CN108949498B - Totally-enclosed integrated reagent extraction and amplification device - Google Patents
Totally-enclosed integrated reagent extraction and amplification device Download PDFInfo
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- CN108949498B CN108949498B CN201810605360.2A CN201810605360A CN108949498B CN 108949498 B CN108949498 B CN 108949498B CN 201810605360 A CN201810605360 A CN 201810605360A CN 108949498 B CN108949498 B CN 108949498B
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- 230000003321 amplification Effects 0.000 title claims abstract description 107
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 107
- 238000000605 extraction Methods 0.000 title claims abstract description 71
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 62
- 238000010828 elution Methods 0.000 claims abstract description 57
- 238000005406 washing Methods 0.000 claims abstract description 47
- 238000005336 cracking Methods 0.000 claims abstract description 44
- 239000007788 liquid Substances 0.000 claims abstract description 43
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 39
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 39
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 39
- 238000005192 partition Methods 0.000 claims abstract description 34
- 239000000126 substance Substances 0.000 claims abstract description 33
- 239000003480 eluent Substances 0.000 claims abstract description 8
- 238000007789 sealing Methods 0.000 claims description 82
- 230000009089 cytolysis Effects 0.000 claims description 18
- 238000001125 extrusion Methods 0.000 claims description 10
- 238000003825 pressing Methods 0.000 claims description 6
- 238000011144 upstream manufacturing Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 2
- 239000012744 reinforcing agent Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 24
- 238000006243 chemical reaction Methods 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- 239000011324 bead Substances 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 5
- 238000012864 cross contamination Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 230000006835 compression Effects 0.000 description 2
- 238000007906 compression Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000013013 elastic material Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 238000011901 isothermal amplification Methods 0.000 description 2
- 230000005389 magnetism Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
Classifications
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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 provides a totally-enclosed integrated reagent extraction and amplification device, which comprises: the extraction bin comprises a cracking mechanism, a washing mechanism and an eluting mechanism which are sequentially distributed according to the flow direction of a sample; the cracking mechanism is internally provided with a cracking reagent and a magnetic substance; the washing mechanism comprises a first oil groove and a washing groove, and washing liquid is arranged in the washing groove; the first oil groove is positioned between the cracking mechanism and the washing groove; the elution mechanism comprises a second oil groove and an elution groove, and eluent is arranged in the elution groove; the second oil groove is positioned between the washing groove and the eluting groove; the first oil groove and the second oil groove are internally provided with oil phases; a movable partition is arranged between two adjacent grooves; a movable partition is arranged between the cracking mechanism and the first oil groove; the driving mechanism comprises a sliding block which can slide according to the flow direction of the sample; the sliding block is provided with a magnet; an amplification tube downstream of the elution mechanism; the amplification tube is communicated with the elution tank. The invention can integrate the extraction and amplification detection of nucleic acid in a fully-closed environment, and has high efficiency, simplicity and convenience and accurate result.
Description
Technical Field
The invention relates to a totally-enclosed integrated reagent extraction and amplification device.
Background
Nucleic acid diagnostic analysis methods based on PCR technology are core technologies in molecular diagnostics. The automatic nucleic acid extraction process and the PCR amplification detection process are mutually integrated to construct an integrated nucleic acid extraction and amplification detection system, and the method has important practical significance for further improving the nucleic acid diagnosis analysis efficiency and realizing the automation of the whole nucleic acid diagnosis analysis process.
Chinese patent 201120449250.5 discloses a nucleic acid amplification and detection reaction tube, which comprises a tube body and a tube cover which are matched with each other, wherein the tube body comprises a liquid storage area positioned above and a nucleic acid amplification area positioned below, and real-time detection means such as reaction, fluorescent signal acquisition and the like are completed in the nucleic acid amplification area; the tube cover is internally provided with a hollow groove, and the volume of the nucleic acid amplification area is respectively smaller than or equal to the volume of the liquid storage area and the volume of the hollow groove.
Chinese patent 201521043284.9 discloses an isothermal nucleic acid amplification device and an experimental method, wherein the isothermal nucleic acid amplification device comprises a flat plate in-situ nucleic acid amplification chip, the chip is composed of an upper layer carrier and a lower layer carrier which are mutually attached and can slide, the upper layer carrier and the lower layer carrier are rectangular with the same size, and when the lower layer is fixed, the upper layer carrier can slide by relative friction. The upper joint surface of the lower carrier is provided with various downward opening groove flow channels and microcavities, and the lower joint surface of the upper carrier is provided with various upward opening groove flow holes and microcavities, and a plurality of inlet and outlet through holes.
Chinese patent 201620284715.9 discloses a multifunctional nucleic acid amplification instrument, which comprises a control system, a switch power supply, an upper computer, a heating circulation system and a sample module, wherein the control system is connected with the switch power supply, the upper computer and the heating circulation system, the sample module is connected above the heating circulation system, and a 384-pore plate, a 96-pore plate, a 48-pore plate and a 12-pore plate with the volume of 0.2mL are arranged on the sample module.
