CN111378574A - Nucleic acid extraction and amplification device - Google Patents
Nucleic acid extraction and amplification device Download PDFInfo
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- CN111378574A CN111378574A CN202010218527.7A CN202010218527A CN111378574A CN 111378574 A CN111378574 A CN 111378574A CN 202010218527 A CN202010218527 A CN 202010218527A CN 111378574 A CN111378574 A CN 111378574A
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- 230000003321 amplification Effects 0.000 title claims abstract description 33
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 33
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- 239000011159 matrix material Substances 0.000 claims description 7
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- 238000012360 testing method Methods 0.000 abstract description 2
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a closed nucleic acid extraction and amplification device.A movable body is arranged in a card box, the length of the movable body is less than the height of an inner cavity of the card box, and the movable body can vertically move and horizontally move in the card box; the movable body is divided into a plurality of independent chambers along the circumferential direction, and different liquids can be contained in each chamber; the movable body is provided with a ferromagnetic material layer, the power magnet is arranged below the card box, the vertical lifting of the movable body is controlled by controlling the magnetic force state of the power magnet, and when the power magnet moves horizontally under the condition that the movable body is kept in a downward adsorption state, the power magnet can drive the movable body to move horizontally relative to the card box; set up the sample hole on the card box, through in the sample hole adds the activity body with the sample, the horizontal motion and the lift of activity body all are through power magnet control, and fundamentally has kept apart external pollution, guarantees the precision of testing result.
Description
Technical Field
The invention relates to the field of molecular biology, and further relates to a nucleic acid extraction and amplification device.
Background
Molecular diagnostics refers to techniques for making a diagnosis by detecting changes in the structure or expression level of genetic material in a patient using molecular biology methods. The samples used for molecular diagnosis are usually tissue cells, hair, anticoagulated blood or dry blood from a subject, formaldehyde-fixed paraffin-embedded tissues, and the like, and nucleic acids (DNA or RNA) in the samples are extracted by processing the samples to perform detection at a molecular level, thereby completing detection of the gene type or detection of pathogenic microorganisms of the subject, or typing tumors, and the like.
For molecular diagnostics, the most common clinical detection method is the fluorescent quantitative PCR (Polymerase chain reaction) method. PCR is a technique for amplifying specific DNA (deoxyribonucleic acid) fragments in vitro. In addition to PCR technology, various isothermal amplification techniques are also used in the field of molecular diagnostics, such as LAMP or RPA.
Various nucleic acid amplification techniques generally require processing of samples, extraction of nucleic acids, construction of reaction systems, amplification reactions, and detection of signals. These processes are very prone to contamination and false positives. Contamination comes from two aspects, on the one hand from the positive sample itself. Since some strongly positive samples themselves contain large amounts of viral, bacterial, cellular or target nucleic acids. These viruses, bacteria, cells or target nucleic acids can contaminate negative samples during the procedure and produce false positives. And the contamination of virus and bacteria in the strong positive sample can also threaten the life health of operators. On the other hand, contamination results from contamination of the product after the amplification reaction. Amplification reactions will replicate the target nucleic acid in the sample in large amounts and detect signals, and these amplified products will contaminate other samples and produce false positives.
In addition, the pretreatment processes such as sample treatment and nucleic acid extraction require complicated steps for performing operations on respective fluids (samples, reagents, and the like) such as quantification, transfer, mixing, and separation, and a phenomenon in which the fluids are mixed and contaminated easily occurs. And once pollution occurs, the detection accuracy is seriously influenced.
Currently, most molecular diagnostic tests require specialists to use very complex instruments and perform complex procedures in specialized laboratories. And doctors or patients often need quick and definite experimental results. Therefore, there is a need for miniaturization of the instrument, integration of the analysis process, and automation. In some application scenarios, medical staff need to go to the site for diagnosis, expensive and complicated instruments are inconvenient to carry, and the site does not have a laboratory environment required for nucleic acid extraction, so that the demand for portability and integration of molecular diagnostic instruments is provided.
Some technicians in foreign countries have provided nucleic acid detection instruments and cartridges that integrate nucleic acid extraction and amplification. But the structure is complex, the cost is high, and the large-scale popularization and application are not facilitated. Moreover, some mechanical rotating shafts of the cartridges move internal structures, and interfaces of the rotating shaft parts are connected with the outside, so that the risk of leakage and pollution is caused.
