nucleic acid in cells is released, then the released nucleic acid is specifically adsorbed on a nucleic acid adsorption membrane (such as a silica gel membrane) of a centrifugal column, the membrane only has strong affinity and adsorption force on the nucleic acid, and basically does not adsorb other biochemical components such as protein, polysaccharide and lipid, so that the nucleic acid can be thrown out of the column during centrifugation, the centrifugal column is rinsed by rinsing liquid to remove impurities, and finally the nucleic acid adsorbed on the nucleic acid adsorption membrane is eluted by the rinsing liquid, so that purified nucleic acid can be obtained for subsequent nucleic acid amplification detection.
Because the nucleic acid extraction process is complicated, time-consuming and labor-consuming, some automatic or semi-automatic nucleic acid extraction equipment and methods appear, and a manipulator is generally adopted to realize the addition of the lysis solution, the binding solution, the rinsing solution and the eluent as required in the nucleic acid extraction process. For example, a method for performing full-automatic nucleic acid extraction on a solid sample disclosed in patent CN112375756A, and a full-automatic nucleic acid purification instrument disclosed in patent CN109628441A all perform addition of reagents at each stage by using a mechanical arm instead of manual work, and although automation can be achieved, the mechanical arm needs to go back and forth to each station for many times, a control procedure of the mechanical arm is cumbersome, and the mechanical arm greatly increases the size and the space occupation of the instrument; in addition, the centrifugal operation needs to be stopped in the reagent adding process, so that a large amount of time is wasted; the operation steps are further increased by replacing the centrifuge tubes before elution (the waste liquid in the operations of lysis solution, binding solution and rinsing solution all enter the centrifuge tubes for collection, and the centrifuge tubes need to be replaced to collect the final nucleic acid extract when elution is finally performed). Therefore, although the existing solutions can realize automatic extraction, there still exist many defects, and there is a need to provide a more reliable solution.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a centrifugal column tube for nucleic acid extraction, which comprises:
centrifuging the tube;
the cracking tube can be inserted in the centrifugal tube in a sliding mode along the Z direction, and the bottom of the cracking tube is provided with a spine structure;
the pre-sealing liquid assembly is arranged in the centrifuge tube and is positioned below the sharp structure, the pre-sealing liquid assembly comprises a support connected with the inner wall of the centrifuge tube and a plurality of pre-sealing liquid bags arranged at intervals along the Z direction, and the plurality of pre-sealing liquid bags can be punctured by the sharp structure in the process that the cracking tube slides downwards along the Z direction so as to enable the internally pre-filled reagent liquid to flow out;
the centrifugal column is connected in the centrifugal tube and is positioned below the pre-sealing liquid assembly and used for adsorbing nucleic acid;
the self-switching channel block is connected to the bottom of the centrifugal tube, a self-switching channel communicated with the space below the centrifugal column is arranged in the self-switching channel block, and the self-switching channel is provided with a first outlet and a second outlet which can be switched on/off;
and a nucleic acid sample bottle detachably connected with the first outlet and a waste liquid bottle detachably connected with the second outlet.
Preferably, a sealing cover is rotatably connected to a pipe orifice of the cracking pipe, a hanging plate is connected to the inner wall of the cracking pipe, a cracking liquid pre-sealing bag is connected to the bottom surface of the hanging plate, at least one easy-to-tear piece is arranged on the cracking liquid pre-sealing bag, a pull rope is connected to the easy-to-tear piece, and the tail end of the pull rope is connected with the bottom of the sealing cover; when the sealing cover is completely opened, the pull rope pulls the easy-to-tear piece open to enable the cracking liquid pre-sealed bag to break and the cracking liquid inside the bag flows out.
Preferably, a first pore plate is connected to an outlet at the bottom of the cracking tube, a microporous filter membrane is arranged at the upper part of the first pore plate, a pre-filter membrane is arranged above the microporous filter membrane, and the spine structure is arranged at the bottom of the first pore plate.
Preferably, the periphery of the upper end of the centrifuge tube is provided with a first edge, the periphery of the upper end of the cracking tube is provided with a second edge, and at least two first springs are connected between the first edge and the second edge at intervals;
the plurality of pre-sealing liquid bags comprise a binding liquid pre-sealing bag, a rinsing liquid pre-sealing bag and an eluent pre-sealing bag which are sequentially arranged from top to bottom along the Z direction;
when the centrifugal tube rotates centrifugally, the first spring is compressed under the action of centrifugal force, so that the cracking tube can slide downwards along the Z direction, and the spine structure moves downwards;
when the centrifugal rotating speed R of the centrifugal tube meets the following conditions: r is1≤R＜R2The spike structure punctures the binding liquid pre-sealing bag and does not puncture the rinsing liquid pre-sealing bag; when less than R2≤R＜R3When the washing liquid is used, the spine structure penetrates through the binding liquid pre-sealed bag and pierces the rinsing liquid pre-sealed bag without piercing the eluent pre-sealed bag; when R is not less than R3When the eluent is used, the spine structure sequentially penetrates through the binding liquid pre-sealing bag and the rinsing liquid pre-sealing bag and pierces the eluent pre-sealing bag;
wherein R is1＜R2＜R3。
Preferably, the support comprises a second porous plate connected with the inner wall of the centrifuge tube, a first supporting pore plate arranged on the second porous plate, a second supporting pore plate arranged on the first supporting pore plate, and a third supporting pore plate arranged on the second supporting pore plate, and gaps are reserved among the first supporting pore plate, the second supporting pore plate and the third supporting pore plate along the Z direction;
the combination liquid pre-sealing bag, the rinsing liquid pre-sealing bag and the eluent pre-sealing bag are all in ring shapes, the combination liquid pre-sealing bag is connected to the third supporting pore plate, the rinsing liquid pre-sealing bag is connected to the second supporting pore plate, and the eluent pre-sealing bag is connected to the first supporting pore plate;
all seted up a plurality of through-holes on first support orifice plate, second support orifice plate and the third support orifice plate, just the position and the size setting of through-hole satisfy: when the spine structure punctures the binding liquid pre-sealing bag, the rinsing liquid pre-sealing bag and the eluent pre-sealing bag, the spine structure can penetrate through the through hole at the corresponding position on the corresponding supporting pore plate;
the surface of the second perforated plate is also provided with a collecting groove.
Preferably, the outer wall of the cracking tube is provided with a guide block, the inner wall of the centrifuge tube is provided with a guide groove matched with the guide block along the Z direction, and the guide block is slidably arranged in the guide groove;
a nucleic acid adsorption membrane is arranged in the centrifugal column, and a conical outlet is formed in the bottom of the centrifugal column.