Chinese patent application 201710163203.6 discloses an integrated nucleic acid extraction and amplification detection system comprising a disposable microfluidic chip and a mating detection device. The microfluidic chip comprises a reaction unit and a waste liquid storage unit, when the chip works, the reaction reagents and the magnetic beads are mutually mixed by means of the vibration module, and the steps of nucleic acid adsorption, purification and the like are sequentially completed by means of the magnetic bead driving module; and (3) flushing the magnetic beads by using the PCR amplification reagent through the fluid driving module, eluting the amplification templates on the magnetic beads, entering a convection PCR reactor constructed by a vertical channel, and realizing PCR amplification reaction and detection through the heating module and the fluorescence acquisition module.
The technical scheme has the defects that:
1. the structure is complex, and a vacuum pump is needed; the pyrolysis liquid is sticky, and bubbles are easy to generate when the vacuum pump operates, so that extraction is influenced;
2. the waste liquid can pass through the nucleic acid amplification detection cavity, and the waste liquid is polluted by residues, so that the nucleic acid amplification detection is affected;
3. the detection process is not closed, so that the environment outside the detection device is contacted with the reagent in the detection device, and the risk of cross contamination exists between the environment outside the detection device and the reagent;
4. the detection process is complicated, and manual operation is required to be added in a plurality of links;
5. the detection time is long.
Therefore, how to provide a vacuum pump-free device with simpler structure; no bubbles are generated, so that the smooth extraction is facilitated, and the nucleic acid with higher purity is obtained; the nucleic acid amplification detection cavity is independent, and no influence of residual waste liquid exists; the reagent extraction and amplification device which has a closed detection process and can effectively avoid cross contamination of an external environment and a detection reagent becomes a problem to be solved in the industry.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a fully-closed integrated reagent extraction and amplification device which has a simple structure, is efficient and accurate, has a closed detection process and can effectively avoid cross contamination of an external environment and a detection reagent.
In order to achieve the above object, the present invention provides a totally enclosed integrated reagent extraction and amplification device, comprising:
the extraction bin comprises a cracking mechanism, a washing mechanism and an eluting mechanism which are sequentially distributed according to the flow direction of a sample; the cracking mechanism is internally provided with a cracking reagent and a magnetic substance; the washing mechanism comprises a first oil groove and a washing groove, and washing liquid is arranged in the washing groove; the first oil groove is positioned between the cracking mechanism and the washing groove; the elution mechanism comprises a second oil groove and an elution groove, and eluent is arranged in the elution groove; the second oil groove is positioned between the washing groove and the eluting groove; the first oil groove and the second oil groove are internally provided with oil phases; a movable partition is arranged between two adjacent grooves; a movable partition is arranged between the cracking mechanism and the first oil groove;
the driving mechanism comprises a sliding block which can slide according to the flow direction of the sample; the sliding block is provided with a magnet;
an amplification tube downstream of the elution mechanism; the amplification tube is communicated with the elution tank.
In the present invention, the extraction cartridge may be made of an elastic material.
In the present invention, the number of the tanks in the extraction chamber may be varied depending on the reagent to be carried or the solid matter to be carried.
In the invention, the magnet is positioned in the center of the sliding block and is a permanent magnet or an electromagnet; the magnetic substance may be a magnetic bead.
In the invention, the driving mechanism further comprises a power component for driving the sliding block to operate, and the power component can be a motor or a cylinder.
In the invention, nucleic acid generated after sample cleavage is attached to a magnetic substance; when the sliding block moves, the magnet drives the magnetic substance to move, so that the nucleic acid is driven to move.
In the invention, each groove is distributed along a straight line; in the use process, the extraction bin can be horizontally placed, vertically placed or obliquely placed.
In the invention, when the sliding block passes through the partition, the driving mechanism can cause the partition to fail, and the grooves on two sides of the partition are communicated; the partial partition is permanently disabled and the partial partition is restored after the slider passes.
The invention solves the technical problems that the nucleic acid extraction and the nucleic acid amplification detection cannot be integrated, the operation of the nucleic acid extraction and the nucleic acid amplification detection is complicated, and has high efficiency, simplicity and convenience and accurate result.
According to another embodiment of the invention, the lysis mechanism comprises a magnetic tank, a lysis tank and a combination tank which are distributed in sequence according to the flow direction of the sample; a movable partition is arranged between two adjacent grooves in the cracking mechanism; a movable partition is arranged between the combining groove and the first oil groove.
Sample cap according to another embodiment of the invention, the lysis mechanism further comprises a sample well upstream of the magnetic well, the sample being located in the sample well; the cracking reagent comprises a cracking reinforcing agent and a cracking binding solution which are respectively arranged in the cracking tank and the binding tank; the magnetic substance is arranged in the magnetic groove.