It is a technical problem to be solved by those skilled in the art to provide a device which has a simple structure, can realize integrated nucleic acid extraction and amplification, and is relatively sealed.
Disclosure of Invention
The invention provides a nucleic acid extraction and amplification device integrating nucleic acid extraction and amplification, which is driven by magnetic force to realize relative isolation from the outside and ensure accurate detection result, and the specific scheme is as follows:
a nucleic acid extraction and amplification device comprises a card box, wherein a movable body is arranged in the card box, the length of the movable body is less than the height of an inner cavity of the card box, and the movable body can move vertically and horizontally in the card box;
the movable body is divided into a plurality of independent chambers; the bottom surface of the movable body is provided with a ferromagnetic material layer;
a power magnet is arranged below the card box, and can adsorb the movable body to enable the movable body to vertically move and drive the movable body to horizontally move relative to the card box;
the card box is provided with at least one sample hole connected with the outside for adding a sample to the movable body.
Optionally, the inner cavity of the cartridge is provided with a suction head protruding downward, and the suction head can be inserted into the cavity of the movable body when the movable body is lifted.
Optionally, the power magnet is an electromagnet.
Optionally, the cartridge is cylindrical and the movable body is cylindrical.
Optionally, the power magnet can rotate circumferentially to drive the movable body to rotate horizontally and circumferentially in the card box.
Optionally, the bottom of the movable body is provided with a jacking spring, and the jacking spring exerts an upward elastic force on the movable body to drive the movable body to move upwards so that the suction head extends into the cavity.
Optionally, a buffer spring is arranged on the top of the movable body, and the buffer spring can be in contact with and buffer the upper surface of the inner cavity of the card box.
Optionally, the jacking spring and the buffer spring are arranged in a cylindrical cavity arranged in the center of the movable body.
Optionally, a reaction chamber attached to a sidewall of the cartridge; when the movable body is at the bottom, the sample is input through the liquid conveying pipeline for butting the inside of the reaction chamber and the inside of the movable body.
Optionally, a sample loading hole is arranged at the top of the reaction chamber, and the sample loading hole is connected with a negative pressure air extraction device to extract a sample into the reaction chamber.
Optionally, an adsorption electromagnet is arranged on the outer side wall of the card box, and the adsorption electromagnet is used for adsorbing magnetic beads placed in the chamber of the movable body.
Optionally, an adsorption matrix is arranged in the middle of the inner cavity of the suction head, a filter membrane is arranged at the top of the inner cavity of the suction head, the adsorption matrix is used for adsorbing nucleic acid, and the filter membrane is used for preventing liquid or nucleic acid from overflowing.
Optionally, an openable or closable lid is rotatably disposed on the sample well.
Optionally, the loading well is provided with a filter for blocking the passage of nucleic acids.
The invention provides a closed nucleic acid extraction and amplification device, wherein a movable body is arranged in a card box, the length of the movable body is less than the height of an inner cavity of the card box, and the movable body can vertically move and horizontally move in the card box; the movable body is divided into a plurality of independent chambers along the circumferential direction, and different liquids can be contained in each chamber; the movable body is provided with a ferromagnetic material layer, the power magnet is arranged below the card box, the vertical lifting of the movable body is controlled by controlling the magnetic force state of the power magnet, and when the power magnet moves horizontally under the condition that the movable body is kept in a downward adsorption state, the movable body can be driven to move horizontally relative to the card box; set up the sample hole on the card box, through in the sample hole adds the activity body with the sample, the horizontal motion and the lift of activity body all are through power magnet control, and fundamentally has kept apart external pollution, guarantees the precision of testing result.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIGS. 1A and 1B are a front view and a rear view, respectively, of an assembly of a nucleic acid extraction and amplification apparatus provided by the present invention;
FIGS. 1C and 1D are left and right side views, respectively, of FIG. 1A;
FIG. 1E is a top view of FIG. 1C;
FIGS. 2A and 2B are an isometric view and an exploded view, respectively, of a closed nucleic acid extraction and amplification apparatus provided by the present invention;
FIG. 3 is an exploded view of the cartridge and the movable body assembled with each other;
FIG. 4A is a cross-sectional view taken along line A-A of FIG. 1C;
FIG. 4B is a cross-sectional view taken along line B-B of FIG. 1E;
FIGS. 4C and 4D are cross-sectional views of the two states in the direction C-C in FIG. 1E, respectively;
FIG. 4E is a cross-sectional view taken along line D-D of FIG. 1E;
FIG. 4F is an enlarged view of a portion of the infusion tube assembly of FIG. 4D;
FIG. 5 is a schematic view of the structure of the suction head;
FIG. 6A is a flow chart of nucleic acid extraction by adsorption;
FIG. 6B is a flow chart of nucleic acid extraction by the magnetic bead method.
