CN111378574B - Nucleic acid extraction and amplification device - Google Patents
Nucleic acid extraction and amplification device Download PDFInfo
- Publication number
- CN111378574B CN111378574B CN202010218527.7A CN202010218527A CN111378574B CN 111378574 B CN111378574 B CN 111378574B CN 202010218527 A CN202010218527 A CN 202010218527A CN 111378574 B CN111378574 B CN 111378574B
- Authority
- CN
- China
- Prior art keywords
- movable body
- nucleic acid
- acid extraction
- sample
- cartridge
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 71
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 71
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 71
- 238000000605 extraction Methods 0.000 title claims abstract description 41
- 230000003321 amplification Effects 0.000 title claims abstract description 36
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 36
- 230000005291 magnetic effect Effects 0.000 claims abstract description 37
- 239000007788 liquid Substances 0.000 claims abstract description 34
- 238000001179 sorption measurement Methods 0.000 claims abstract description 30
- 239000003302 ferromagnetic material Substances 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims description 41
- 239000011324 bead Substances 0.000 claims description 21
- 239000012528 membrane Substances 0.000 claims description 12
- 239000011159 matrix material Substances 0.000 claims description 8
- 230000000903 blocking effect Effects 0.000 claims description 4
- 210000001503 joint Anatomy 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 22
- 230000033001 locomotion Effects 0.000 abstract description 10
- 238000000034 method Methods 0.000 description 29
- 238000001802 infusion Methods 0.000 description 24
- 239000000243 solution Substances 0.000 description 14
- 239000003480 eluent Substances 0.000 description 11
- 230000009089 cytolysis Effects 0.000 description 9
- 238000011109 contamination Methods 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 239000006166 lysate Substances 0.000 description 7
- 238000003752 polymerase chain reaction Methods 0.000 description 7
- 230000009471 action Effects 0.000 description 5
- 238000007664 blowing Methods 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- 238000005336 cracking Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000003139 buffering effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000010808 liquid waste Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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]
Abstract
The invention discloses 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 smaller 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 each chamber can contain different liquids; the movable body is provided with a ferromagnetic material layer, the power magnet is arranged below the clamping 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 clamping box; the upper part of the cartridge is provided with a sample hole, a sample is added into the movable body through the sample hole, the horizontal movement and lifting of the movable body are controlled by the power magnet, the external pollution is fundamentally isolated, and the accuracy of the detection result is ensured.
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 diagnosis refers to a technique of detecting changes in the structure or expression level of genetic material in a patient using molecular biological methods to make a diagnosis. Samples used for molecular diagnosis are usually tissue cells, hair, anticoagulants or dried blood stains from a subject, formaldehyde-fixed, paraffin-embedded tissues, and the like, and nucleic acids (DNA or RNA) therein are extracted by processing the samples to detect molecular levels, thereby completing detection of the genotype of the subject or detection of pathogenic microorganisms, typing of 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 techniques, various isothermal amplification techniques are also applied in the field of molecular diagnostics, such as LAMP or RPA.
Various nucleic acid amplification techniques generally require sample processing, nucleic acid extraction, reaction system construction, amplification reactions, signal detection, and the like. Contamination is very likely to occur in these processes to produce false positives. Contamination comes from two aspects, on the one hand from the positive sample itself. Since some strongly positive samples themselves contain a large amount of virus, bacteria, cells or target nucleic acids. During manipulation, these viruses, bacteria, cells or target nucleic acids can contaminate negative samples and produce false positives. And the contamination of viruses 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 products after the amplification reaction. Amplification reactions can cause substantial replication of target nucleic acids in a sample and detection of signals, and these amplified products can contaminate other samples and produce false positives.
In addition, the pretreatment process such as sample processing and nucleic acid extraction requires complicated steps for performing operations of respective fluids (sample, reagent, etc.) such as quantification, transfer, mixing, separation, etc., and the phenomenon that contamination occurs due to mixing of fluids is likely to occur. And once contamination occurs, the accuracy of the detection is severely affected.
Currently, most molecular diagnostic tests require a professional to perform in a professional laboratory through complex procedures using very complex instrumentation. And doctors or patients often need quick and definite experimental results. Therefore, there is a need for miniaturization of the apparatus, and integration and automation of the analysis process. In some application scenarios, medical staff need to rush to the field to perform diagnosis, expensive and complex instruments are inconvenient to carry, and the field does not have laboratory environments required by nucleic acid extraction, so that the demand for portability and integration of molecular diagnostic instruments is raised.