In this scheme, be equipped with the feed inlet that is used for adding the sample on the sample groove, be equipped with the sample lid on the feed inlet for the sample adds back closed sample groove.
According to another embodiment of the invention, the extraction bin further comprises a buffer tank, wherein the buffer tank is positioned at the downstream of the elution tank and is provided with a movable partition with the elution tank; the buffer tank is positioned at the upstream of the amplification tube and is communicated with the amplification tube.
In the scheme, a buffer liquid tank is connected with an amplification tube through a connector, and a discharge hole is arranged on the connector; the sample flows into the amplification tube from the buffer liquid tank through the discharge port.
In addition, the amplification reaction may be performed in the buffer tank without providing an amplification tube; at this time, a connector is not required to be arranged, and a discharge hole is directly arranged on the buffer tank and used for flowing out the sample after the amplification reaction.
According to another specific embodiment of the invention, a sealing piece capable of moving up and down is arranged between the first oil groove and the washing groove; and a sealing piece capable of moving up and down is arranged between the second oil groove and the elution groove.
According to another specific embodiment of the invention, a detachable fixed card is arranged between two adjacent grooves in the cracking mechanism; a fixing card which can fall off is arranged between the combining groove and the first oil groove; a fixing card which can fall off is arranged between the washing tank and the second oil tank; a fixing card which can fall off is arranged between the eluting groove and the buffer liquid groove.
According to another embodiment of the invention, the extraction cartridge further comprises an elastic sealing membrane, which is located below the sealing plate and the fixing card and is pressed by the sealing plate and the fixing card.
According to another embodiment of the invention, the sliding block is provided with a wedge-shaped block for jacking up the sealing piece or enabling the fixing card to fall off.
In this scheme, the reagent extracts the amplification device and further includes upper cover, support and bottom cover; the bracket is fixed on the extraction bin, and the bracket and the extraction bin are combined in a bonding mode, a cementing mode, a thermal bonding mode and the like; the upper cover and the bottom cover are detachably connected, a cavity is formed by the upper cover and the bottom cover, and the extraction bin and the bracket are positioned in the cavity; the bracket is provided with an upper cover mounting hole for being connected with the upper cover.
The bottom of the extraction bin is sealed by a sealing film, and the sealing film can seal liquid or solid in each groove in the extraction bin; the sealing film and the extraction bin can be combined in a bonding, cementing, thermal bonding and other modes; the elastic sealing film is positioned above the sealing film, and is attached to the sealing film under the compression action of the fixing card and the sealing sheet to isolate liquid or solid between the grooves.
The fixed card is used for blocking the reagent, so that the reagent in two adjacent grooves is prevented from being mixed, and the quantity of the fixed card is determined according to the reaction requirement; two sides of the fixed card are respectively connected with the bottom of the upper cover through weak supports; the weak support is used for connecting the fixed card and the upper cover, and is easily cut off.
The support is provided with a limit groove for limiting the degree of freedom of the sealing piece, and the limit groove is arranged corresponding to the sealing piece; the sealing sheet is used for preventing the reagents in the two adjacent tanks from being mixed; the top of the inner side of the upper cover is provided with a spring mounting hole which is arranged corresponding to the sealing piece; the sealing piece comprises a protrusion for pressing the elastic sealing film and a pin shaft for being matched with the spring, the protrusion is positioned at the bottom of the sealing piece, and the pin shaft is positioned at the top of the sealing piece; one side of the spring is arranged in the spring mounting hole, and the other side of the spring is sleeved on the pin shaft.
The number of the wedge-shaped blocks is two, and the wedge-shaped blocks are respectively positioned at two sides of the magnet; the sliding block is positioned at the outer side of the bottom cover.
The middle part of the bottom cover is provided with a driving groove for moving the magnet, and two sides of the bottom cover are provided with slide ways for moving the wedge-shaped blocks; when the sliding block moves, the extraction bin is positioned between the two wedge-shaped blocks; the wedge block can cut off the weak support, so that the fixed card falls off; or the sealing plate is jacked up by the inclined plane at the top of the sealing plate.
The upper cover is provided with an extrusion hole, and the extrusion hole is positioned above the buffer tank; the extrusion hole is connected with a pressure plate which can be opened and closed, and the pressure plate can extrude the elution tank and the buffer tank when moving downwards.
According to another embodiment of the invention, a rotatable sealing shaft is arranged between two adjacent grooves, and a channel is arranged on the sealing shaft.
According to another specific embodiment of the invention, a sealing groove matched with the sealing shaft is arranged between two adjacent grooves, and the sealing groove is arranged corresponding to the sealing shaft.
In the scheme, the reagent extraction and amplification device further comprises a top cover, wherein the top cover is detachably connected with the top end of the extraction bin and seals each groove of the extraction bin; the size and shape of each groove of the extraction bin can be customized according to the needs, and the quantity is determined according to the reaction needs.
The sealing shaft is provided with a knob which is positioned above the channel; the top cover is provided with a plurality of rotating holes which are arranged corresponding to the sealing shafts; the knob passes through the corresponding rotation hole.