The figure includes:
the kit comprises a card box 1, a suction head 11, an adsorption matrix 111, a filter membrane 112, a sample hole 12, a sample loading hole 13, a movable body 2, a power magnet 3, a jacking spring 4, a buffer spring 5, a reaction chamber 6, a liquid conveying pipeline 7 and an adsorption electromagnet 8.
Detailed Description
The core of the invention is to provide a nucleic acid extraction and amplification device, which is driven by magnetic force to realize relative isolation with the outside, thus ensuring the convenience of the detection process and the accuracy of the detection result.
In order to make the technical solution of the present invention better understood by those skilled in the art, the nucleic acid extracting and amplifying apparatus of the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
FIGS. 1A and 1B are front and rear views, respectively, of an assembly of a closed nucleic acid extraction and amplification apparatus and a detection instrument provided by the present invention, FIGS. 1C and 1D are left and right side views, respectively, of FIG. 1A, and FIG. 1E is a top view of FIG. 1C; the nucleic acid extracting and amplifying device provided by the invention comprises a cartridge 1 with a hollow interior, wherein the cartridge 1 comprises an upper surface, a lower surface and peripheral outer walls, and the interior is a relatively closed space. Set up movable body 2 in card box 1, the length of movable body 2 is less than the inner chamber height that card box 1, and movable body 2 can be in card box 1 vertical removal and horizontal movement, and the motion of movable body 2 includes two kinds of circumstances, and one kind is along the vertical removal of the axial of card box 1, and another kind moves or rotates along the horizontal direction.
The movable body 2 is divided into a plurality of independent chambers along the circumferential direction, each chamber is used for containing respective liquid, each chamber is provided with a top opening, liquid is taken from the top opening, and the stored liquid comprises a sample, lysate, binding liquid, washing liquid, eluent and the like.
The bottom surface of the movable body 2 is provided with a ferromagnetic material layer, the lower part of the card box 1 is provided with a power magnet 3, and fig. 2A and 2B are an axonometric view and an exploded view of the nucleic acid extraction and amplification device and the detection instrument provided by the invention respectively; the power magnet 3 is a structure below the card box and can move relative to the card box 1, and the power magnet 3 is positioned outside the card box 1 and is relatively isolated from the inner space of the card box 1; the movement of the movable body 2 is controlled by changing the magnetic force state of the power magnet 3; when the power magnet 3 adsorbs the movable body 2, the movable body 2 moves downwards and is close to the bottom surface of the card box 1; and the power magnet 3 can drive the movable body 2 to move horizontally relative to the card box 1 when moving horizontally.
The upper surface of card box 1 sets up sample hole 12, and sample hole 12 sets up one at least, and sample hole 12 communicates with the external world relatively, and sample hole 12 is used for making the application of sample device pass, and the application of sample can adopt manual operation, and the sample can be liquid also can be oral swab. The sample adding device stretches into the cavity of the movable body 2 for storing the sample from outside to inside, the sample hole 12 is sealed after the sample is put in, the liquid in the inner container of the movable body 2 is isolated from the outside relatively, and the rotation and the lifting of the movable body 2 are controlled by the power magnet 3, so that the external pollution is isolated fundamentally, and the precision of the detection result is ensured.
The invention realizes the extraction and molecular diagnosis of the sample by driving the movable body in the card box to move through magnetic force, and the movable body in the card box is driven by adopting a mechanical rotating shaft mode in the prior art, so that a gap is inevitably formed between the rotating shaft and the card box, and the leakage risk of the internal sample in the processing process is caused.