Preferably, the self-switching channel comprises a switching cavity positioned in the middle, a plurality of liquid inlet holes for communicating the switching cavity with a space below the centrifugal column, a first liquid discharge channel for communicating the switching cavity with the first outlet, and a second liquid discharge channel for communicating the switching cavity with the second outlet;
a first channel port is formed at the joint of the first liquid discharge channel and the switching cavity, and a second channel port is formed at the joint of the second liquid discharge channel and the switching cavity;
the first liquid discharge channel and the second liquid discharge channel are obliquely arranged, so that the first channel opening is closer to the central axis of the centrifugal tube than the first outlet, and the second channel opening is closer to the central axis of the centrifugal tube than the second outlet;
the switching cavity is internally provided with a second spring along the X direction, one end of the second spring is connected with the inner wall of the switching cavity, the other end of the second spring is connected with a sealing block which can slide along the X direction in the switching cavity, and the sealing block is used for sealing the first passage port or the second passage port.
Preferably, the centrifuge tube is rotated in the first direction or the second direction but at a speed less than R 'while the tube is stationary'3When the first channel opening is closed, the second channel opening is opened;
when the centrifugal tube rotates towards the second direction and the rotating speed is not less than R3When the first spring is stretched, the sealing block is positioned above the first passage opening and completely covers the first passage opening, the first passage opening is opened, and the second passage opening is closed;
wherein, R is1＜R2＜R'3＜R3。
Preferably, a top column is further arranged above the sealing block along the Z direction, and the top column comprises a hollow column connected to the sealing block, a third spring connected to the inside of the hollow column, and a top ball connected to the upper end of the third spring and movably arranged in the hollow column;
the knob is in contact with the upper inner wall of the switching cavity, and the liquid inlet hole above the switching cavity is kept away from the movement track of the knob.
The invention also provides a nucleic acid extraction device which comprises the centrifugal column tube.
The invention also provides a nucleic acid extraction method, which adopts the centrifugal column tube or the nucleic acid extraction device to extract nucleic acid, and comprises the following steps:
s1, completely opening the sealing cover, pulling the easy-to-tear piece on the lysate pre-sealing bag by the pull rope, so that the lysate in the lysate pre-sealing bag flows out and enters the lysis tube, and simultaneously, a certain volume of sample is added into the lysis tube, and the sealing cover is covered;
s2, placing the centrifugal column tube into a centrifuge, fixing the centrifugal column tube on a centrifugal station of the centrifuge through the centrifugal tube, and controlling the centrifuge to rotate forward and backward alternately and to rotate at a rotating speed R less than R0Working in a manner to perform sample lysis;
s3, controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R satisfies the following conditions: r0≤R＜R1So that the lysate in the lysate passes through the microfiltration membrane and enters a centrifugal column below the microfiltration membrane, and solid impurities are intercepted by the prefiltration membrane and the microfiltration membrane;
s4, controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R satisfies the following conditions: r1≤R＜R2The spine structure punctures the binding solution pre-sealing bag, the internal binding solution flows into a centrifugal column below to be mixed with the lysis solution, and the nucleic acid is adsorbed by a nucleic acid adsorption membrane in the centrifugal column;
s5, controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R meets the following requirements: r2≤R＜R'3The spine structure is made to pierce the rinsing liquid pre-sealing bag, the rinsing liquid inside flows out to rinse the centrifugal column, and then the rinsing liquid is discharged from a conical outlet below the centrifugal column;
in the processes of steps S1-S5, the sealing block is always located above the first channel port and completely covers the first channel port, the first channel port is closed, the second channel port is opened, so that the first outlet is always closed, the second outlet is opened, and all the waste liquid discharged from the bottom of the centrifuge tube enters the waste liquid bottle for collection;
s6, controlling the centrifugal machine to rotate reversely, wherein the rotating speed R meets the following requirements: r is not less than R3So that the spine structure punctures the eluent pre-sealing bag, the internal eluent flows out to elute the centrifugal column, and the nucleic acid in the centrifugal column is separated from the nucleic acid adsorption membrane; and simultaneously, the sealing block moves to the position above the second channel opening and completely covers the second channel opening, the first channel opening is opened, the second channel opening is closed, and the eluted nucleic acid and the eluent flow into the nucleic acid sample bottle together for collection to obtain the nucleic acid extracting solution.
The invention has the beneficial effects that:
the invention provides a nucleic acid extraction centrifugal column tube and a nucleic acid extraction method based on the same, which are characterized in that a lysis solution, a binding solution, a rinsing solution and an eluent which are required to be used in a nucleic acid extraction process are pre-packaged in the centrifugal column tube, the addition of the lysis solution is realized by the linkage of uncapping during sample addition, the binding solution, the rinsing solution and the eluent can be sequentially and correspondingly released in each operation process by controlling the centrifugal force, and the automatic function of the extraction process can be realized; meanwhile, the defects of complex control, large equipment volume, incapability of effectively shortening the time and the like caused by the adoption of a manipulator for automatic reagent adding operation in the existing scheme are overcome, the extraction flow can be greatly simplified, the reagent is added without stopping a centrifugal machine in the extraction process, the whole operation process is smoother, and the nucleic acid extraction time is shortened;
according to the invention, through the matching design of the self-switching channel and structures such as a pre-sealing liquid assembly, a centrifugal tube and a cracking tube, a second channel flowing to a waste liquid bottle can be conducted in the cracking, combining and rinsing processes of nucleic acid extraction, so that waste liquid is collected through the waste liquid bottle; and the first channel which flows to the nucleic acid sample bottle is conducted in the elution process of nucleic acid extraction, and the required nucleic acid extracting solution is collected, so that a liquid storage bottle for collecting reagents is not required to be replaced in the nucleic acid extraction process, and the nucleic acid extraction step is further simplified.
Drawings
FIG. 1 is a schematic structural view of a nucleic acid extraction centrifugation column tube according to the present invention;
FIG. 2 is a schematic structural view of a cracking tube of the present invention;
FIG. 3 is a schematic structural diagram of a pre-sealing liquid assembly of the present invention;
FIG. 4 is a schematic top view of the pre-sealing liquid assembly of the present invention;
FIG. 5 is a schematic top view of a spike structure according to the present invention;
FIG. 6 is a diagram illustrating a self-switching channel block according to an embodiment of the present invention;
FIG. 7 is a schematic structural view showing a state in which a sealing cap is opened in a nucleic acid extraction centrifugation column according to the present invention;
FIG. 8 is a schematic structural view of the puncture structure of the centrifugal column tube for nucleic acid isolation according to the present invention puncturing the binding liquid pre-sealing bag;
FIG. 9 is a schematic structural diagram of a nucleic acid extraction centrifugation column tube according to the present invention, in which a spike structure pierces an eluent pre-sealing bag;
FIG. 10 is a schematic diagram of a disassembled centrifuge tube of a nucleic acid extraction centrifuge column according to an embodiment of the present invention;
fig. 11 is a schematic structural diagram of a self-switching channel block in another embodiment of the present invention.