The top cover further comprises two piston holes which are respectively positioned above the elution tank and the buffer tank; each piston bore has a piston passing therethrough.
According to another specific embodiment of the invention, an amplification reagent for realizing nucleic acid incremental amplification is arranged in the amplification tube, and the amplification reagent is a PCR platform reagent or a isothermal amplification reagent.
The operating principle of the invention is as follows (the magnet is an electromagnet):
1. adding a sample into the sample tank, and sealing the feed inlet; simultaneously, the sliding block is arranged below the sample groove; at this time, the magnet is not electrified and has no magnetism;
2. the slide block moves towards the amplification tube direction, the partition is invalid, and the liquid flows into the magnetic groove;
3. the sliding block continues to move, the partition fails, and the liquid flows into the cracking groove;
4. the sliding block continues to move, the partition fails, and the liquid flows into the combining groove;
5. the magnetic substance is uniformly diffused into the cracking binding solution; the sample is lysed, releasing the nucleic acids; the nucleic acid is combined with the magnetic substance;
6. the magnet is electrified and starts to have magnetism; the sliding block continues to move, the partition fails, and the magnetic substance flows into the first oil groove;
7. the sliding block continues to move, the temporary failure is blocked, and the magnetic substance flows into the washing tank; when the sliding block is positioned below the washing tank, the partition between the first oil groove and the washing tank is restored, and the first oil groove and the washing tank are continuously partitioned;
8. the magnet is powered off, and magnetic substances are uniformly diffused into the washing liquid; impurities on the magnetic substance are washed away and transferred into the washing liquid;
9. the magnet is electrified, the sliding block continues to move, the failure is blocked, and the magnetic substance flows into the second oil groove;
10. the sliding block continues to move, the temporary failure is blocked, and the magnetic substance flows into the elution groove; when the sliding block is positioned below the elution groove, the partition between the second oil groove and the elution groove is restored, and the second oil groove and the elution groove are continuously partitioned;
11. the magnet is powered off, and magnetic substances are uniformly diffused into the eluent; nucleic acid on the magnetic substance is washed off and transferred into the eluent;
12. the magnet is electrified, the sliding block continues to move, the partition is invalid, and the liquid flows into the buffer liquid tank; the sliding block moves back and stops below the elution tank, so that the magnetic substances are kept in the elution tank; the nucleic acid is left in the buffer tank;
13. under external extrusion, the liquid in the buffer tank flows into the amplification tube, and nucleic acid amplification is carried out in the amplification tube.
Compared with the prior art, the invention has the following beneficial effects:
the invention can seamlessly combine nucleic acid extraction and nucleic acid amplification detection, and can obtain a result within 1 hour only by adding a sample; the whole process is carried out in a totally-enclosed environment without adding operation steps, so that the cross contamination of the external environment and the detection reagent can be effectively avoided; the cracking liquid, the washing liquid and the eluent are respectively fixed in the independent liquid storage cavities, the liquids are separated by one another by one by using the oil phase, and the liquids are not in direct contact with one another, so that cross contamination is avoided; the amplification tubes are also arranged independently, which is more beneficial to the amplification reaction.
The present invention will be described in further detail with reference to the accompanying drawings.
Drawings
FIG. 1 is an exploded view of a reagent extraction amplification device of example 1;
FIG. 2 is a cross-sectional view of the reagent extraction amplification device of example 1;
fig. 3 is a schematic structural view of the stent of embodiment 1;
fig. 4 is a schematic structural view of the upper cover of embodiment 1;
FIG. 5 is a schematic view of the structure of the slider of embodiment 1;
FIG. 6 is a schematic structural view of a sealing sheet of embodiment 1;
FIG. 7 is a schematic diagram showing the overall structure of a reagent extraction amplification apparatus of example 1;
FIG. 8 is an exploded view of the reagent extraction amplification device of example 2;
FIG. 9 is a cross-sectional view of a reagent extraction and amplification apparatus of example 2;
fig. 10 is a schematic view of the structure of the extraction cartridge of embodiment 2;
fig. 11 is a schematic structural view of the top cover of embodiment 2;
FIG. 12 is a schematic view of the structure of a slider of embodiment 2;
FIG. 13 is a schematic structural view of a seal shaft of embodiment 2;
FIG. 14 is a schematic diagram showing the overall structure of a reagent extraction amplification apparatus according to example 2.
Detailed Description
Example 1
The embodiment provides a totally-enclosed integrated reagent extraction and amplification device, which comprises an extraction bin 1, a cracking mechanism 2, a washing mechanism 3, an elution mechanism 4, a driving mechanism 5, an amplification tube 6, an upper cover 7, a bracket 8 and a bottom cover 9 as shown in figures 1-7.