The upper surface of the card box 1 protrudes downwards to be provided with a suction head 11, the suction head 11 is butted with a suction head hole on the card box 1, the suction head 11 is connected with a pneumatic device of a detection instrument and provides power for the suction head 11, and the suction head 11 is always butted with the detection instrument and is always kept closed; when the movable body 2 is raised, the suction head 13 can be inserted into the chamber of the movable body 2, and liquid in the corresponding chamber is sucked or a blow-and-beat mixing is performed by the suction head 13.
Preferably, the power magnet 3 of the present invention is configured as an electromagnet, the position of the power magnet 3 is kept relatively fixed, and the control is more flexible and convenient by changing the power-on state to control the existence and magnitude of the magnetic force.
The cartridge 1 of the present invention has a cylindrical shape, and the movable body 2 is also provided in a cylindrical shape.
The power magnet 3 can rotate in the circumferential direction, and the power magnet 3 drives the movable body 2 to move horizontally in the card box 1 in the circumferential direction. The power magnet 3 rotates along the axis of the power magnet, the power magnet 3 is arranged on the rotating shaft, the power magnet 3 and the motor are in transmission through gears or belts, the motor drives the power magnet 3 to rotate around the rotating shaft, and the power magnet 3 further drives the movable body 2 to rotate through magnetic force; the movable body 2 is sucked to the position of the card box 1 close to the lower surface to rotate, and after rotating a proper angle, the movable body 2 moves upwards again, so that the suction head is inserted into different chambers.
The power magnet 3 can also adopt a motion form of horizontal linear motion, and the power magnet 3 drives the movable body 2 to horizontally and transversely displace
The downward movement of the movable body 2 can be achieved not only by the attraction force of the electromagnet but also by the attraction force of the permanent magnet. The attraction and release of the movable body 2 are achieved by changing the position of the permanent magnet, and these specific embodiments are all included in the scope of the present invention.
As shown in fig. 3, which is an exploded view of the cartridge 1 and the movable body 2 assembled with each other, based on the above solution, the present invention has a jacking spring 4 at the bottom of the movable body 2, and fig. 4A is a cross-sectional view taken along the direction a-a in fig. 1C; FIG. 4B is a cross-sectional view taken along line B-B of FIG. 1E; fig. 4C and 4D are sectional views in the direction C-C in fig. 1E, respectively, fig. 4C showing a state where the movable body 2 is above, and fig. 4D showing a state where the movable body 2 is below; FIG. 4E is a cross-sectional view taken along line D-D of FIG. 1E; the top of the jacking spring 4 contacts with the movable body 2, the bottom contacts with the lower surface of the card box 1, the jacking spring 4 exerts upward elastic force on the movable body 2 when compressed, the movable body 2 moves upwards through the jacking spring 4, and the movable body 2 is jacked on the upper surface of the card box 1, so that the suction head 13 extends into the cavity. The downward movement of the movable body 2 is realized by the attraction of the power magnet 3, and the attraction of the power magnet 3 to the cartridge 1 is larger than the elastic force of the jacking spring 4.
In addition to providing upward power to the movable body 2 by using the jacking spring 4, a permanent magnet may be disposed at a specific position at the bottom of the movable body 2, that is, the ferromagnetic material layer is a magnet; by changing the magnetic pole direction of the power magnet 3, these specific embodiments are all included in the protection scope of the present invention.
Preferably, still can set up buffer spring 5 at the top of the activity body 2, buffer spring 5's elastic coefficient is less than the elastic coefficient of jacking spring 4, buffer spring 5's top and the upper surface contact of card box 1, and the bottom contacts with the activity body 2, and buffer spring 5 is compressed when activity body 2 rebound, and buffer spring 5 plays the effect of buffering to activity body 2, produces the striking with the upper surface of card box 1 when avoiding card box 1 rebound.
The jacking spring 4 and the buffer spring 5 are arranged in a cylindrical cavity arranged at the center of the movable body 2, all cavities of the movable body 2 are distributed in a central symmetry manner around a cylinder at the center, and a spacing plate is horizontally arranged in the cylindrical cavity in the middle of the movable body 2 and used for spacing the jacking spring 4 from the buffer spring 5.