Description of reference numerals:
1, centrifuging a tube; 10 — a first edge; 11-a first spring; 12-a guide groove; 13-upper half of the tube; 14-lower half pipe; 15-a threaded hole; 16-a connecting head;
2-cracking tube; 20-spike structure; 21-sealing cover; 22-hanging the board; 23-pre-sealing a bag with a lysate; 230-easy-to-tear pieces; 231-a pull rope; 24-a first orifice plate; 25-microporous filter membrane; 26-pre-filtration membrane; 27 — second edge; 28-a guide block; 29-hanging ring;
3-pre-sealing liquid component; 30-a bracket; 31-pre-sealing a bag with binding liquid; 32-pre-sealing the bag by using rinsing liquid; 33-pre-sealing the bag with the eluent; 300-a second perforated plate; 301 — a first support aperture plate; 302 — a second support aperture plate; 303 — a third supporting aperture plate; 304-a through hole; 305-a collection recess;
4-centrifugal column; 40-nucleic acid adsorption membrane; 41-a conical outlet;
5-self-switching channel block; 50-self-switching channel; 500-liquid inlet hole; 501, switching a cavity; 502 — first passage port; 503 — second access; 504-first drain channel; 505 — a second drainage channel; 506 — a first outlet; 507 — a second outlet; 51-a second spring; 52-a sealing block; 53-top post; 530-hollow column; 531 — third spring; 532-heading;
6-nucleic acid sample bottle; 7-waste liquid bottle.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
Referring to fig. 1 to 7, a nucleic acid rapid extraction centrifugation column of the present embodiment includes:
centrifuging a tube 1;
the cracking tube 2 can be inserted into the centrifuge tube 1 in a sliding manner along the Z direction, and the bottom of the cracking tube 2 is provided with a spine structure 20;
the pre-sealing liquid assembly 3 is arranged in the centrifuge tube 1 and is positioned below the prick structure 20, the pre-sealing liquid assembly 3 comprises a support 30 connected with the inner wall of the centrifuge tube 1 and a plurality of pre-sealing liquid bags arranged at intervals along the Z direction, and the plurality of pre-sealing liquid bags can be pricked by the prick structure 20 in the process that the cracking tube 2 slides downwards along the Z direction so as to enable the internally pre-filled reagent liquid to flow out;
the centrifugal column 4 is connected in the centrifuge tube 1 and is positioned below the pre-sealing liquid component 3 and used for adsorbing nucleic acid;
the self-switching channel block 5 is connected to the bottom of the centrifugal tube 1, a self-switching channel 50 communicated with the space below the centrifugal column 4 is arranged in the self-switching channel block 5, and the self-switching channel 50 is provided with a first outlet 506 and a second outlet 507 which can be switched on/off;
and a nucleic acid sample bottle 6 detachably connected to the first outlet 506 and a waste liquid bottle 7 detachably connected to the second outlet 507.
In this embodiment, a sealing cover 21 is rotatably connected to a pipe orifice of the cracking pipe 2, a hanging plate 22 is connected to the inner wall of the cracking pipe 2, a cracking liquid pre-sealing bag 23 is connected to the bottom surface of the hanging plate 22, at least one easy-to-tear piece 230 is arranged on the cracking liquid pre-sealing bag 23, a pull rope 231 is connected to the easy-to-tear piece 230, and the tail end of the pull rope 231 is connected to the bottom of the sealing cover 21; when the sealing lid 21 is completely opened, the pull cord 231 pulls the tear tab 230 open to break the lysate prepack 23 and allow the lysate to flow out of the lysate inside.
In a preferred embodiment, a hanging ring 29 for connecting the pulling rope 231 is arranged at the bottom of the sealing cover 21, when the cracking tube 2 is produced and packaged, after the cracking liquid pre-sealing bag 23 in which the cracking liquid is packaged is arranged on the hanging plate 22, the sealing cover 21 is half-opened, so that the tail end of the pulling rope 231 can be fixedly connected to the hanging ring 29, and then the sealing cover 21 is buckled and sealed, as shown in fig. 1. The length of the pull rope 231 is suitable, and the length of the pull rope can be approximately equal to the linear distance between the hanging ring 29 and the easy-tear piece 230 closest to the hanging ring 29 when the sealing cover 21 is half-opened, so that all the easy-tear pieces 230 cannot be torn off when the sealing cover 21 is half-opened, and all the easy-tear pieces 230 can be torn off when the sealing cover 21 is completely opened, so that the lysate in the lysate pre-sealed bag 23 is released, as shown in fig. 7; through the linkage setting of sealed lid 21 and easy tear piece 230 for when this centrifugation column pipe used, can add the lysate by automatic when opening sealed lid 21 and adding the sample completely, can simplify the step of a adding the lysate. After the sample is added, the sealing cover 21 is buckled to wait for the next operation.
Referring to fig. 2, in this embodiment, a first pore plate 24 is connected to the bottom outlet of the cracking tube 2, a microfiltration membrane 25 is disposed on the upper portion of the first pore plate 24, a pre-filtration membrane 26 is disposed above the microfiltration membrane 25, and the spike structure 20 is disposed at the bottom of the first pore plate 24.
The pre-filtering membrane 26 is arranged above and is used for filtering out some large-particle impurities generated after the sample is cracked, and the micro-porous filtering membrane 25 below can be prevented from being blocked. The microfiltration membrane 25 is a hydrophobic membrane having a micropore array thereon, so that when no external force is applied, liquid above the microfiltration membrane 25 cannot or cannot substantially flow into the space below through the microfiltration membrane 25, and when a certain external force (such as centrifugal force) is applied, the liquid above the microfiltration membrane 25 can flow into the space below through the microfiltration membrane 25, and most of solid impurities are retained above the microfiltration membrane 25, thereby forming a space above the microfiltration membrane 25 for fully mixing a sample and a lysate. For example, in an alternative embodiment, the microfiltration membrane 25 can be a Polytetrafluoroethylene (PTFE) membrane, a polypropylene (PP) membrane, or a Polyethersulfone (PES) membrane, and the like, and the size of the micropores in the array of micropores is 0.05um to 100 um. When it is understood that the smaller the pore size is, the more external force is required for the liquid above the microporous filter membrane 25 to pass through the microporous filter membrane 25, and the larger the centrifugal rotation speed is required to be provided corresponding to the centrifugal force as the external force, so that by setting the appropriate pore size, the centrifugal rotation speed (for example, R) not less than a certain value can be satisfied (for example, R is0) The lower liquid may pass through the microfiltration membrane 25, whereas below this centrifugation speed the liquid cannot pass or substantially cannot pass through the microfiltration membrane 25.
In this embodiment, a nucleic acid adsorption membrane 40 is disposed in the centrifugal column 4, and a conical outlet 41 is disposed at the bottom of the centrifugal column 4. The same nucleic acid adsorbing membrane 40 may be made of a material having affinity or adsorbing ability for nucleic acid and having an array of micropores with a size of 0.02um to 50um, the size of the micropores being set so as to rotate at a centrifugal rotation speed (for example, R) of not less than a certain value2) The lower liquid may pass through the microfiltration membrane 25, whereas below this centrifugation speed the liquid cannot pass or substantially cannot pass through the microfiltration membrane 25.