Wherein, the extraction bin 1 is made of elastic material and comprises a cracking mechanism 2, a washing mechanism 3, an eluting mechanism 4, a buffer tank 101 and an elastic sealing film 104 which are distributed in sequence according to the flow direction of a sample; the bracket 8 is fixed on the extraction bin 1, and the bracket and the extraction bin are bonded through an adhesive; the upper cover 7 and the bottom cover 9 are detachably connected, a cavity is formed by the upper cover and the bottom cover, and the extraction bin 1 and the bracket 8 are positioned in the cavity; the bracket 8 is provided with an upper cover mounting hole 801 for connection with the upper cover 7.
The cracking mechanism 2 is internally provided with a cracking reagent and a magnetic substance; the magnetic substance is magnetic beads.
The washing mechanism 3 comprises a first oil groove 301 and a washing groove 302, and washing liquid is arranged in the washing groove 302; the first oil sump 301 is located between the lysis mechanism 2 and the wash sump 302.
The elution mechanism 4 comprises a second oil groove 401 and an elution groove 402, and an eluent is arranged in the elution groove 402; a second oil sump 401 is located between the wash tank 302 and the elution tank 402; an oil phase is arranged in each of the first oil groove 301 and the second oil groove 401; a movable partition is arranged between two adjacent grooves; a movable partition is arranged between the cracking mechanism 2 and the first oil groove 301.
The lysis mechanism 2 comprises a sample tank 201, a magnetic tank 202, a lysis tank 203 and a combination tank 204 which are distributed in sequence according to the flow direction of the sample; a movable partition is arranged between two adjacent grooves in the cracking mechanism 2; a movable partition is arranged between the combining groove 204 and the first oil groove 301; the sample tank 201 is provided with a feed port 205 for adding a sample, and the feed port 205 is provided with a sample cover 206 for closing the sample tank 201 after the sample is added.
The cracking reagent comprises a cracking enhancer and a cracking binding solution which are respectively arranged in the cracking tank 203 and the binding tank 204; the magnetic substance is disposed in the magnetic groove 202.
The driving mechanism 5 comprises a sliding block 501 capable of sliding according to the flow direction of a sample and a power component (not shown in the figure) for driving the sliding block 501 to operate, wherein the power component is a motor; a magnet 502 is arranged on the slide block 501; a magnet 502, which is an electromagnet, is located in the center of the slider 501.
The amplification tube 6 is positioned downstream of the elution mechanism 4; the amplification tube 6 is communicated with the elution tank 402; the amplification tube is internally provided with an amplification reagent for realizing the incremental amplification of nucleic acid, and the amplification reagent is a PCR platform reagent.
The buffer tank 101 is positioned at the downstream of the elution tank 402, and a movable partition is arranged between the buffer tank 101 and the elution tank 402; the buffer tank 101 is positioned upstream of the amplification tube 6 and is communicated with the amplification tube 6; the buffer tank 101 is connected with the amplification tube 6 through a connector 102, and a discharge port 103 is arranged on the connector 102; the sample flows into the amplification tube 6 from the buffer liquid tank 101 through the discharge port 103; the upper cover 7 is provided with an extrusion hole 701, and the extrusion hole 701 is positioned above the buffer tank 101; the extrusion hole 701 is connected with a pressure plate 702 which can be opened and closed, and the pressure plate 702 can extrude the elution tank 402 and the buffer tank 101 when moving downwards.
A sealing piece 11 which can move up and down is arranged between the first oil groove 301 and the washing groove 302; a sealing piece 11 which can move up and down is arranged between the second oil groove 401 and the elution groove 402; the bracket 8 is provided with a limit groove 802 for limiting the degree of freedom of the sealing piece 11, and the limit groove 802 is correspondingly arranged with the sealing piece 11; the sealing sheet 11 is used for preventing the reagents in the two adjacent tanks from being mixed; the top of the inner side of the upper cover 7 is provided with a spring mounting hole 703 which is arranged corresponding to the sealing piece 11; the sealing plate 11 comprises a protrusion 1101 for pressing the elastic sealing film 104 and a pin 1102 for matching with the spring 13, the protrusion 1101 is positioned at the bottom of the sealing plate 11, and the pin 1102 is positioned at the top of the sealing plate 11; one side of the spring 13 is arranged in the spring mounting hole 703, and the other side is sleeved on the pin 1102.
A detachable fixed card 12 is arranged between two adjacent grooves in the cracking mechanism 2; a detachable fixed card 12 is arranged between the combining groove 204 and the first oil groove 301; a detachable fixed card 12 is arranged between the washing tank 302 and the second oil tank 401; a detachable fixed card 12 is arranged between the elution groove 402 and the buffer tank 101; the fixing card 12 is used for reagent blocking and preventing the reagents in two adjacent grooves from being mixed; the two sides of the fixed card 12 are respectively connected with the bottom of the upper cover 7 through the weak supports 1201.
The elastic sealing film 104 is located below the sealing sheet 11 and the fixing card 12, and is pressed by the sealing sheet 11 and the fixing card 12.