On the basis of any one of the above technical solutions and the combination thereof, the invention further comprises a reaction chamber 6 connected to the side wall of the cartridge 1; as shown in fig. 4F, which is a partially enlarged view of the fitting of the transfusion tube 7 in fig. 4D, when the movable body 2 is at the bottom, the transfusion tube 7 in which the inside of the reaction chamber 6 and the inside of the movable body 2 are butted inputs a sample, and the flow direction of the sample is shown by an arrow in the figure; the infusion line 7 comprises two parts, one inside the reaction chamber 6 and the other on the mobile body 2, the two parts of the infusion line 7 being staggered or abutting each other when the mobile body is moved to different positions. The infusion pipeline 7 on the movable body 2 is positioned in the cavity where the sample is positioned, and the conduction can be realized only when the interface of the infusion pipeline 7 on the movable body 2 is aligned with the interface of the infusion pipeline 7 on the card box 1, namely, the movable body 2 rotates to a specific angle and is positioned at a specific vertical height, and liquid flows in an S shape when the infusion pipeline 7 is conducted.
The movable body 2 moves upwards under the elastic force of the jacking spring 4, at the moment, the interface of the infusion pipeline 7 on the movable body 2 and the infusion pipeline 7 on the card box 1 are mutually staggered, and the liquid in the corresponding cavity is sucked by the suction head 13; the movable body 2 moves to the lower part under the attraction of the power magnet 3, at the moment, the interface of the liquid conveying pipeline 7 on the movable body 2 is mutually butted with the liquid conveying pipeline 7 on the card box 1 to form a complete channel, and the processed sample in the corresponding chamber is upwards drawn into the reaction chamber.
Specifically, the top of the reaction chamber 6 is provided with a sample loading hole 13, the sample loading hole 13 is connected with a negative pressure pumping device to pump a sample to the reaction chamber 6, the negative pressure pumping device is of a structure of a detection instrument, and when the reaction chamber 6 is communicated with the segmented liquid conveying pipeline 7 on the movable body 2, negative pressure is formed at the sample loading hole 13, so that the sample in the cavity flows upwards along the liquid conveying pipeline 7 and enters the reaction chamber 6.
The invention provides another preferred embodiment, an adsorption electromagnet 8 is arranged on the outer side wall of the card box 1, and the adsorption electromagnet 8 can be fixed on the outer surface of the card box 1 and can also be arranged on a detection instrument; when the adsorption electromagnet 8 is powered on, the adsorption electromagnet 8 adsorbs magnetic beads placed in the chamber of the movable body 2, and the adsorption electromagnet 8 is used for the nucleic acid detection process by the magnetic bead method.
As shown in FIG. 5, it is a schematic view of the structure of the suction head 11; the middle part of the inner cavity of the suction head 11 is provided with an adsorption matrix 111, the top part is provided with a filter membrane 112, the adsorption matrix 111 is used for adsorbing nucleic acid, and the filter membrane is used for preventing liquid or nucleic acid from overflowing.
A seal cover which can be opened or closed is rotatably arranged on the sample hole 12, the seal cover is opened before the sample is injected into the movable body 2, the seal cover is closed after the sample is injected, and the seal cover is rotatably connected to the upper surface of the card box 1 through a rotating shaft.
The loading holes 13 are provided with a membrane for blocking the passage of nucleic acids, and the operation of sampling the loading holes 13 is normally stopped after the sample is drawn into the reaction chamber 6, but in order to prevent the nucleic acids from being drawn out, a filter membrane is provided at the loading holes 13, and the filter membrane blocks the nucleic acids, so that the nucleic acids cannot pass through the filter membrane.
FIG. 6A is a flowchart of nucleic acid extraction by adsorption, and FIG. 6B is a flowchart of nucleic acid extraction by magnetic bead method; when extracting nucleic acid by an adsorption method, firstly putting a sample into a lysis solution for lysis, adding a detergent after centrifugation, adding an eluent after the centrifugation again, and finally obtaining a purified product. When extracting nucleic acid by a magnetic bead method, adding lysis solution into a sample for lysis, then adding magnetic beads to combine the magnetic beads with the nucleic acid, then adding washing solution, adsorbing the magnetic beads by an iron-absorbing agent, extracting waste liquid, and then adding eluent for elution operation to obtain purified nucleic acid.