Some technicians abroad 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. In addition, some mechanical rotating shafts of the cartridges move the inner structure, and the interface of the rotating shaft part of the mechanical rotating shaft is connected with the outside, so that the risk of leakage and pollution exists.
It is a technical problem to be solved at present that a device which has a simple structure and can realize integrated nucleic acid extraction and amplification and is relatively sealed is provided for a person skilled in the art.
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 results, and the specific scheme is as follows:
the nucleic acid extraction and amplification device comprises a cartridge, wherein a movable body is arranged in the cartridge, the length of the movable body is smaller than the height of an inner cavity of the cartridge, and the movable body can vertically move and horizontally move in the cartridge;
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 absorb the movable body to make the movable body vertically move and drive the movable body to horizontally move relative to the card box;
the cartridge 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 in a protruding manner, and when the movable body is lifted, the suction head can be inserted into the cavity of the movable body.
Optionally, the power magnet is an electromagnet.
Optionally, the cartridge is cylindrical, and the movable body is cylindrical.
Optionally, the power magnet can circumferentially rotate to drive the movable body to horizontally and circumferentially rotate in the clamping box.
Optionally, a lifting spring is arranged at the bottom of the movable body, and applies upward elastic force to the movable body by the lifting spring to drive the movable body to move upward, so that the suction head stretches into the cavity.
Optionally, a buffer spring is arranged at the top of the movable body, and the buffer spring can be in contact with the upper surface of the inner cavity of the card box for buffering.
Optionally, the jacking spring and the buffer spring are disposed in a cylindrical cavity centrally disposed in the movable body.
Optionally, the reaction chamber is connected to the side wall of the cartridge; when the movable body is at the bottom, the infusion pipeline in which the inside of the reaction chamber is in butt joint with the inside of the movable body inputs a sample.
Optionally, a sample loading hole is formed in the top of the reaction chamber, and the sample loading hole is connected with a negative pressure air extracting device to extract a sample to the reaction chamber.
Optionally, an adsorption electromagnet is arranged on the outer side wall of the cartridge, and the adsorption electromagnet is used for adsorbing the magnetic beads placed in the cavity of the movable body.
Optionally, an adsorption matrix is arranged in the middle of the cavity of the suction head, a filter membrane is arranged at the top of the 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 cover is rotatably disposed on the sample hole.
Optionally, the loading well is provided with a filter membrane for blocking 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 smaller 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 each chamber can contain different liquids; the movable body is provided with a ferromagnetic material layer, the power magnet is arranged below the clamping box, the vertical lifting of the movable body is controlled by controlling the magnetic force state of the power magnet, and when the movable body keeps the power magnet to move horizontally under the condition of being adsorbed downwards, the movable body can be driven to move horizontally relative to the clamping box; the upper part of the cartridge is provided with a sample hole, a sample is added into the movable body through the sample hole, the horizontal movement and lifting of the movable body are controlled by the power magnet, the external pollution is fundamentally isolated, and the accuracy of the detection result is ensured.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIGS. 1A and 1B are front and rear views, 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 device provided by the present invention;
FIG. 3 is an exploded view of the cartridge and the mobile body assembled with each other;
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 the direction B-B in FIG. 1E;
FIGS. 4C and 4D are cross-sectional views of the two states in the direction C-C of FIG. 1E, respectively;
FIG. 4E is a cross-sectional view taken along the direction D-D in FIG. 1E;
FIG. 4F is an enlarged view of a portion of the infusion tubing set 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 drawings include:
cartridge 1, suction head 11, adsorption matrix 111, filter membrane 112, sample hole 12, sample loading hole 13, movable body 2, power magnet 3, jack spring 4, buffer spring 5, reaction chamber 6, infusion pipeline 7, 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 from the outside, thereby ensuring the convenience of the detection process and the accurate detection result.