The slide block 501 is provided with a wedge block 503 for jacking up the sealing piece 11 or falling off the fixing card 12; the number of the wedge blocks 503 is two, and the wedge blocks are respectively positioned at two sides of the magnet 502; the slider 501 is located outside the bottom cover 9; the middle part of the bottom cover 9 is provided with a driving groove 901 for the movement of the magnet 502, and two sides of the bottom cover are provided with slide ways 902 for the movement of the wedge-shaped blocks 503; when the slide block 501 moves, the extraction bin 1 is positioned between the two wedge blocks 503; the wedge block 503 can cut off the weak support 1201 to enable the fixed card 12 to fall off; or the sealing plate 11 is lifted up by the slope of the top thereof.
In the embodiment, the bottom of the extraction bin 1 is sealed by a sealing film 105, and the sealing film 105 can seal liquid or solid in each groove in the extraction bin 1; the sealing film 105 is adhered to the extraction bin 1 through an adhesive; the elastic sealing film 104 is located above the sealing film 105, and is attached to the sealing film 105 under the compression action of the fixing card 12 and the sealing sheet 11, so as to isolate the liquid or solid between the grooves.
The application method of the embodiment is as follows:
1. removing sample cap 206, adding sample to sample well 201, and capping sample cap 206;
2. the slide block 501 moves towards the direction of the amplification tube 6 under the drive of the power component; when the slider 501 passes over the fixed card 12, the wedge 503 cuts off the weak supports 1201 on both sides of the fixed card 12; the fixing card 12 falls off, and the sealing effect is invalid; the elastic sealing film 104 tightly supported and sealed by the fixed card 12 is sprung up under the self elastic action, and never returns to the sealing state; at this time, a passage is formed, and the liquid in the grooves at both sides of the passage is communicated;
3. the sample flows into the magnetic groove 202, and the magnetic substance carries sample information; under the action of the magnet 502, the magnetic substance carrying the sample information moves continuously along with the slider 501; repeating step 2 while the card 12 is being held;
4. when the slider 501 passes the sealing piece 11, the sealing piece 11 springs up when the wedge-shaped block 503 passes, and the elastic sealing film 104 tightly pressed and sealed by the sealing piece 11 springs up under the self elastic action; the magnetic substance passes along with the slider 501; after the wedge-shaped block 503 passes, the sealing piece 11 compresses the elastic sealing film 104 again under the action of the spring 13, and the liquid on two sides of the sealing piece is blocked again;
5. when the magnetic substance remains in the elution tank 402 and the nucleic acid remains in the buffer tank 101, the pressing plate 702 moves downward, pressing the elution tank 402 and the buffer tank 101; the liquid in the buffer tank 101 flows into the amplification tube 6, and PCR amplification is performed in the amplification tube 6.
Example 2
This embodiment provides a totally enclosed integrated reagent extraction amplification device, as shown in FIGS. 8-14, comprising an extraction cartridge 21, a lysis mechanism 22, a washing mechanism 23, an elution mechanism 24, a driving mechanism 25, an amplification tube 26 and a top cover 27.
Wherein the extraction cartridge 21 comprises a lysis mechanism 22, a washing mechanism 23, an elution mechanism 24 and a buffer tank 2101 which are distributed in sequence according to the flow direction of the sample.
The cracking mechanism 22 is internally provided with a cracking reagent and a magnetic substance; the magnetic substance is magnetic beads.
The washing mechanism 23 comprises a first oil groove 2301 and a washing groove 2302, and washing liquid is arranged in the washing groove 2302; a first oil sump 2301 is located between the lysing mechanism 22 and the wash sump 2302.
The elution mechanism 24 comprises a second oil groove 2401 and an elution groove 2402, and eluent is arranged in the elution groove 2402; the second oil sump 2401 is located between the wash tank 2302 and the elution tank 2402; an oil phase is arranged in each of the first oil groove 2301 and the second oil groove 2401; a movable partition is arranged between two adjacent grooves; a movable partition is provided between the cracking mechanism 22 and the first oil sump 2301.
The lysis mechanism 22 includes a sample cell 2201, a magnetic cell 2202, a lysis cell 2203, and a binding cell 2204, which are sequentially arranged in the sample flow direction; a movable partition is arranged between two adjacent grooves in the cracking mechanism 22; a movable partition is arranged between the combining groove 2204 and the first oil groove 2301; the sample slot 2201 is provided with a feed port 2205 for adding a sample, and the feed port 2205 is provided with a sample cover 2206 for closing the sample slot 2201 after the sample is added.
The lysis reagent comprises a lysis enhancer and a lysis binding solution which are respectively arranged in the lysis trough 2203 and the binding trough 2204; the magnetic substance is disposed in the magnetic groove 2202.
The driving mechanism 25 includes a slide 2501 slidable in a sample flow direction and a power member (not shown) for driving the slide 2501 to operate, the power member being a motor; a magnet 2502 is arranged on the slide block 2501; a magnet 2502 is centrally located in the slide 2501, which is an electromagnet.