Before detection, the card box 1 is placed at a corresponding position on a detection instrument, and according to different adopted principles, the closed nucleic acid extraction and amplification device provided by the invention is used, and the specific operation process is as follows:
extraction of nucleic acid by adsorption
1. The power magnet 3 is in a non-magnetic state, and a sample is added into the chamber containing the lysate through the sample hole 12 to carry out the lysis process.
2. After the cracking is completed, the power magnet 3 is powered on, the whole movable body 2 is adsorbed by the magnetic force and moves downwards, and the movable body 2 is not obstructed by the suction head 11 when rotating.
3. The power magnet 3 outside the card box 1 is rotated by mechanical force, the power magnet 3 drives the movable body 2 inside the card box 1 to rotate, and the cavity filled with the lysate and the sample is rotated to the position below the sucker 11.
4. The power magnet 3 is powered off, the power magnet 3 is in a non-magnetic state, and the movable body 2 moves towards the upper part of the card box 1 under the action of the jacking spring 4, so that the suction head 11 is inserted into a chamber filled with lysate and a sample.
5. The pneumatic device on the detection instrument absorbs the mixed solution of the lysate and the sample through the suction head 11. The tip 11 is inserted into each chamber containing the lysis solution and the sample binding solution one by one in accordance with the procedures 2 to 4 described above. Repeatedly blowing and sucking air through an external pneumatic device, blowing and beating the mixed solution of the lysate and the sample, and uniformly mixing the lysate, the mixed solution of the sample and the binding solution through repeatedly blowing and beating; when the nucleic acid in the sample is adsorbed on the adsorption substrate 131 in the tip 11 and the liquid in the tip 11 is evacuated, a large amount of nucleic acid is adsorbed in the tip 11.
6. According to the above-mentioned procedures 2 to 4, the nucleic acid-adsorbing tip 13 is inserted into the chamber containing the washing solution, the tip 11 is repeatedly blown so that the adsorption substrate 111 in the tip 11 is washed with the washing solution, and the liquid in the tip 11 is evacuated.
7. According to the above 2 to 4 processes, the tip 11 is inserted into the chamber containing the eluent, the tip is repeatedly blown to elute the nucleic acid adsorbed on the adsorption substrate 111 in the tip 11 into the chamber containing the eluent, and the liquid in the tip 11 is emptied.
8. According to the method of 2-3 above, the chamber containing the eluent and nucleic acid in the movable body 2 is rotated to a position close to the reaction chamber 6, and at this time, the power magnet 3 is kept in a magnetic state, and the movable body 2 is communicated with the liquid feeding tube 7 of the cartridge 1.
9. The eluent is filled into the reaction chamber 6 through the suction effect of the pneumatic device connected to the sample loading hole 13, and the freeze-drying reagent required for PCR reaction is pre-filled in the reaction chamber 6.
10. When the power magnet 3 is in a non-magnetic state, the movable body 2 moves to the upper part of the card box 1 under the action of the jacking spring 4, and the movable body 2 and the infusion pipeline 7 on the card box 1 are staggered, so that the reaction chamber 6 is relatively isolated from the movable body 2.
11. The heating and refrigerating device outside the reaction chamber 6 controls the temperature of the reaction chamber 6, and the corresponding optical element monitors the PCR reaction in the reaction chamber 6.
Magnetic bead method for extracting nucleic acid, and structure shown in FIG. 3 for driving adsorption electromagnet
1. The adsorption electromagnet 8 and the bottom electromagnet 3 are both in a non-magnetic state, the whole body is in a state shown in figure 3, and a sample is added into a chamber containing lysis solution through the sample hole 12 for lysis.
2. After the cracking is finished, the power magnet 3 is electrified and magnetized, and the whole movable body 2 is sucked down.
3. The power magnet 3 at the bottom is rotated by mechanical force to drive the movable body 2 in the card box 1 to rotate circumferentially, so that the chamber filled with the lysate and the sample rotates to the position below the suction head 11.
4. The power magnet 3 at the bottom is powered off and is in a non-magnetic state, the movable body 2 moves upwards under the action of the jacking spring 4, and the sucker 11 is inserted into a chamber filled with lysate and a sample.