Referring to fig. 3, in the embodiment, a first edge 10 is arranged on the outer periphery of the upper end of the centrifuge tube 1, a second edge 27 is arranged on the outer periphery of the upper end of the lysis tube 2, and at least two first springs 11 are connected between the first edge 10 and the second edge 27 at intervals;
the plurality of pre-sealing liquid bags comprise a binding liquid pre-sealing bag 31, a rinsing liquid pre-sealing bag 32 and an eluent pre-sealing bag 33 which are sequentially arranged from top to bottom along the Z direction (namely the axial direction of the centrifuge tube 1). Specifically, the rack 30 includes a second porous plate 300 connected to the inner wall of the centrifuge tube 1, a first supporting pore plate 301 disposed on the second porous plate 300, a second supporting pore plate 302 disposed on the first supporting pore plate 301, and a third supporting pore plate 303 disposed on the second supporting pore plate 302, and a gap is left between the first supporting pore plate 301, the second supporting pore plate 302, and the third supporting pore plate 303 along the Z direction;
the binding liquid pre-sealing bag 31, the rinsing liquid pre-sealing bag 32 and the eluent pre-sealing bag 33 are all in a ring shape, the binding liquid pre-sealing bag 31 is connected to the third supporting pore plate 303, the rinsing liquid pre-sealing bag 32 is connected to the second supporting pore plate 302, and the eluent pre-sealing bag 33 is connected to the first supporting pore plate 301;
a plurality of through holes 304 have all been seted up on first support orifice plate 301, second support orifice plate 302 and the third support orifice plate 303, and the position and the size setting of through hole 304 satisfy: when the spike structure 20 punctures the bonding liquid pre-sealing bag 31, the rinsing liquid pre-sealing bag 32, and the eluent pre-sealing bag 33, the spike structure 20 can pass through the through hole 304 at the corresponding position on the corresponding support pore plate, that is, on the track of the spike structure 20 moving along the axial direction, the through holes 304 for each spike structure 20 to pass through are formed on the three layers of support pore plates, so as to ensure that the spike structure 20 can smoothly puncture the bonding liquid pre-sealing bag 31, the rinsing liquid pre-sealing bag 32, and the eluent pre-sealing bag 33, referring to fig. 9.
Referring to fig. 3-5, in a preferred embodiment, the binding liquid pre-sealing bag 31 is located at the inner circle and has the highest position in the Z direction for the prick structure 20 to pierce first when moving, and the rinsing liquid pre-sealing bag 32 and the eluent pre-sealing bag 33 are located at the outer circle of the binding liquid pre-sealing bag 31 and have the sequentially lower positions in the Z direction for the prick structure 20 to pierce sequentially; correspondingly, the spine structure 20 comprises an inner circle and an outer circle, each circle is provided with a plurality of spines at intervals, and the heights of the positions below all the spines are the same; when the spike structure 20 moves downward, the binding liquid pre-sealing bag 31 is punctured by the inner ring spike, and then the rinsing liquid pre-sealing bag 32 and the eluent pre-sealing bag 33 are punctured by the inner ring spike and the outer ring spike together.
Wherein, the middle part of the bonding liquid pre-sealing bag 31 is provided with a cavity, so that the liquid flowing down from the upper part can not be influenced to pass through the pre-sealing liquid component 3. The surface of the second porous plate 300 is further provided with a collection groove 305 which collects the liquid flowing down from above and then uniformly discharges the liquid downward through a plurality of liquid discharge holes of the inner uniform step, thereby facilitating the sufficient and uniform contact of each reagent with the nucleic acid adsorbing membrane 40 of the lower centrifugal column 4.
When the centrifugal tube 1 rotates centrifugally, the first spring 11 is compressed under the action of centrifugal force, so that the lysis tube 2 can slide downwards along the Z direction, and the spike structure 20 can move downwards.
When the centrifugal column tube is arranged on a centrifugal machine, the centrifugal tube 1 passing through the centrifugal column tube is fixed on a centrifugal station of the centrifugal machine, and the cracking tube 2 is movably inserted in the centrifugal tube 1, so when the centrifugal column tube is wholly centrifugally rotated, the centrifugal tube 1 is fixed along the axial direction, the cracking tube 2 can move outwards relative to the centrifugal tube 1 along the axial direction (the cracking tube 2 is inserted into the centrifugal tube 1 deeper, namely below in figure 1), so that the first spring 11 is compressed, the spine structure 20 at the bottom of the cracking tube 2 moves towards the direction close to the pre-sealing liquid component 3, the centrifugal rotating speed is larger, the compression amount of the first spring 11 is larger, the moving distance of the cracking tube 2 is larger, and therefore the binding liquid pre-sealing bags 31, the rinsing liquid pre-sealing bags 32 and the eluent pre-sealing bags 33 which are arranged along the axial direction of the centrifugal tube 1 can be sequentially punctured through the spine structure 20 at sequentially increasing centrifugal rotating speed, the method comprises the following specific steps:
when the centrifugal rotating speed R of the centrifugal tube 1 meets the following requirements: r is more than 0 and less than R1In the process, the cracking tube 2 moves to a certain extent, but the puncture structure 20 cannot move to the position contacting with the binding liquid pre-sealing bag 31 and cannot puncture the binding liquid pre-sealing bag 31; under the centrifugal speed, the lysate and the sample can be uniformly mixed by controlling the forward and reverse centrifugal rotation;
when the centrifugal rotating speed R of the centrifugal tube 1 meets the following requirements: r1≤R＜R2At this time, the spike structure 20 pierces the bonding liquid pre-sealing bag 31 without piercing the rinsing liquid pre-sealing bagEnvelope 32, at which point the bonding operation can be completed: DNA or RNA is efficiently adsorbed on the nucleic acid adsorption film 40; due to the design of the micropore array of the nucleic acid adsorption membrane 40 and the structural design of the conical outlet 41 of the centrifugal column 4, the liquid above the nucleic acid adsorption membrane 40 can not pass through the nucleic acid adsorption membrane 40 basically at the centrifugal rotation speed, so that a space for mixing the binding solution and the lysate to a certain degree can be formed in the centrifugal column 4;
when less than R2≤R＜R3In the meantime, the spike structure 20 penetrates the binding liquid pre-sealed bag 31 and pierces the rinsing liquid pre-sealed bag 32 without piercing the eluent pre-sealed bag 33; at this time, the centrifugal column 4 can be rinsed by the rinsing liquid to remove impurities, and the nucleic acid still remains on the nucleic acid adsorption membrane 40;
when R is not less than R3In this process, the spike structure 20 passes through the binding liquid pre-sealing bag 31, the rinsing liquid pre-sealing bag 32 and punctures the eluent pre-sealing bag 33 in sequence. At this time, nucleic acid can be collected by separating nucleic acid from the nucleic acid adsorbing membrane 40 by the elution action of the eluent, thereby completing nucleic acid extraction. Wherein R is1＜R2＜R3。
The reagents in the lysate pre-sealing bag 23, the binding solution pre-sealing bag 31, the rinsing solution pre-sealing bag 32 and the eluent pre-sealing bag 33 are prepared from the lysate, the binding solution, the rinsing solution and the eluent which are respectively used in each stage of nucleic acid extraction, and are respectively pre-sealed and arranged at corresponding positions in the centrifugal column tube, the amounts of the reagents are matched according to a preset certain sample addition amount, and the range of the sample addition amount can be marked on the outer wall of the centrifugal column tube, or the sample addition amount range can be explained in other manners. For example, in one embodiment, the amount of standard sample added to the sealing cap 21 of the spin column is in the range of 1-5mL, matching the pre-load of each reagent; therefore, when in use, only a certain amount of sample is required to be added according to requirements, and the rest of the reagent amount can be matched with the sample, and is not required to be added. Of course, the centrifugal column tube with a proper size is selected and each reagent amount matched with the centrifugal column tube is selected according to requirements, so that the larger or smaller sample adding amount can be matched.