The amplification tube 26 is downstream of the elution mechanism 24; the amplification tube 26 is in communication with the elution tank 2402; the amplification tube is internally provided with an amplification reagent for realizing the incremental amplification of nucleic acid, and the amplification reagent is a isothermal amplification reagent.
Buffer tank 2101 is located downstream of elution tank 2402 and is provided with a movable partition from elution tank 2402; the buffer tank 2101 is located upstream of the amplification tube 26 and communicates with the amplification tube 26; the buffer tank 2101 is connected with the amplification tube 26 through a connector 2102, and a discharge port 2103 is arranged on the connector 2102; the sample flows from the buffer tank 2101 into the amplification tube 26 through the outlet port 2103.
In the embodiment, a rotatable sealing shaft 28 is arranged between two adjacent grooves, a channel 2801 and a knob 2802 are arranged on the sealing shaft 28, and the knob 2802 is positioned above the channel 2801; the top cover 27 is detachably connected with the top end of the extraction bin 21 and seals each groove of the extraction bin 21; a sealing groove 29 matched with the sealing shaft 28 is arranged between the two adjacent grooves, and the sealing groove 29 is arranged corresponding to the sealing shaft 28.
The top cover 27 is provided with a plurality of rotating holes 2701 and two piston holes 2702, which are arranged corresponding to the sealing shaft 28; knob 2802 passes through corresponding rotation hole 2701; two piston bores 2702 are located above elution tank 2402 and buffer tank 2101, respectively; each piston bore 2702 has a piston 2703 extending therethrough.
The application method of the embodiment is as follows:
1. removing the sample cap 2206, adding the sample, and covering the sample cap 2206; at this time, the connection line between the channel 2801 and the grooves at the two sides is vertical, so as to prevent the substances in the two grooves from flowing, and play a role in isolation;
2. the slide 2501 moves towards the amplification tube 26 under the drive of the power unit; when the slide 2501 passes the seal shaft 28, the seal shaft 28 rotates 90 ° and the channel 2801 communicates the adjacent two slots;
3. sample flows into the magnetic groove 2202 through the channel 2801, and the magnetic substance carries sample information; under the influence of the magnet 2502, the magnetic substance carrying the sample information continues to move with the slider 2501; step 2 is repeated while passing through the seal shaft 28; after the slide 2501 passes, the sealing shaft 28 between the first oil groove 2301 and the washing groove 2302 and the sealing shaft 28 between the second oil groove 2401 and the elution groove 2402 are rotated by 90 degrees to reseal the grooves on both sides; the remaining seal shaft 28 is no longer rotating;
4. when the magnetic substance remains in the elution tank 2402 and the nucleic acid remains in the buffer tank 2101, the piston 2703 is pushed downward, squeezing the buffer tank 2101; the liquid in the buffer tank 2101 flows into the amplification tube 26, and PCR amplification is performed in the amplification tube 26.
Example 3
This embodiment differs from embodiment 1 in that: the amplification reaction is carried out in the buffer liquid tank without an amplification pipe; at this time, a connector is not required to be arranged, and a discharge hole is directly arranged on the buffer tank and used for flowing out the sample after the amplification reaction.
Example 4
This embodiment differs from embodiment 2 in that: the extraction bin is vertically placed.
Example 5
This embodiment differs from embodiment 1 in that: a third oil groove is arranged between the buffer liquid groove and the amplification pipe, and an oil phase is arranged in the third oil groove; a fixing card capable of falling off is arranged between the buffer tank and the third oil tank, and a fixing card capable of falling off is arranged between the amplification tube and the third oil tank.
While the invention has been described in terms of preferred embodiments, it is not intended to limit the scope of the invention. It is intended that all modifications within the scope of the invention, i.e., all equivalents thereof, be embraced by the invention as they come within their scope without departing from the invention.
Claims (10)
1. A totally enclosed integrated reagent extraction amplification device, wherein the reagent extraction amplification device comprises:
the extraction bin comprises a cracking mechanism, a washing mechanism and an eluting mechanism which are sequentially distributed according to the flow direction of a sample; the cracking mechanism is internally provided with a cracking reagent and a magnetic substance; the washing mechanism comprises a first oil groove and a washing groove, and washing liquid is arranged in the washing groove; the first oil groove is positioned between the cracking mechanism and the washing groove; the elution mechanism comprises a second oil groove and an elution groove, and eluent is arranged in the elution groove; the second oil groove is positioned between the washing groove and the eluting groove; an oil phase is arranged in each of the first oil groove and the second oil groove; a movable partition is arranged between two adjacent grooves; a movable partition is arranged between the cracking mechanism and the first oil groove; the extraction bin further comprises a buffer liquid tank, wherein the buffer liquid tank is positioned at the downstream of the elution tank, and a movable partition is arranged between the buffer liquid tank and the elution tank;
the driving mechanism comprises a sliding block which can slide according to the flow direction of the sample; the sliding block is provided with a magnet;
an amplification tube downstream of the elution mechanism; the amplification tube is communicated with the elution groove; the buffer tank is positioned at the upstream of the amplification tube and is communicated with the amplification tube;
the upper cover is provided with an extrusion hole, and the extrusion hole is positioned above the buffer tank; the extrusion hole is connected with an openable pressing plate, and the pressing plate can extrude the elution groove and the buffer groove when moving downwards.