5. The pneumatic device connected to the hole of the suction head sucks the mixed solution of the lysate and the sample through the suction head 11. Repeating the process of 2-4, inserting the tip 11 into the chamber containing the mixture of magnetic beads and binding solution; in addition, the magnetic beads may be placed in a chamber separately, and the magnetic beads are sucked out by the tip 11 and transferred to the chamber containing the binding solution. And blowing the mixed solution of the lysate and the sample by a pneumatic device of the detection instrument. And mixing the lysate, the sample mixed solution, the binding solution and the magnetic beads uniformly by repeatedly blowing and beating. At this time, the nucleic acid released from the lysate is adsorbed on the surface of the magnetic beads by the binding solution.
6. Standing, electrifying and magnetizing an adsorption electromagnet 8 on the side surface of the card box 1, adsorbing magnetic beads on the inner side wall of the chamber, then absorbing liquid in the chamber by a suction head 11, and transferring the absorbed waste liquid to a waste liquid containing and collecting chamber according to the 2-4 process.
7. According to the process 2-4, the suction head 11 is inserted into the chamber filled with the cleaning liquid, the cleaning liquid is sucked and transferred into the cavity where the magnetic beads are located, the adsorption electromagnet 8 is powered off, the magnetic beads are mixed with the cleaning liquid, and the cleaning liquid is blown and beaten repeatedly through the suction head 11. Then the adsorption electromagnet 8 is electrified and magnetized again, the magnetic beads are adsorbed on the side wall of the chamber, the liquid in the chamber is sucked out, and the chamber is transferred to a waste liquid containing collecting chamber.
8. Following the procedure of 2-4, the tip 11 is inserted into the chamber containing the eluent to aspirate the eluent and transferred to the chamber where the magnetic beads are located. The adsorption electromagnet 8 on the side wall of the card box 1 is powered off to remove magnetism, and the suction head 11 blows, beats and mixes repeatedly. The adsorption electromagnet 8 is electrified and magnetically adsorbs the magnetic beads again, and liquid is sucked up through the sucker 11. The eluent was transferred to another chamber following the procedure of 2-4.
9. According to the procedure of 2-3, the chamber containing the eluent and the nucleic acid in the movable body 2 is rotated to a position close to the reaction chamber 6. At this time, the power magnet 3 is kept energized and magnetized, and the infusion tube 7 of the movable body 2 is conducted to the infusion tube 7 of the cassette 1.
10. The eluent is filled into the reaction chamber 6 through the suction effect of the pneumatic device connected to the sample loading hole 13, and the freeze-drying reagent required for PCR reaction is pre-filled in the reaction chamber 6.
11. The power magnet 3 is in a non-magnetic state when being powered off, the movable body 2 moves towards the upper part of the card box 1 under the action of the jacking spring 4, the infusion pipeline 7 on the movable body 2 is disconnected with the infusion pipeline 7 on the card box 1, and the reaction chamber 6 is relatively isolated from the cavity in the movable body 2.
12. The heating and refrigerating device outside the reaction chamber 6 controls the temperature of the reaction chamber, and the corresponding optical element monitors the PCR reaction in the reaction chamber.
Cartridges of prior art designs tend to be complex and unsealed, which often involve mechanical movement within the cartridge in order to change the position of the cavity within the container or to drive the movement of the liquid within the container, but the gaps between moving and non-moving parts are prone to sample leakage contamination. The present invention utilizes magnetic force to drive moving parts. The entire cartridge is sealed against the transmission, greatly reducing the possibility of leakage contamination.
The device provided by the invention can be used for extracting and amplifying nucleic acid in a sample, and has the advantages of simple and reliable structure, low manufacturing cost and convenience for popularization and use. Particularly, the invention adopts electromagnetic adsorption to change the position between the suction head and the cavity in the closed card box, thereby ensuring the tightness of the whole card box and preventing the occurrence of pollution. In addition, the transfusion pipeline is controlled by electromagnetic force, so that the tightness of the reaction chamber is ensured.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (14)
1. A nucleic acid extraction and amplification device is characterized by comprising a card box (1), wherein a movable body (2) is arranged in the card box (1), the length of the movable body (2) is less than the height of an inner cavity of the card box (1), and the movable body (2) can move vertically and horizontally in the card box (1);
the movable body (2) is divided into a plurality of independent chambers; the bottom surface of the movable body (2) is provided with a ferromagnetic material layer;
a power magnet (3) is arranged below the card box (1), and the power magnet (3) can adsorb the movable body (2) to enable the movable body to vertically move and can drive the movable body (2) to horizontally move relative to the card box (1);
the card box (1) is at least provided with a sample hole connected with the outside for adding a sample to the movable body (2).