Wherein a sufficient amount of rinse is packaged by the rinse pre-bag 32 such that a single rinse is sufficient. It will be appreciated that the number of layers of rinse solution pre-sealed bags 32 may be increased, with the corresponding addition of a centrifuge control step to puncture multiple layers of rinse solution pre-sealed bags 32, but this would add a control step and is therefore preferred.
In a preferred embodiment, the outer wall of the lysis tube 2 is provided with a guide block 28, the inner wall of the centrifuge tube 1 is provided with a guide groove 12 matching with the guide block 28 along the Z direction, and the guide block 28 is slidably arranged in the guide groove 12. In a further preferred embodiment, the guide slot 12 is a rectangular slot, and the guide block 28 is shaped to fit into the guide slot 12, so that the tube 2 is prevented from rotating relative to the tube 1 by the fit of the guide block 28 into the slot 12, while only the tube 2 is allowed to slide axially.
In the preferred embodiment, sealing elements such as sealing rings are arranged between the lysis tube 2 and the centrifuge tube 1 to realize sealing, so as to prevent liquid leakage.
In this embodiment, through the design and the cooperation of above-mentioned centrifuging tube 1, cracking tube 2, seal liquid subassembly 3 isotructures in advance, can realize the preservation of sealing in advance of all reagents in the nucleic acid extraction process, and in the schizolysis of nucleic acid extraction, combine, rinsing, elution process, by to centrifugal force control, correspond release lysate, bonding solution, rinsing liquid and eluant in proper order, can realize the full-automatic function of extraction process, and need not to carry out reagent interpolation through the manipulator that control is complicated, the size is big, with high costs like in the conventional automatic extraction scheme, can simplify the flow greatly, shorten nucleic acid extraction time.
Example 2
As a further improvement on the basis of embodiment 1, in this embodiment, the automatic distribution and collection function of the waste liquid and the nucleic acid liquid in the nucleic acid extraction process is realized through the self-switching channel 50, and the need of the collection tube replacement operation in the nucleic acid extraction process as in the conventional scheme can be avoided.
Specifically, referring to fig. 6, in the present embodiment, the self-switching channel 50 includes a switching cavity 501 located in the middle, a plurality of liquid inlet holes 500 communicating the switching cavity 501 with the space below the centrifugal column 4, a first liquid drainage channel 504 communicating the switching cavity 501 with a first outlet 506, and a second liquid drainage channel 505 communicating the switching cavity 501 with a second outlet 507;
a first channel opening 502 is formed at the joint of the first drainage channel 504 and the switching cavity 501, and a second channel opening 503 is formed at the joint of the second drainage channel 505 and the switching cavity 501;
the first drainage channel 504 and the second drainage channel 505 are both arranged obliquely so that the first passage opening 502 is closer to the central axis of the centrifuge tube 1 than the first outlet 506, and the second passage opening 503 is closer to the central axis of the centrifuge tube 1 than the second outlet 507; thereby bringing the first passage opening 502 and the second passage opening 503 closer to each other to reduce the distance required for the seal block 52 to move when switching the passage opening and closing.
A second spring 51 is arranged in the switching cavity 501 along the X direction, one end of the second spring 51 is connected with the inner wall of the switching cavity 501, the other end is connected with a sealing block 52 which can slide in the switching cavity 501 along the X direction, and the sealing block 52 is used for sealing the first passage opening 502 or the second passage opening 503.
While the centrifuge tube 1 is still rotating in the first direction or in the second direction but at a rotation speed less than R'3When the sealing block 52 is located above the first passage opening 502 and completely covers the first passage opening 502, the first passage opening 502 is closed, and the second passage opening 503 is opened;
when the centrifugal tube 1 rotates in the second direction and the rotating speed is not less than R3When the second spring 51 is stretched, the sealing block 52 is positioned above the second passage opening 503 and completely covers the second passage opening 503, the first passage opening 502 is opened, and the second passage opening 503 is closed;
wherein R is1＜R2＜R'3＜R3。
Referring to fig. 6 to 9, the automatic switching process of the sealing block 52 and the second spring 51 to open/close the first port 502 and the second port 503 at different centrifugal rotational speeds will be described in more detail. In the figure, the arrow indicates the centrifugal rotation direction, and O indicates the rotation center.
Referring to fig. 6, when the centrifugal column tube rotates centrifugally, the sealing block 52 moves left and right due to the centrifugal force, and the moving direction depends on the rotating direction, for example, if it is clockwiseWhen the needle is rotated, the sealing block 52 will move to the left, and when the needle is rotated counterclockwise, the sealing block 52 will move to the right; while the sealing block 52 is fixed by the second spring 51, the movement of the sealing block 52 depends on the combined centrifugal and spring action to which the sealing block 52 is subjected. When rotating clockwise, as shown in fig. 8, the sealing block 52 moves leftwards to compress the second spring 51, and the lengths of the second spring 51 and the sealing block 52 are designed such that when the second spring 51 is compressed to the shortest, the first passage opening 502 is still completely covered under the sealing block 52, so that when rotating clockwise, the first passage opening 502 is always closed. When rotating counterclockwise, the sealing block 52 moves rightwards to stretch the second spring 51, and the sealing block 52 gradually moves rightwards as the rotating speed increases, and when the rotating speed reaches R'3At this time, the seal block 52 starts to disengage from the first passage port 502, and the first passage port 502 starts to open; as the speed continues to increase, when increasing to R3At this time, the sealing block 52 is completely separated from the first opening 502 and completely covers the second opening 503, so that the first opening 502 is opened and the second opening 503 is closed, as shown in fig. 9. And due to the limitation of the right inner wall of the switching cavity 501 on the sealing block 52, when the rotating shaft rotates in the anticlockwise direction, the rotating speed R is more than R3Then, the sealing block 52 cannot move any further because it is blocked by the inner wall on the right side of the switching cavity 501, and the second opening 503 still remains closed.