In order to make the technical scheme of the present invention better understood by those skilled in the art, the following detailed description will be given of the nucleic acid extraction and amplification apparatus of the present invention 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 according to 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 extraction and amplification device provided by the invention comprises a cartridge 1 with a hollow inside, wherein the cartridge 1 comprises an upper surface, a lower surface and peripheral outer walls, and the inside is a relatively closed space. The movable body 2 is arranged in the card box 1, the length of the movable body 2 is smaller than the height of the inner cavity of the card box 1, the movable body 2 can vertically move and horizontally move in the card box 1, the movement of the movable body 2 comprises two conditions, one is vertically moved along the axial direction of the card box 1, and the other is moved or rotated along the horizontal direction.
The movable body 2 is circumferentially divided into a plurality of independent chambers, each chamber is used for containing respective liquid, each chamber is provided with a top opening, liquid is taken and placed from the top opening, and the stored liquid comprises a sample, a lysate, a binding liquid, a washing liquid, an eluent and the like.
A ferromagnetic material layer is arranged on the bottom surface of the movable body 2, a power magnet 3 is arranged below the cartridge 1, and fig. 2A and 2B are an isometric 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 of a structure below the cartridge and can move relative to the cartridge 1, and the power magnet 3 is positioned outside the cartridge 1 and is isolated relative to the inner space of the cartridge 1; the motion 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 horizontally move relative to the card box 1 when moving horizontally.
The upper surface of the cartridge 1 is provided with sample holes 12, at least one sample hole 12 is arranged, the sample holes 12 are communicated with the outside relatively, the sample holes 12 are used for enabling a sample adding device to pass through, manual operation can be adopted for sample adding, and the sample can be liquid or an oral swab. The sample adding device stretches into the cavity of the movable body 2 for storing samples from outside to inside, the sample hole 12 is closed after sample feeding is completed, liquid contained in the movable body 2 is isolated from the outside relatively, the rotation and lifting of the movable body 2 are controlled by the power magnet 3, the outside pollution is fundamentally isolated, and the accuracy of a detection result is ensured.
The invention drives the movable body in the cartridge to move by magnetic force to realize the extraction and molecular diagnosis of the sample, and the prior art adopts a mechanical rotating shaft mode to drive the movable body in the cartridge, so that a gap is necessarily formed between the rotating shaft and the cartridge, and the leakage risk of the internal sample in the treatment process is caused.
The upper surface of the cartridge 1 is provided with a suction head 11 in a protruding way downwards, the suction head 11 is butted with a suction head hole on the cartridge 1, the suction head 11 is connected with a pneumatic device of a detection instrument, power is provided for the suction head 11, and the suction head 11 is always butted with the detection instrument and always kept closed; when the movable body 2 is lifted, the suction head 13 can be inserted into the chamber of the movable body 2, and the liquid in the corresponding chamber can be sucked up through the suction head 13 or blown and mixed.
Preferably, the power magnet 3 is arranged as an electromagnet, the position of the power magnet 3 is kept relatively fixed, and the existence and the magnitude of magnetic force are controlled by changing the electrified state, so that the control is more flexible and convenient.
The cartridge 1 of the present invention is cylindrical, and the movable body 2 is also provided in a cylindrical shape.
The power magnet 3 can rotate circumferentially, and the power magnet 3 drives the movable body 2 to horizontally move in the card box 1 to horizontally rotate circumferentially. The power magnet 3 rotates along the axis, the power magnet 3 is arranged on the rotating shaft, the power magnet 3 and the motor are driven by a gear or belt, 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 adsorbed to the position of the card box 1 near the lower surface and rotates, and after rotating by 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 mode 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 may be realized by the attractive force of a permanent magnet, in addition to the attractive force of an electromagnet. The attraction and release of the movable body 2 are achieved by changing the position of the permanent magnet, and these specific embodiments are included in the scope of the present invention.
As shown in fig. 3, which is an exploded view of the assembly of the cartridge 1 and the movable body 2, on the basis of the above scheme, the present invention is provided with a jack spring 4 at the bottom of the movable body 2, and fig. 4A is a sectional view in A-A direction in fig. 1C; FIG. 4B is a cross-sectional view taken along the direction B-B in FIG. 1E; fig. 4C and 4D are sectional views of fig. 1E in the direction C-C, respectively, fig. 4C showing the state where the movable body 2 is above, and fig. 4D showing the state where the movable body 2 is below; FIG. 4E is a cross-sectional view taken along the direction D-D in FIG. 1E; the top end of the jacking spring 4 is contacted with the movable body 2, the bottom end is contacted with the lower surface of the cartridge 1, when the jacking spring 4 is compressed, upward elastic force is applied to the movable body 2, the movable body 2 is moved upward through the jacking spring 4, the movable body 2 is jacked on the upper surface of the cartridge 1, and thus the suction head 13 stretches into the cavity. The downward movement of the movable body 2 is realized by the attractive force of the power magnet 3, and the attractive force of the power magnet 3 to the card box 1 is larger than the elastic force of the jacking spring 4.