2. The nucleic acid extraction and amplification apparatus according to claim 1, wherein a tip (11) is provided to protrude downward from an inner cavity of the cartridge (1), and the tip (11) is insertable into a cavity of the movable body (2) when the movable body (2) is raised.
3. The nucleic acid extraction and amplification apparatus according to claim 1, wherein the power magnet (3) is an electromagnet.
4. The nucleic acid extraction and amplification apparatus according to claim 1, wherein the cartridge (1) has a cylindrical shape and the movable body (2) has a cylindrical shape.
5. The nucleic acid extraction and amplification apparatus according to claim 4, wherein the power magnet (3) is capable of rotating circumferentially to drive the movable body (2) to rotate horizontally and circumferentially in the cartridge (1).
6. The nucleic acid extraction and amplification device according to claim 1, wherein a lift-up spring (4) is disposed at the bottom of the movable body (2), and the lift-up spring (4) exerts an upward elastic force on the movable body (2) to drive the movable body (2) to move upward, so that the suction head (11) extends into the chamber.
7. The nucleic acid extraction and amplification apparatus according to claim 6, wherein a buffer spring (5) is provided on the top of the movable body (2), and the buffer spring (5) is capable of buffering in contact with the upper surface of the inner cavity of the cartridge (1).
8. The nucleic acid extraction and amplification apparatus according to claim 7, wherein the lift-up spring (4) and the buffer spring (5) are disposed in a cylindrical cavity provided in the center of the movable body (2).
9. The nucleic acid extraction and amplification apparatus of any one of claims 1 to 4, further comprising a reaction chamber (6) attached to a side wall of the cartridge (1); when the movable body (2) is at the bottom, the sample is input through a liquid conveying pipeline (7) which is butted between the inside of the reaction chamber (6) and the inside of the movable body (2).
10. The nucleic acid extracting and amplifying device according to claim 9, wherein a sample loading hole (13) is disposed at the top of the reaction chamber (6), and the sample loading hole (13) is connected to a negative pressure pumping device for pumping a sample to the reaction chamber (6).
11. The nucleic acid extraction and amplification apparatus according to any one of claims 1 to 4, wherein an adsorption electromagnet (8) is disposed on an outer side wall of the cartridge (1), and the adsorption electromagnet (8) is configured to adsorb magnetic beads disposed in the chamber of the movable body (2).
12. The nucleic acid extraction and amplification apparatus according to any one of claims 1 to 4, wherein an adsorption matrix (111) is disposed in the middle of the inner cavity of the tip (11), and a filter (112) is disposed on the top of the inner cavity of the tip, wherein the adsorption matrix (111) is used for adsorbing nucleic acids, and the filter is used for preventing liquid or nucleic acids from overflowing.
13. The nucleic acid extraction and amplification apparatus according to any one of claims 1 to 4, wherein the sample well (12) is rotatably provided with an openable or closable lid.
14. The nucleic acid extraction and amplification apparatus according to any one of claims 1 to 4, wherein the loading well (13) is provided with a filter for blocking passage of nucleic acid.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010218527.7A CN111378574B (en) | 2020-03-25 | 2020-03-25 | Nucleic acid extraction and amplification device |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010218527.7A CN111378574B (en) | 2020-03-25 | 2020-03-25 | Nucleic acid extraction and amplification device |
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| CN111378574B CN111378574B (en) | 2023-10-27 |
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| CN112226358A (en) * | 2020-12-11 | 2021-01-15 | 博奥生物集团有限公司 | Nucleic acid analysis card box and nucleic acid analysis equipment |
| CN114032173A (en) * | 2022-01-11 | 2022-02-11 | 至美时代生物智能科技(北京)有限公司 | Closed air sampling bottle |
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| CN111378574B (en) | 2023-10-27 |
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