In a preferred embodiment, the centrifuge tube 1 comprises an upper tube half 13 and a lower tube half 14 which are detachably connected. In a further preferred embodiment, a threaded hole 15 is formed in the bottom end of the upper half pipe 13, a connector 16 which is inserted into the threaded hole 15 in a threaded fit manner is formed at the upper end of the lower half pipe 14, and a sealing element such as a sealing ring can be arranged between the upper half pipe 13 and the lower half pipe 14 to ensure the sealing performance of connection and prevent liquid leakage. Further, the pre-sealing liquid component 3 and the centrifugal column 4 can be detachably connected in the centrifuge tube 1, so that the centrifuge column tube can be reused, the upper half tube 13 and the lower half tube 14 are disassembled, and then the internal pre-sealing liquid component 3, the centrifugal column 4, the cracking tube 2 and the like are cleaned and replaced by pre-sealing bags and/or the microporous filter membrane 25 and the nucleic acid filter membrane, so that the centrifuge column tube can be reused.
Referring to fig. 11, in a preferred embodiment, a top pillar 53 is further disposed above the sealing block 52 along the Z direction, and the top pillar 53 includes a hollow pillar 530 connected to the sealing block 52, a third spring 531 connected inside the hollow pillar 530, and a top ball 532 connected to an upper end of the third spring 531 and movably disposed inside the hollow pillar 530; wherein the top ball 532 has a diameter greater than the diameter of the top opening of the hollow cylinder 530 such that the top ball 532 can rotate without being disengaged from the inside of the hollow cylinder 530.
The knob 532 contacts with the upper inner wall of the switching cavity 501, and the liquid inlet hole 500 above the switching cavity 501 avoids the movement track of the knob 532, that is, the knob 532 does not have the liquid inlet hole 500 at the contact part with the upper inner wall of the switching cavity 501 during the movement process, so that the knob 532 is prevented from being clamped into the liquid inlet hole 500.
When the sealing block 52 moves, the jacking ball 532 is in rolling contact with the upper inner wall of the switching cavity 501, the ground of the sealing block 52 can be kept in closer contact with the bottom inner wall of the switching cavity 501 through jacking action, and meanwhile, the sealing block 52 can still move smoothly under the centrifugal action through rolling contact.
In a further preferred embodiment, the sealing layer (e.g. teflon layer) is disposed on the bottom surface of the sealing block 52, and the surface is flat and smooth, and the sealing layer (e.g. teflon layer) may also be disposed on the inner wall of the bottom of the switching cavity 501 to ensure that the surface is flat and smooth, so that the sealing block 52 can move smoothly and achieve a good sealing when moving to a proper position.
Through the matching design of the self-switching channel 50, the pre-sealing liquid assembly 3, the centrifuge tube 1, the cracking tube 2 and other structures, a second channel flowing to the waste liquid bottle 7 can be conducted in the cracking, combining and rinsing processes of nucleic acid extraction, so that waste liquid is collected through the waste liquid bottle 7; the first channel flowing to the nucleic acid sample bottle 6 is conducted in the elution process of nucleic acid extraction, and the required nucleic acid extracting solution is collected, so that the liquid storage bottle for collecting the reagent is not required to be replaced in the nucleic acid extraction process; this is further illustrated in example 3 below.
Wherein, each component such as the self-switching channel 50, the pre-sealing liquid component 3 and the like is made of anti-corrosion and anti-rust materials.
Example 3
This example provides a method for nucleic acid extraction using the spin column of example 2, comprising the steps of:
s1, opening the sealing cover 21 completely, pulling the easy-to-tear tab 230 on the lysate pre-sealing bag 23 by the pull rope 231, so that the lysate in the lysate pre-sealing bag 23 flows out into the lysis tube 2, as shown in fig. 7; simultaneously, adding a certain volume of sample into the cracking tube 2, and covering a sealing cover 21; at the moment, the centrifugal column tube can be manually shaken to preliminarily mix the lysate and the sample;
s2, placing the centrifugal column tube into a centrifuge, fixing the centrifugal column tube on a centrifugal station of the centrifuge through a centrifugal tube 1, and controlling the centrifuge to rotate forward and backward alternately and to rotate at a rotating speed R less than R0Working for a certain time in the mode of (1), and carrying out sample cracking; at the rotating speed, the liquid above the microporous filter membrane 25 can not pass through or can not pass through the microporous filter membrane 25 basically, so that the lysate and the sample can be well and uniformly mixed;
s3, controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R satisfies the following conditions: r is0≤R＜R1So that the lysate in the lysate passes through the microporous filter membrane 25 and enters the centrifugal column 4 below, and the solid impurities are intercepted by the pre-filtering membrane 26 and the microporous filter membrane 25;
wherein, at the rotation speed of S2 and S3, the spike structure 20 will move to a certain extent, but cannot move to the position contacting with the binding liquid pre-sealing bag 31, and will not puncture the binding liquid pre-sealing bag 31;
s4, controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R satisfies the following conditions: r is1≤R＜R2So that the prick structure 20 pierces the binding solution pre-sealing bag 31, referring to fig. 8, the internal binding solution flows into the centrifugal column 4 below to mix with the lysis solution, and the nucleic acid is adsorbed by the nucleic acid adsorption membrane 40 in the centrifugal column 4; and at this time, the liquid above the nucleic acid adsorbing membrane 40 cannot pass or substantially cannot pass;
s5, controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R satisfies the following conditions: r2≤R＜R'3So that the prick structure 20 pierces the rinsing liquid pre-sealing bag 32, the rinsing liquid inside flows out to rinse the centrifugal column 4, and the liquid above the nucleic acid adsorption membrane 40 can smoothly pass through at the rotating speed at the moment; so that the rinsing liquid can flow fromThe conical outlet 41 below the centrifugal column 4 is discharged;
in the processes of steps S1-S5, the sealing block 52 is always located above the first channel port 502 and completely covers the first channel port 502, the first channel port 502 is closed, the second channel port 503 is opened, so that the first outlet 506 is always closed, the second outlet 507 is opened, and all the lysis waste liquid, the binding waste liquid and the rinsing waste liquid discharged from the bottom of the centrifuge tube 1 enter the waste liquid bottle 7 for collection;
s6, controlling the centrifugal machine to rotate reversely, wherein the rotating speed R meets the following requirements: r is not less than R3So that the prick structure 20 pierces the pre-sealed bag 33 of the eluent, referring to fig. 9, the internal eluent flows out to elute the centrifugal column 4, so as to separate the nucleic acid in the centrifugal column 4 from the nucleic acid adsorption membrane 40;
at the same time, at the rotation speed, the sealing block 52 moves to the position above the second channel opening 503 and completely covers the second channel opening 503, the first channel opening 502 is opened, the second channel opening 503 is closed, and the eluted nucleic acid and the eluent flow into the nucleic acid sample bottle 6 together for collection, so as to obtain the nucleic acid extracting solution.