In addition to the use of the jack spring 4 to provide upward force to the movable body 2, a permanent magnet may be disposed at a specific position at the bottom of the movable body 2, that is, the ferromagnetic material layer described above uses a magnet; by changing the pole direction of the power magnet 3, these specific embodiments are intended to be included in the scope of the present invention.
Preferably, a buffer spring 5 can be further arranged at the top of the movable body 2, the elastic coefficient of the buffer spring 5 is smaller than that of the jacking spring 4, the top end of the buffer spring 5 is in contact with the upper surface of the card box 1, the bottom end of the buffer spring 5 is in contact with the movable body 2, when the movable body 2 moves upwards, the buffer spring 5 is compressed, the buffer spring 5 plays a role in buffering the movable body 2, and the card box 1 is prevented from being impacted with the upper surface of the card box 1 when moving upwards.
In the cylindrical cavity that jacking spring 4 and buffer spring 5 set up in the movable body 2 center set up, each cavity of movable body 2 is central symmetry around the drum in center and distributes, sets up the division board in the cylindrical cavity in the middle of the movable body 2 level for with jacking spring 4 and buffer spring 5 interval.
On the basis of any one of the technical schemes and the mutual combination, the invention also 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 infusion tube 7 in fig. 4D, when the movable body 2 is at the bottom, the infusion tube 7, in which the inside of the reaction chamber 6 and the inside of the movable body 2 are butted, inputs a sample, the flow direction of which is shown by an arrow in the figure; the infusion line 7 comprises two parts, one part being inside the reaction chamber 6 and the other part being on the movable body 2, the two parts of the infusion line 7 being staggered or butted with each other when the movable 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 cartridge 1, namely, the movable body 2 rotates to a specific angle, the conduction is realized when the movable body is positioned at a specific vertical height, and the liquid flows in an S shape when the infusion pipeline 7 is conducted.
The movable body 2 moves to the upper side by the elasticity of the jacking spring 4, at the moment, the interface of the infusion pipeline 7 on the movable body 2 is staggered with the infusion pipeline 7 on the cartridge 1, and the liquid in the corresponding cavity is sucked by the suction head 13; the movable body 2 moves to the lower side under the attractive force of the power magnet 3, at the moment, the interface of the infusion pipeline 7 on the movable body 2 is mutually butted with the infusion pipeline 7 on the cartridge 1 to form a complete channel, and the samples processed in the corresponding chamber are upwards drawn out 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 air extractor to extract a sample to the reaction chamber 6, the negative pressure air extractor is of a detection instrument structure, and when the reaction chamber 6 is communicated with the infusion pipeline 7 segmented on the movable body 2, negative pressure is formed at the sample loading hole 13, so that the sample in the chamber flows upwards along the infusion pipeline 7 and enters the reaction chamber 6.
The invention provides another preferred embodiment, wherein the adsorption electromagnet 8 is arranged on the outer side wall of the cartridge 1, and the adsorption electromagnet 8 can be fixed on the outer surface of the cartridge 1 or can be arranged on a detection instrument; when the adsorption electromagnet 8 is electrified, the adsorption electromagnet 8 adsorbs magnetic beads placed in the cavity of the movable body 2, and the adsorption electromagnet 8 is used for extracting nucleic acid in a magnetic bead method.
As shown in fig. 5, a schematic structural view of the suction head 11 is shown; the middle part of the cavity of the suction head 11 is provided with an adsorption matrix 111, the top 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.
An openable or closable cover is rotatably provided on the sample hole 12, and is opened before injecting the sample into the movable body 2, and is closed after injecting the sample, and is rotatably connected to the upper surface of the cartridge 1 by a rotation shaft.
The loading hole 13 is provided with a membrane for blocking the passage of nucleic acid, and in general, the sample is drawn into the reaction chamber 6 and then the extraction operation of the loading hole 13 is stopped, but in order to prevent the nucleic acid from being drawn out, the loading hole 13 is provided with a filter membrane for blocking the nucleic acid, and the nucleic acid cannot pass through the filter membrane.