2. The reagent extraction amplification device according to claim 1, wherein the lysis mechanism comprises a magnetic tank, a lysis tank and a binding tank which are sequentially arranged in a sample flow direction; a movable partition is arranged between two adjacent grooves in the cracking mechanism; a movable partition is arranged between the combining groove and the first oil groove.
3. The reagent extraction amplification device of claim 2, wherein the lysis mechanism further comprises a sample well upstream of the magnetic well, a sample being located in the sample well; the cracking reagent comprises a cracking reinforcing agent and a cracking binding solution which are respectively arranged in the cracking groove and the binding groove; the magnetic substance is arranged in the magnetic groove.
4. The reagent extraction amplification device according to claim 3, wherein a sealing sheet movable up and down is provided between the first oil tank and the washing tank; and a sealing piece capable of moving up and down is arranged between the second oil groove and the elution groove.
5. The reagent extraction and amplification device according to claim 4, wherein a releasable fixing card is arranged between two adjacent grooves in the lysis mechanism; a fixing card capable of falling off is arranged between the combining groove and the first oil groove; a fixing card which can fall off is arranged between the washing groove and the second oil groove; and a fixing card which can fall off is arranged between the eluting groove and the buffer liquid groove.
6. The reagent extraction amplification device of claim 5, wherein the extraction cartridge further comprises an elastic sealing membrane positioned below and compressed by the sealing sheet and the fixing card.
7. The reagent extraction amplification device according to claim 6, wherein a wedge for lifting up the sealing sheet or releasing the fixing card is provided on the slider.
8. The reagent extraction amplification device according to claim 3, wherein a rotatable sealing shaft is provided between two adjacent cells, and a passage is provided in the sealing shaft.
9. The reagent extraction amplification device according to claim 8, wherein a seal groove is provided between two adjacent grooves, the seal groove being provided in correspondence with the seal shaft.
10. The reagent extraction amplification device according to claim 1, wherein an amplification reagent for performing incremental amplification of nucleic acids is provided in the amplification tube.
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| CN201810605360.2A CN108949498B (en) | 2018-06-12 | 2018-06-12 | Totally-enclosed integrated reagent extraction and amplification device |
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| CN108949498A CN108949498A (en) | 2018-12-07 |
| CN108949498B true CN108949498B (en) | 2024-01-30 |
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| CN108642049B (en) * | 2018-06-12 | 2023-09-29 | 广州和实生物技术有限公司 | Cut-off type reagent extraction and amplification device |
| CN110373309A (en) * | 2019-06-12 | 2019-10-25 | 广州知芯科技有限公司 | A kind of nucleic acid extraction amplification system and molecular detector arrangement |
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| CN1767897A (en) * | 2003-02-05 | 2006-05-03 | 伊库姆公司 | Sample processing tubule |
| CN105562132A (en) * | 2016-01-04 | 2016-05-11 | 上海医脉赛科技有限公司 | Device for extracting and detecting biological sample |
| CN107541462A (en) * | 2017-09-13 | 2018-01-05 | 北京理工大学 | One kind is used for the system and application process of nucleic acid purification, amplification and genetic test |
| CN107709992A (en) * | 2015-04-30 | 2018-02-16 | 新加坡科技研究局 | The new design and its application method of enzyme linked immunosorbent assay (ELISA) plate and system |
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| EP2418018B1 (en) * | 2004-12-23 | 2013-05-22 | Abbott Point of Care Inc. | Methods for the separation nucleic acids |
| DE102005029809B4 (en) * | 2005-06-27 | 2007-04-26 | Siemens Ag | Apparatus and method for preparing a sample for analysis and apparatus and method for analyzing a sample |
| US8728410B2 (en) * | 2010-02-26 | 2014-05-20 | Wisconsin Alumni Research Foundation | Device for and method of extracting a fraction from a biological sample |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1767897A (en) * | 2003-02-05 | 2006-05-03 | 伊库姆公司 | Sample processing tubule |
| CN107709992A (en) * | 2015-04-30 | 2018-02-16 | 新加坡科技研究局 | The new design and its application method of enzyme linked immunosorbent assay (ELISA) plate and system |
| CN105562132A (en) * | 2016-01-04 | 2016-05-11 | 上海医脉赛科技有限公司 | Device for extracting and detecting biological sample |
| CN107541462A (en) * | 2017-09-13 | 2018-01-05 | 北京理工大学 | One kind is used for the system and application process of nucleic acid purification, amplification and genetic test |
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