Example 4
As a further improvement on the basis of example 3, this example provides a nucleic acid extraction method comprising the steps of:
s1, completely opening the sealing cover 21, pulling open the easy-to-tear piece 230 on the lysate pre-sealing bag 23 through the pull rope 231, so that the lysate in the lysate pre-sealing bag 23 flows out and enters the lysis tube 2, meanwhile, 2mL of sample is added into the lysis tube 2, and the sealing cover 21 is well covered; at the moment, the centrifugal column tube can be manually shaken for 10s, so that the lysate and the sample are primarily mixed uniformly;
s2, placing the centrifugal column tube into a centrifugal machine, fixing the centrifugal column tube on a centrifugal station of the centrifugal machine through a centrifugal tube 1, controlling the centrifugal machine to work for 20S in a mode of forward and reverse alternate rotation and with the rotating speed R being 600R/min, and carrying out sample cracking; at the rotating speed, the liquid above the microporous filter membrane 25 can not pass through or can not pass through the microporous filter membrane 25 basically, so that the lysate and the sample can be well and uniformly mixed;
s3, controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R is 1200R/min, and working for 2min, so that lysate in a cracking product passes through the microporous filter membrane 25 and enters the centrifugal column 4 below, and solid impurities are intercepted by the pre-filtering membrane 26 and the microporous filter membrane 25;
wherein, at the rotation speed of S2 and S3, the piercing structure 20 will move to a certain extent but cannot move to the position contacting with the binding liquid pre-sealed bag 31, and will not pierce the binding liquid pre-sealed bag 31;
s4, controlling the centrifugal machine to rotate in the forward direction, enabling the spine structure 20 to pierce the binding solution pre-sealing bag 31 when the rotation speed R is 2500R/min and working for 4min, enabling the inside binding solution to flow into the centrifugal column 4 below to be mixed with the lysis solution, and adsorbing nucleic acid through the nucleic acid adsorption membrane 40 in the centrifugal column 4; and at this time, the liquid above the nucleic acid adsorbing membrane 40 cannot pass or substantially cannot pass;
s5, controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R is 5000R/min, and working for 5min to enable the prick structure 20 to prick the rinsing liquid pre-sealing bag 32, the rinsing liquid in the bag flows out to rinse the centrifugal column 4, and the liquid above the nucleic acid adsorption membrane 40 can smoothly pass through at the rotating speed at the moment; so that the rinsing liquid will be discharged from the conical outlet 41 below the centrifugal column 4;
in the processes of steps S1-S5, the sealing block 52 is always located above the first channel port 502 and completely covers the first channel port 502, the first channel port 502 is closed, the second channel port 503 is opened, so that the first outlet 506 is always closed, the second outlet 507 is opened, and all the lysis waste liquid, the binding waste liquid and the rinsing waste liquid discharged from the bottom of the centrifuge tube 1 enter the waste liquid bottle 7 for collection;
s6, controlling the centrifuge to rotate reversely, wherein the rotating speed R is 8500R/min, working for 5min, so that the spine structure 20 pierces the eluent pre-sealing bag 33, the eluent inside flows out to elute the centrifugal column 4, and the nucleic acid in the centrifugal column 4 is separated from the nucleic acid adsorption film 40;
at the same time, at the rotation speed, the sealing block 52 moves to the position above the second channel opening 503 and completely covers the second channel opening 503, the first channel opening 502 is opened, the second channel opening 503 is closed, and the eluted nucleic acid and the eluent flow into the nucleic acid sample bottle 6 together for collection, so as to obtain the nucleic acid extracting solution.
It should be understood that the specific parameters such as the rotation speed and the reaction time are selected and designed according to specific conditions, and the above rules are only required to be satisfied.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (10)
1. A nucleic acid extraction centrifugation column, comprising:
centrifuging the tube;
the cracking tube can be inserted in the centrifugal tube in a sliding mode along the Z direction, and the bottom of the cracking tube is provided with a spine structure;
the pre-sealing liquid assembly is arranged in the centrifugal tube and is positioned below the sharp-prick structure, the pre-sealing liquid assembly comprises a bracket connected with the inner wall of the centrifugal tube and a plurality of pre-sealing liquid bags arranged at intervals along the Z direction, and the plurality of pre-sealing liquid bags can be pierced by the sharp-prick structure in the process that the cracking tube slides downwards along the Z direction so as to enable the internally pre-installed reagent liquid to flow out;
the centrifugal column is connected in the centrifugal tube and is positioned below the pre-sealing liquid assembly and used for adsorbing nucleic acid;
the self-switching channel block is connected to the bottom of the centrifugal tube, a self-switching channel communicated with the space below the centrifugal column is arranged in the self-switching channel block, and the self-switching channel is provided with a first outlet and a second outlet which can be switched on/off;
and a nucleic acid sample bottle detachably connected with the first outlet and a waste liquid bottle detachably connected with the second outlet.
2. The nucleic acid extraction centrifugation column tube of claim 1, wherein a sealing cover is rotatably connected to a tube opening of the lysis tube, a hanging plate is connected to the inner wall of the lysis tube, a lysate pre-sealing bag is connected to the bottom surface of the hanging plate, at least one easy-to-tear piece is arranged on the lysate pre-sealing bag, a pull rope is connected to the easy-to-tear piece, and the tail end of the pull rope is connected to the bottom of the sealing cover; when the sealing cover is completely opened, the pull rope pulls the easy-to-tear piece open to enable the cracking liquid pre-sealing bag to be broken and the cracking liquid inside the bag to flow out.
3. The nucleic acid extraction centrifugation column tube of claim 2, wherein a first pore plate is connected to the bottom outlet of the lysis tube, a microfiltration membrane is disposed on the upper portion of the first pore plate, a pre-filtration membrane is disposed above the microfiltration membrane, and the spine structure is disposed at the bottom of the first pore plate.