FIG. 6A is a flow chart of nucleic acid extraction by adsorption, and FIG. 6B is a flow chart of nucleic acid extraction by magnetic bead; when the adsorption method is used for extracting nucleic acid, the sample is firstly put into a lysate for pyrolysis, the detergent is added after centrifugation, and the eluent is added after centrifugation again, so that the purified product is finally obtained. When extracting nucleic acid by magnetic bead method, adding cracking liquid into sample for cracking, then adding magnetic beads to make them combined with nucleic acid, then adding washing liquid, adsorbing magnetic beads by iron-absorbing, extracting waste liquid, then adding eluent for eluting operation so as to obtain purified nucleic acid.
Before detection, the cartridge 1 is placed at a corresponding position on a detection instrument, and according to the difference of the adopted principles, the closed nucleic acid extraction and amplification device provided by the invention is used, and the specific operation process is as follows:
adsorption method for extracting nucleic acid
1. The power magnet 3 is in a non-magnetic state, and a sample is added into the chamber containing the lysis solution through the sample hole 12 to carry out the lysis process.
2. After the splitting is completed, the power magnet 3 is electrified, the whole movable body 2 is adsorbed by magnetic force to move downwards, and the movable body 2 is not blocked by the suction head 11 when rotating.
3. The power magnet 3 outside the cartridge 1 is rotated by mechanical force, the power magnet 3 drives the movable body 2 inside the cartridge 1 to rotate, and the cavity containing the lysis solution and the sample is rotated to a position below the suction head 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 to the upper side of the cartridge 1 under the action of the jacking spring 4, so that the suction head 11 is inserted into a chamber filled with the lysis solution and the sample.
5. A pneumatic device on the detecting instrument sucks the lysate and the sample mixed solution through the suction head 11. The tips 11 are sequentially inserted into the respective chambers containing the lysis solution and the sample-binding solution according to the procedure of 2 to 4 described above. Repeatedly blowing and sucking air through an external pneumatic device, blowing the mixed solution of the cracking solution and the sample, and uniformly mixing the cracking solution with the mixed solution of the sample and the combined solution through repeated blowing; the nucleic acid in the sample is adsorbed on the adsorption matrix 131 in the tip 11, and the liquid in the tip 11 is discharged, and at this time, a large amount of nucleic acid is adsorbed in the tip 11.
6. According to the above-described procedures 2 to 4, the nucleic acid-adsorbing tip 13 is inserted into the chamber containing the washing liquid, and the tip 11 is repeatedly blown so that the adsorbing substrate 111 in the tip 11 is washed with the washing liquid, and the liquid in the tip 11 is evacuated.
7. According to the procedure of 2 to 4 described above, the tip 11 is inserted into the chamber containing the eluent, and the tip is repeatedly blown so that the nucleic acid adsorbed on the adsorption substrate 111 in the tip 11 elutes into the chamber containing the eluent, and the liquid in the tip 11 is evacuated.
8. According to the method 2-3 described above, the chamber of the movable body 2 containing the eluent and nucleic acid is rotated to a position close to the reaction chamber 6, and the magnet 3 is kept in a magnetic state at this time, and the movable body 2 is communicated with the infusion line 7 on the cartridge 1.
9. The eluent is poured into the reaction chamber 6 by the suction effect of the pneumatic device connected to the loading hole 13, and the reaction chamber 6 is pre-filled with the freeze-dried reagent required for the PCR reaction.
10. When the power magnet 3 is powered off and is in a non-magnetic state, the movable body 2 moves to the upper side of the cartridge 1 under the action of the jacking spring 4, and the movable body 2 is staggered with the infusion pipeline 7 on the cartridge 1, so that the reaction chamber 6 is relatively isolated from the movable body 2.
11. The heating and cooling 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.
The magnetic bead method is used for extracting nucleic acid, and the structure of the adsorption electromagnet is driven as shown in figure 3
1. The adsorption electromagnet 8 and the bottom electromagnet 3 are in a non-magnetic state, the whole body is in a state shown in fig. 3, and a sample is added into a chamber containing a lysis solution through a sample hole 12 for lysis.
2. After the splitting is completed, the power magnet 3 is electrified and magnetized to suck the whole movable body 2 down.
3. The dynamic magnet 3 at the bottom is rotated by mechanical force to drive the movable body 2 in the cartridge 1 to rotate circumferentially, so that the chamber filled with the lysis solution and the sample rotates to a position below the suction head 11.
4. The bottom power magnet 3 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 suction head 11 is inserted into the chamber containing the lysis solution and the sample.
5. A pneumatic device connected to the tip hole sucks the lysate and the sample mixture through the tip 11. Repeating the steps of inserting the suction head 11 into the chamber containing the mixture of the magnetic beads and the binding liquid according to the steps of 2-4; alternatively, the beads may be placed in a single chamber, and the beads may be sucked out through the suction head 11 and transferred to the chamber containing the binding liquid. And blowing the mixed solution of the lysate and the sample by a pneumatic device of the detection instrument. And (3) uniformly mixing the lysate with the sample mixed solution, the binding solution and the magnetic beads by repeated blowing. At this time, the nucleic acid released from the lysate is adsorbed on the surface of the magnetic beads under the action of the binding solution.
6. Standing, electrifying and magnetizing the adsorption electromagnet 8 on the side surface of the card box 1, adsorbing the magnetic beads on the inner side wall of the chamber, sucking the liquid in the chamber by the suction head 11, and transferring the sucked liquid waste into the liquid waste collecting chamber according to the process of 2-4.
7. According to the procedure of 2-4, the suction head 11 is inserted into a chamber containing a cleaning liquid, the cleaning liquid is sucked up, transferred into the chamber where the magnetic beads are located, the suction electromagnet 8 is powered off, the magnetic beads are mixed with the cleaning liquid, and repeatedly blown through the suction head 11. Then electrifying and magnetizing the adsorption electromagnet 8 again, adsorbing the magnetic beads on the side wall of the chamber, sucking out the liquid in the chamber, and transferring the liquid into a waste liquid collecting chamber.
8. According to the procedure of 2-4, the tip 11 is inserted into a chamber containing an eluent to aspirate the eluent and transferred to the chamber in which the magnetic beads are located. The electromagnet 8 on the side wall of the card box 1 is powered off to remove magnetism, and the suction head 11 repeatedly blows and mixes. The adsorption electromagnet 8 is electrified and magnetized again to adsorb the magnetic beads, and the liquid is sucked through the suction head 11. The eluate was transferred to another chamber according to the procedure of 2-4.
9. According to the procedure of 2-3, the cavity containing the eluent and 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 keeps an electrified magnetic state, and the infusion pipeline 7 on the movable body 2 is conducted relatively to the infusion pipeline 7 on the cartridge 1.
10. The eluent is poured into the reaction chamber 6 by the suction effect of the pneumatic device connected to the loading hole 13, and the reaction chamber 6 is pre-filled with the freeze-dried reagent required for the PCR reaction.
11. The power magnet 3 is powered off and is in a non-magnetic state, the movable body 2 moves to the upper side of the card box 1 under the action of the jacking spring 4, the infusion pipeline 7 on the movable body 2 is disconnected from the infusion pipeline 7 on the card box 1, and the reaction chamber 6 is relatively isolated from the chamber in the movable body 2.
12. The heating and cooling 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.
The cartridges of the prior art designs tend to be complex in structure and not sealed, and in order to change the position of the interior of the container or drive the movement of the liquid within the container, mechanical movement within the cartridge is often involved, but gaps between moving and non-moving parts tend to cause sample leakage contamination. The present invention uses magnetic force to drive the moving parts. The whole cartridge is sealed relative to the transmission device, so that the possibility of leakage pollution is greatly reduced.
The device provided by the invention can be used for extracting and amplifying the nucleic acid in the sample, has simple and reliable structure and low manufacturing cost, and is convenient to popularize and use. In particular, the electromagnetic adsorption is adopted to change the position between the suction head and the cavity in the closed cartridge, so that the tightness of the whole cartridge is ensured, and pollution is prevented. In addition, the electromagnetic force is used for controlling the infusion pipeline, 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. The 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 smaller than the height of an inner cavity of the card box (1), and the movable body (2) can vertically move and horizontally move 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 move vertically and can drive the movable body (2) to move horizontally relative to the card box (1);
the cartridge (1) is provided with at least one sample hole connected with the outside for adding a sample into the movable body (2).