4. The nucleic acid extraction centrifugal column tube of claim 3, wherein a first edge is arranged on the periphery of the upper end of the centrifugal tube, a second edge is arranged on the periphery of the upper end of the lysis tube, and at least two first springs are connected between the first edge and the second edge at intervals;
the plurality of pre-sealing liquid bags comprise a binding liquid pre-sealing bag, a rinsing liquid pre-sealing bag and an eluent pre-sealing bag which are sequentially arranged from top to bottom along the Z direction;
when the centrifugal tube rotates centrifugally, the first spring is compressed under the action of centrifugal force, so that the cracking tube can slide downwards along the Z direction, and the spine structure moves downwards;
when the centrifugal rotating speed R of the centrifugal tube meets the following requirements: r1≤R＜R2The spike structure punctures the binding liquid pre-sealing bag and does not puncture the rinsing liquid pre-sealing bag; when less than R2≤R＜R3While the spike structure is passing through the binding liquid pre-sealing bag and piercing the rinsing liquid pre-sealing bag without piercing the eluent pre-sealing bag; when R is not less than R3When the eluent is used, the spine structure sequentially penetrates through the binding liquid pre-sealing bag and the rinsing liquid pre-sealing bag and pierces the eluent pre-sealing bag;
wherein R is1＜R2＜R3。
5. The nucleic acid extraction spin column of claim 4, wherein the rack comprises a second porous plate connected to an inner wall of the centrifuge tube, a first support pore plate disposed on the second porous plate, a second support pore plate disposed on the first support pore plate, and a third support pore plate disposed on the second support pore plate, and a gap is left between the first support pore plate, the second support pore plate, and the third support pore plate along the Z direction;
the combination liquid pre-sealing bag, the rinsing liquid pre-sealing bag and the eluent pre-sealing bag are all in ring shapes, the combination liquid pre-sealing bag is connected to the third supporting pore plate, the rinsing liquid pre-sealing bag is connected to the second supporting pore plate, and the eluent pre-sealing bag is connected to the first supporting pore plate;
a plurality of through-holes have all been seted up on first support orifice plate, second support orifice plate and the third support orifice plate, just the position and the size setting of through-hole satisfy: when the spine structure punctures the binding liquid pre-sealing bag, the rinsing liquid pre-sealing bag and the eluent pre-sealing bag, the spine structure can penetrate through the through hole at the corresponding position on the corresponding supporting pore plate;
the surface of the second porous plate is also provided with a collecting groove;
the outer wall of the cracking tube is provided with a guide block, the inner wall of the centrifuge tube is provided with a guide groove matched with the guide block along the Z direction, and the guide block is slidably arranged in the guide groove;
a nucleic acid adsorption membrane is arranged in the centrifugal column, and a conical outlet is formed in the bottom of the centrifugal column.
6. The nucleic acid extraction centrifugation column tube of claim 5, wherein the self-switching channel comprises a switching cavity in the middle, a plurality of liquid inlet holes communicating the switching cavity with the space below the centrifugation column, a first liquid discharge channel communicating the switching cavity with the first outlet, and a second liquid discharge channel communicating the switching cavity with the second outlet;
a first channel port is formed at the joint of the first liquid discharge channel and the switching cavity, and a second channel port is formed at the joint of the second liquid discharge channel and the switching cavity;
the first liquid discharge channel and the second liquid discharge channel are obliquely arranged, so that the first channel opening is closer to the central axis of the centrifugal tube than the first outlet, and the second channel opening is closer to the central axis of the centrifugal tube than the second outlet;
the switching cavity is internally provided with a second spring along the X direction, one end of the second spring is connected with the inner wall of the switching cavity, the other end of the second spring is connected with a sealing block which can slide along the X direction in the switching cavity, and the sealing block is used for sealing the first passage port or the second passage port.
7. The nucleic acid extraction spin column of claim 6, wherein the spin tube rotates in the first direction or the second direction at a speed less than R 'while the spin tube remains stationary'3When the first channel opening is closed, the second channel opening is opened;
when the centrifuge tube rotates towards the second direction and the rotating speed is not less than R3When the first spring is stretched, the sealing block is positioned above the first passage opening and completely covers the first passage opening, the first passage opening is opened, and the second passage opening is closed;
wherein, R is1＜R2＜R'3＜R3。
8. The nucleic acid extraction centrifugal column tube of claim 7, wherein a top column is further arranged above the sealing block along the Z direction, and the top column comprises a hollow column connected to the sealing block, a third spring connected to the inside of the hollow column, and a top ball connected to the upper end of the third spring and movably arranged in the hollow column;
the knob is in contact with the upper inner wall of the switching cavity, and the liquid inlet hole above the switching cavity is kept away from the movement track of the knob.
9. A nucleic acid extraction apparatus comprising the centrifugal column according to any one of claims 1 to 8.
10. A method for extracting nucleic acid using the centrifugal column according to any one of claims 1 to 8 or the nucleic acid extraction apparatus according to claim 9, comprising the steps of:
s1, completely opening the sealing cover, pulling the easy-to-tear piece on the lysate pre-sealing bag by the pull rope, so that the lysate in the lysate pre-sealing bag flows out and enters the lysis tube, and simultaneously, a certain volume of sample is added into the lysis tube, and the sealing cover is covered;
s2, placing the centrifugal column tube into a centrifuge, fixing the centrifugal column tube on a centrifugal station of the centrifuge through the centrifugal tube, and controlling the centrifuge to rotate forward and backward alternately and to rotate at a rotating speed R less than R0Working in a manner to perform sample lysis;
s3, controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R satisfies the following conditions: r0≤R＜R1So that the lysate in the lysate passes through the microfiltration membrane and enters a centrifugal column below the microfiltration membrane, and solid impurities are intercepted by the pre-filtration membrane and the microfiltration membrane;
s4, controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R satisfies the following conditions: r1≤R＜R2So that the spine structure punctures the binding solution pre-sealing bag, the internal binding solution flows into a centrifugal column below to be mixed with a lysis solution, and nucleic acid is adsorbed by a nucleic acid adsorption membrane in the centrifugal column;
s5, controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R satisfies the following conditions: r is2≤R＜R'3So that the spine structure punctures the rinsing liquid pre-sealed bag, the rinsing liquid in the spine structure flows out to rinse the centrifugal column, and then the rinsing liquid is discharged from a conical outlet below the centrifugal column;
in the processes of steps S1-S5, the sealing block is always located above the first channel port and completely covers the first channel port, the first channel port is closed, the second channel port is opened, so that the first outlet is always closed, the second outlet is opened, and all the waste liquid discharged from the bottom of the centrifuge tube enters the waste liquid bottle for collection;
s6, controlling the centrifugal machine to rotate reversely, wherein the rotating speed R meets the following requirements: r is not less than R3So that the spine structure punctures the eluent pre-sealing bag, the internal eluent flows out to elute the centrifugal column, and the nucleic acid in the centrifugal column is separated from the nucleic acid adsorption membrane; and simultaneously, the sealing block moves to the position above the second channel opening and completely covers the second channel opening, the first channel opening is opened, the second channel opening is closed, and the eluted nucleic acid and the eluent flow into the nucleic acid sample bottle together for collection to obtain the nucleic acid extracting solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115339768A (en) * | 2022-08-30 | 2022-11-15 | 重庆医药高等专科学校 | Storage device of medical science inspection sample |
| CN117363451A (en) * | 2023-08-28 | 2024-01-09 | 北京航空航天大学 | Nucleic acid extraction device and method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115339768A (en) * | 2022-08-30 | 2022-11-15 | 重庆医药高等专科学校 | Storage device of medical science inspection sample |
| CN117363451A (en) * | 2023-08-28 | 2024-01-09 | 北京航空航天大学 | Nucleic acid extraction device and method |
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