2. The nucleic acid extraction and amplification apparatus according to claim 1, wherein a tip (11) is provided so as to protrude downward from an inner cavity of the cartridge (1), and the tip (11) is insertable into a chamber 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 2, wherein a jack spring (4) is provided at the bottom of the movable body (2), and the jack spring (4) applies an upward elastic force to 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 device according to claim 6, wherein a buffer spring (5) is provided at the top of the movable body (2), and the buffer spring (5) is capable of being buffered 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 jack 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 according to 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, a transfusion pipeline (7) which is in butt joint with the inside of the reaction chamber (6) and the inside of the movable body (2) inputs a sample.
10. The nucleic acid extraction and amplification device according to claim 9, wherein a sample loading hole (13) is formed in the top of the reaction chamber (6), and the sample loading hole (13) is connected with a negative pressure air suction device to draw a sample into 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 provided on an outer side wall of the cartridge (1), the adsorption electromagnet (8) being for adsorbing magnetic beads placed in a chamber of the movable body (2).
12. The nucleic acid extraction and amplification device according to claim 2, wherein an adsorption matrix (111) is disposed in the middle of the inner cavity of the suction head (11), and a filter membrane (112) is disposed on the top, the adsorption matrix (111) is used for adsorbing nucleic acid, and the filter membrane is used for preventing liquid or nucleic acid from overflowing.
13. The nucleic acid extraction and amplification apparatus according to any one of claims 1 to 4, wherein an openable or closable closure is rotatably provided on the sample well (12).
14. The nucleic acid extraction and amplification apparatus as set forth in claim 10, wherein the loading well (13) is provided with a filter membrane for blocking passage of nucleic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010218527.7A CN111378574B (en) | 2020-03-25 | 2020-03-25 | Nucleic acid extraction and amplification device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010218527.7A CN111378574B (en) | 2020-03-25 | 2020-03-25 | Nucleic acid extraction and amplification device |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111378574A CN111378574A (en) | 2020-07-07 |
| CN111378574B true CN111378574B (en) | 2023-10-27 |
Family
ID=71213889
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010218527.7A Active CN111378574B (en) | 2020-03-25 | 2020-03-25 | Nucleic acid extraction and amplification device |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111378574B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226358B (en) * | 2020-12-11 | 2021-03-23 | 博奥生物集团有限公司 | Nucleic acid analysis card box and nucleic acid analysis equipment |
| CN114032173B (en) * | 2022-01-11 | 2022-03-15 | 至美时代生物智能科技(北京)有限公司 | Closed air sampling bottle |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1521398A (en) * | 2003-01-28 | 2004-08-18 | �廪��ѧ | Fluid transmission method and minisize peristaltic pump for realizing the same |
| CN103571740A (en) * | 2013-10-15 | 2014-02-12 | 周国华 | Device integrated with extraction, amplification and detection of nucleic acid |
| JP2015019590A (en) * | 2013-07-16 | 2015-02-02 | セイコーエプソン株式会社 | Nucleic acid amplification reaction apparatus |
| CN104673625A (en) * | 2015-02-13 | 2015-06-03 | 西安交通大学 | Automatic reaction device and method for pretreating cells |
| CN105021835A (en) * | 2015-07-28 | 2015-11-04 | 东南大学 | Magnetically driven biological sample treatment device and treatment method thereof |
| CN205317791U (en) * | 2015-12-09 | 2016-06-15 | 中国科学院苏州生物医学工程技术研究所 | Blood group card centrifuge with card box position appearance guiding mechanism |
| CN107034134A (en) * | 2017-06-08 | 2017-08-11 | 湖南工业大学 | A kind of rotary detection of nucleic acids cartridge |
| CN107271703A (en) * | 2016-04-08 | 2017-10-20 | 爱-森新株式会社 | The circular cartridge that can centrifuge and utilize its modular automatic analyzer |
| CN107653301A (en) * | 2017-10-16 | 2018-02-02 | 南京中科拜尔医学技术有限公司 | A kind of hermetic multiple fluorescent PCR detection cartridge |
| CN107760602A (en) * | 2017-11-06 | 2018-03-06 | 南京中科拜尔医学技术有限公司 | A kind of cartridge for PCR primer purifying |
| CN109370896A (en) * | 2018-11-21 | 2019-02-22 | 南京中科拜尔医学技术有限公司 | Cartridge is used in a kind of closed four-way multiple fluorescence PCR detection |
-
2020
- 2020-03-25 CN CN202010218527.7A patent/CN111378574B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1521398A (en) * | 2003-01-28 | 2004-08-18 | �廪��ѧ | Fluid transmission method and minisize peristaltic pump for realizing the same |
| JP2015019590A (en) * | 2013-07-16 | 2015-02-02 | セイコーエプソン株式会社 | Nucleic acid amplification reaction apparatus |
| CN103571740A (en) * | 2013-10-15 | 2014-02-12 | 周国华 | Device integrated with extraction, amplification and detection of nucleic acid |
| CN104673625A (en) * | 2015-02-13 | 2015-06-03 | 西安交通大学 | Automatic reaction device and method for pretreating cells |
| CN105021835A (en) * | 2015-07-28 | 2015-11-04 | 东南大学 | Magnetically driven biological sample treatment device and treatment method thereof |
| CN205317791U (en) * | 2015-12-09 | 2016-06-15 | 中国科学院苏州生物医学工程技术研究所 | Blood group card centrifuge with card box position appearance guiding mechanism |
| CN107271703A (en) * | 2016-04-08 | 2017-10-20 | 爱-森新株式会社 | The circular cartridge that can centrifuge and utilize its modular automatic analyzer |
| CN107034134A (en) * | 2017-06-08 | 2017-08-11 | 湖南工业大学 | A kind of rotary detection of nucleic acids cartridge |
| CN107653301A (en) * | 2017-10-16 | 2018-02-02 | 南京中科拜尔医学技术有限公司 | A kind of hermetic multiple fluorescent PCR detection cartridge |
| CN107760602A (en) * | 2017-11-06 | 2018-03-06 | 南京中科拜尔医学技术有限公司 | A kind of cartridge for PCR primer purifying |
| CN109370896A (en) * | 2018-11-21 | 2019-02-22 | 南京中科拜尔医学技术有限公司 | Cartridge is used in a kind of closed four-way multiple fluorescence PCR detection |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111378574A (en) | 2020-07-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8062846B2 (en) | Apparatus for isolating a nucleic acid from a sample | |
| CN107249746B (en) | Systems and methods for collecting nucleic acid samples | |
| CN111378574B (en) | Nucleic acid extraction and amplification device | |
| EP3216855A1 (en) | Cassette for sample preparation | |
| EP2871460B1 (en) | Kit for nucleic acid extraction, and nucleic acid extractor | |
| CN111909835A (en) | Closed micro-fluidic nucleic acid detection card box | |
| CN112538414A (en) | Full-sealed intelligent nucleic acid extraction device with micro-fluidic structure | |
| CN111575152A (en) | Closed card box integrating nucleic acid extraction, amplification and detection functions and detection method | |
| CN113817600A (en) | Sample processing and detecting device and application thereof | |
| CN110241008A (en) | A kind of device for extracting and detecting for large biological molecule | |
| US7449326B2 (en) | System for simple nucleic acid analysis | |
| CN115161188A (en) | Air microorganism detection system and method | |
| US11565233B2 (en) | Integrated tubular reaction device | |
| CN214937358U (en) | Full-sealed intelligent nucleic acid extraction device with micro-fluidic structure | |
| CN219032123U (en) | Nucleic acid purification and detection reaction liquid preparation device | |
| CN115651835A (en) | Gene detection processing device and method | |
| CN112342128B (en) | Nucleic acid extraction amplification test tube and working method thereof | |
| CN210945545U (en) | High-flux micro-fluidic rapid nucleic acid extraction and detection device | |
| CN109072230B (en) | Method and system for magnetically extracting components in liquid sample | |
| CN213506849U (en) | Magnetic needle and cannula system and apparatus for extracting biologically active substances | |
| CN213327539U (en) | Magnetic needle and cannula system and apparatus for extracting biologically active substances | |
| CN220317751U (en) | Nucleic acid extraction and purification device | |
| CN218491735U (en) | Full-automatic magnetic control multiple nested PCR detection test tube | |
| CN215947252U (en) | Liquid sample processing device | |
| CN113583826A (en) | Magnetic needle and cannula system for nucleic acid extractor and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |