CN110257240B - Nucleic acid extraction, amplification and detection integrated reagent card - Google Patents
Nucleic acid extraction, amplification and detection integrated reagent card Download PDFInfo
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- CN110257240B CN110257240B CN201910643050.4A CN201910643050A CN110257240B CN 110257240 B CN110257240 B CN 110257240B CN 201910643050 A CN201910643050 A CN 201910643050A CN 110257240 B CN110257240 B CN 110257240B
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- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 191
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 191
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 191
- 238000001514 detection method Methods 0.000 title claims abstract description 94
- 230000003321 amplification Effects 0.000 title claims abstract description 78
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 78
- 238000000605 extraction Methods 0.000 title claims abstract description 72
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 61
- 239000007788 liquid Substances 0.000 claims abstract description 151
- 239000000523 sample Substances 0.000 claims description 88
- 238000007789 sealing Methods 0.000 claims description 59
- 239000002699 waste material Substances 0.000 claims description 44
- 238000009396 hybridization Methods 0.000 claims description 42
- 239000006249 magnetic particle Substances 0.000 claims description 40
- 238000003860 storage Methods 0.000 claims description 40
- 238000004090 dissolution Methods 0.000 claims description 32
- 238000001179 sorption measurement Methods 0.000 claims description 32
- 238000004140 cleaning Methods 0.000 claims description 27
- 238000010828 elution Methods 0.000 claims description 18
- 239000012528 membrane Substances 0.000 claims description 18
- 238000005336 cracking Methods 0.000 claims description 15
- 239000003480 eluent Substances 0.000 claims description 15
- 238000004108 freeze drying Methods 0.000 claims description 13
- 239000000758 substrate Substances 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 8
- 230000009089 cytolysis Effects 0.000 claims description 7
- 239000000243 solution Substances 0.000 description 63
- 238000010586 diagram Methods 0.000 description 3
- 239000002122 magnetic nanoparticle Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 238000007599 discharging Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000005465 channeling Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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- 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
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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
- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The invention relates to the technical field of biological detection, in particular to a nucleic acid extraction, amplification and detection integrated reagent card. The integrated nucleic acid extraction, amplification and detection reagent card comprises a reagent card main body, a liquid package component, a sample placement region, a driving component, a nucleic acid extraction region, a nucleic acid amplification region, a nucleic acid detection region and a micro-channel system, wherein the liquid package component, the sample placement region, the driving component, the nucleic acid extraction region, the nucleic acid amplification region, the nucleic acid detection region and the micro-channel system are arranged on the reagent card main body; the sample tube is arranged in the sample placing area, and the sample placing area is also provided with a puncturing assembly for puncturing the sample tube; the microchannel system communicates the drive assembly with the nucleic acid extraction region, the nucleic acid amplification region, and the nucleic acid detection region, and valves are provided on each microchannel between the drive assembly and the nucleic acid extraction region, the nucleic acid amplification region, and the nucleic acid detection region to enable the drive assembly to communicate with any one of the nucleic acid extraction region, the nucleic acid amplification region, and the nucleic acid detection region separately. The three steps of nucleic acid extraction, amplification and detection in the prior art are complicated and complicated in independent operation, and the technical problem of easiness in pollution is solved.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a nucleic acid extraction, amplification and detection integrated reagent card.
Background
At present, the nucleic acid detection is widely applied to the clinical medicine field, such as the common detection of viral nucleic acid, SNP (single nucleotide polymorphisms) detection and the like, the nucleic acid detection is generally divided into three steps of nucleic acid extraction, nucleic acid amplification and nucleic acid detection, the nucleic acid detection equipment on the market at present can only perform the two steps of nucleic acid amplification and nucleic acid detection, and the nucleic acid extraction needs to be performed manually or by using other equipment, so that the whole nucleic acid detection process has long period, complicated operation and higher cost, and the detection result is also easily influenced by external environmental pollution or pollutes the external environment in the detection process.
Disclosure of Invention
The invention aims to provide a nucleic acid extraction, amplification and detection integrated reagent card, which is used for solving the technical problems that three steps of nucleic acid extraction, amplification and detection in the prior art are complicated and complicated in independent operation and easy to pollute.
In a first aspect, an embodiment of the present invention provides a nucleic acid extraction, amplification and detection integrated reagent card, including: the reagent card comprises a reagent card main body, a liquid package component, a sample placement area, a driving component, a nucleic acid extraction area, a nucleic acid amplification area and a nucleic acid detection area and a micro-channel system;
the extending direction of the liquid pack assembly is the same as the length direction of the reagent card main body, the driving assembly and the liquid pack assembly are arranged in parallel, and the sample placing area is arranged between the liquid pack assembly and the driving assembly;
the sample tube is arranged in the sample placing area, and the sample placing area is also provided with a puncturing assembly for puncturing the sample tube;
the liquid package component, the sample placement region, the driving component, the nucleic acid extraction region, the nucleic acid amplification region and the nucleic acid detection region are all arranged on the reagent card main body, the liquid package component is respectively communicated with the nucleic acid extraction region, the nucleic acid amplification region and the nucleic acid detection region, the micro-channel system is used for communicating the driving component with the nucleic acid extraction region, the nucleic acid amplification region, the nucleic acid detection region and the liquid package component, and valves are arranged on all micro-channels between the driving component and the nucleic acid extraction region, the nucleic acid amplification region, the nucleic acid detection region and the liquid package component, so that the driving component can be independently communicated with any one of the nucleic acid extraction region, the nucleic acid amplification region and the nucleic acid detection region;
the drive assembly is for liquid flow within the microchannel system;
in an alternative embodiment, the drive assembly includes a pump chamber, a waste reservoir, a seal cap, a flexible seal cap, and a ram;
the pump chamber is communicated with the waste liquid storage tank, the flexible sealing cover and the sealing top cover are sequentially arranged above the pump chamber and the waste liquid storage tank, the pressure head is arranged above the sealing top cover and can squeeze the sealing top cover, the pump chamber and the waste liquid storage tank are respectively communicated with the nucleic acid extraction area, the nucleic acid amplification area, the nucleic acid detection area and the liquid package component through the micro-channel system, and valves are arranged in micro-channels between the pump chamber and the waste liquid storage tank and between the pump chamber and the nucleic acid extraction area, between the pump chamber and the waste liquid storage tank and the waste liquid package component.
In an alternative embodiment, the nucleic acid extraction area comprises a sample cracking chamber arranged in the liquid package assembly and an adsorption elution bin communicated with the sample cracking chamber, and a nucleic acid capturing column is arranged in the adsorption elution bin;
the sample cracking chamber is communicated with the sample placing area, the adsorption eluting bin is communicated with the waste liquid storage pool, valves are arranged on the sample cracking chamber and the micro-channels between the sample placing area and the adsorption eluting bin, and the valves are arranged on the micro-channels between the adsorption eluting bin and the waste liquid storage pool.
In an alternative embodiment, the nucleic acid amplification area is respectively communicated with the adsorption eluting bin and the waste liquid storage pool is communicated with the adsorption eluting bin through a micro-channel system, a valve is arranged on a micro-channel between the nucleic acid amplification area and the waste liquid storage pool, and a valve is arranged on the micro-channel between the nucleic acid amplification area and the adsorption eluting bin.
In an alternative embodiment, the liquid pack assembly further comprises a magnetic particle complex solution chamber, a hybridization solution chamber, a third wash solution chamber, an eluent chamber, a second wash solution chamber, and a first wash solution chamber;
the eluent chamber, the second cleaning liquid chamber and the first cleaning liquid chamber are communicated with the adsorption elution bin through a micro-channel system, and valves are arranged in micro-channels for respectively communicating the eluent chamber, the second cleaning liquid chamber and the first cleaning liquid chamber with the adsorption elution bin;
the magnetic particle complex solution chamber, the hybridization solution chamber and the third cleaning solution chamber are communicated with the nucleic acid detection area through a micro-channel system.
In an alternative embodiment, the reagent card body includes a magnetic particle lyophilization zone, a hybridization solution lyophilization zone, a magnetic particle reconstitution buffer channel, and a hybridization solution reconstitution buffer channel;
the hybridization solution re-dissolution buffer channel is sequentially communicated with the hybridization solution chamber and the hybridization solution chamber, and the magnetic particle re-dissolution buffer channel is sequentially communicated with the magnetic particle freeze-drying area and the magnetic particle re-solution chamber;
the waste liquid storage pool can be respectively communicated with the magnetic particle re-dissolution buffer channel and the hybridization solution re-dissolution buffer channel through a micro-channel system, and a valve is arranged in the micro-channel for respectively communicating the waste liquid storage pool with the magnetic particle re-dissolution buffer channel and the hybridization solution re-dissolution buffer channel;
the nucleic acid detection area is respectively communicated with the magnetic particle re-dissolution buffer channel and the hybridization solution re-dissolution buffer channel through a micro-channel system, and a valve is arranged in the micro-channel for respectively communicating the nucleic acid detection area with the magnetic particle re-dissolution buffer channel and the hybridization solution re-dissolution buffer channel.
In an alternative embodiment, the nucleic acid detection zone comprises a chip;
the surface of the chip is provided with a plurality of probes, and the probes are used for detecting amplified target nucleic acid for hybridization.
In an alternative embodiment, the magnetic particle complex solution chamber, the hybridization solution chamber, the third cleaning solution chamber, the eluent chamber, the second cleaning solution chamber, the first cleaning solution chamber and the sample lysis chamber are sequentially arranged at intervals.
In an alternative embodiment, the liquid package assembly includes a plurality of liquid package substrates, a semipermeable membrane and a tearable sealing membrane disposed above the liquid package substrates, a bottom sealing membrane disposed below the liquid package substrates, and a cavity formed between two adjacent liquid package substrates.
In an alternative embodiment, the nucleic acid extraction, amplification and detection integrated reagent card further comprises a microchannel sealing top die, a mounting groove, a bottom microchannel sealing film, a circuit board, a liquid package sealing film and a bottom cover;
the mounting groove is fixed on the reagent card main body, the mounting groove is used for mounting the liquid package assembly, the liquid package sealing film is arranged between the mounting groove and the liquid package assembly, the micro-channel sealing top die and the bottom micro-channel sealing film are arranged at the upper end and the lower end of the reagent card, the bottom cover is arranged below the reagent card main body, the chip is arranged on the circuit board, and the circuit board is arranged between the bottom micro-channel sealing film and the bottom cover.
The invention provides a nucleic acid extraction, amplification and detection integrated reagent card, which comprises the following components: the reagent card comprises a reagent card main body, a liquid package component, a sample placement area, a driving component, a nucleic acid extraction area, a nucleic acid amplification area and a nucleic acid detection area and a micro-channel system; the extending direction of the liquid pack assembly is the same as the length direction of the reagent card main body, the driving assembly and the liquid pack assembly are arranged in parallel, and the sample placing area is arranged between the liquid pack assembly and the driving assembly; the sample tube is arranged in the sample placing area, and the sample placing area is also provided with a puncturing assembly for puncturing the sample tube; the liquid package component, the sample placement region, the driving component, the nucleic acid extraction region, the nucleic acid amplification region and the nucleic acid detection region are all arranged on the reagent card main body, the micro-channel system is used for communicating the driving component with the nucleic acid extraction region, the nucleic acid amplification region, the nucleic acid detection region and the liquid package component, and valves are arranged on all micro-channels between the driving component and the nucleic acid extraction region, the nucleic acid amplification region, the nucleic acid detection region and the liquid package component, so that the driving component can be independently communicated with any one of the nucleic acid extraction region, the nucleic acid amplification region and the nucleic acid detection region; the drive assembly is for liquid flow within the microchannel system. The driving component can drive the sample in the sample placing region to enter the nucleic acid extracting region, the nucleic acid amplifying region and the nucleic acid detecting region respectively so as to extract, amplify and detect the nucleic acid, so that the technical problems that three steps of nucleic acid extraction, amplification and detection in the prior art are complicated and complex in independent operation and easy to pollute are solved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a reagent card for integrating nucleic acid extraction, amplification and detection according to an embodiment of the present invention;
FIG. 2 is a cross-sectional view of a drive assembly in a nucleic acid extraction, amplification and detection integrated reagent card according to an embodiment of the present invention;
FIG. 3 is a partial cross-sectional view of a liquid pack assembly in a nucleic acid extraction, amplification and detection integrated reagent card according to an embodiment of the present invention;
FIG. 4 is a top view of a nucleic acid extraction, amplification and detection integrated reagent card provided in an embodiment of the present invention without a microchannel sealing top mold;
FIG. 5 is a cross-sectional view of a liquid pack assembly in an integrated nucleic acid extraction, amplification and detection reagent card according to an embodiment of the present invention;
FIG. 6 is a schematic diagram of a liquid pack assembly in a nucleic acid extraction, amplification and detection integrated reagent card according to an embodiment of the present invention;
FIG. 7 is a bottom view of a reagent card body in a nucleic acid extraction, amplification and detection integrated reagent card according to an embodiment of the present invention;
FIG. 8 is a perspective view of a nucleic acid extraction, amplification and detection integrated reagent card according to an embodiment of the present invention;
FIG. 9 is a schematic diagram of a sample lysis chamber of a reagent card for integrated nucleic acid extraction, amplification and detection according to an embodiment of the present invention.
Icon: 1-a reagent card body; 11-microchannel system; 12-magnetic particle lyophilization zone; 13-a lyophilization zone of the hybridization solution; 14-a magnetic particle reconstitution buffer channel; 15-a hybridization solution re-dissolution buffer channel; 16-nucleic acid amplification region; 2-a liquid pack assembly; 21-a liquid pack matrix; 22-semipermeable membrane; 23-a tearable sealing film; 24-a bottom sealing film; 210-a magnetic particle reconstitution solution chamber; 211-hybridization solution chamber; 212-a third cleaning solution chamber; 213-eluent chamber; 214-a second cleaning solution chamber; 215-a first cleaning solution chamber; 216-sample lysis chamber; 2160—sample outlet; 2161—sample introduction needle; 3-a drive assembly; 31-a pump chamber; 32-a waste liquid storage tank; 33-sealing the top cap; 34-a flexible sealing cover; 35-pressing head; 4-a sample placement area; 5-micro-channel sealing top mold; 6-bottom microchannel sealing membrane; 7-a circuit board; 71-chip; 8-a bottom cover; 9-liquid pack sealing film.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.
In the description of the present invention, it should be noted that, directions or positional relationships indicated by terms such as "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., are directions or positional relationships based on those shown in the drawings, or are directions or positional relationships conventionally put in use of the inventive product, are merely for convenience of describing the present invention and simplifying the description, and are not indicative or implying that the apparatus or element to be referred to must have a specific direction, be constructed and operated in a specific direction, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," "third," and the like are used merely to distinguish between descriptions and should not be construed as indicating or implying relative importance.
Furthermore, the terms "horizontal," "vertical," "overhang," and the like do not denote a requirement that the component be absolutely horizontal or overhang, but rather may be slightly inclined. As "horizontal" merely means that its direction is more horizontal than "vertical", and does not mean that the structure must be perfectly horizontal, but may be slightly inclined.
In the description of the present invention, it should also be noted that, unless explicitly specified and limited otherwise, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
Some embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.
As shown in fig. 1-9, an embodiment of the present invention provides a nucleic acid extraction, amplification and detection integrated reagent card, comprising: a reagent card body 1, a liquid pack assembly 2, a sample placement section 4, a drive assembly 3, a nucleic acid extraction section, a nucleic acid amplification section 16, and a nucleic acid detection section and a microchannel system 11; the extending direction of the liquid pack assembly 2 is the same as the length direction of the reagent card main body 1, the driving assembly 3 is arranged in parallel with the liquid pack assembly 2, and the sample placing area 4 is arranged between the liquid pack assembly 2 and the driving assembly 3; the sample tube is arranged in the sample placement area 4, and the sample placement area 4 is also provided with a puncturing component for puncturing the sample tube; the liquid pack assembly 2, the sample placement area 4, the driving assembly 3, the nucleic acid extraction area, the nucleic acid amplification area 16 and the nucleic acid detection area are all arranged on the reagent card main body 1, the liquid pack assembly 2 is respectively communicated with the nucleic acid extraction area, the nucleic acid amplification area 16 and the nucleic acid detection area, the micro-channel system 11 is used for communicating the driving assembly 3 with the nucleic acid extraction area, the nucleic acid amplification area 16 and the nucleic acid detection area and the liquid pack assembly, and valves are arranged on each micro-channel between the driving assembly 3 and the nucleic acid extraction area, the nucleic acid amplification area 16, the nucleic acid detection area and the liquid pack assembly 2 so that the driving assembly 3 can be independently communicated with any one of the nucleic acid extraction area, the nucleic acid amplification area 16 and the nucleic acid detection area; the drive assembly 3 is used for the flow of liquid within the microchannel system 11.
Wherein the microchannel system 11 is provided on both upper and lower sides of the reagent card body 1.
In this embodiment, the driving component 3 is provided with valves in the micro-channels communicating with the sample placing region 4, the nucleic acid extracting region, the nucleic acid amplifying region 16, the nucleic acid detecting and liquid packaging component, so that the driving component can independently drive the flow direction of the liquid, and further the samples in the sample tubes arranged in the sample placing region 4 are sequentially subjected to the calculation extraction, amplification and detection, so that the technical problems that three steps of nucleic acid extraction, amplification and detection in the prior art are complicated and complicated in independent operation and are easy to pollute are solved.
In an alternative embodiment, the drive assembly 3 comprises a pump chamber 31, a waste reservoir 32, a seal cap 33, a flexible seal cap 34 and a ram 35; the pump chamber 31 is communicated with the waste liquid storage tank 32, the flexible sealing cover 34 and the sealing top cover 33 are sequentially arranged above the pump chamber 31 and the waste liquid storage tank 32, the pressure head 35 is arranged above the sealing top cover 33 and can squeeze the sealing top cover 33, the pump chamber 31 and the waste liquid storage tank 32 are respectively communicated with the nucleic acid extraction area, the nucleic acid amplification area 16, the nucleic acid detection area and the liquid package component through the micro-channel system 11, and valves are arranged in micro-channels between the pump chamber 31 and the waste liquid storage tank 32 and the nucleic acid extraction area, the nucleic acid amplification area 16, the nucleic acid detection area and the liquid package component.
In this embodiment, the pump chamber 31 is communicated with the waste liquid storage tank 32 through a channel port, when the pressure head 35 moves downwards or upwards, the pressure head 35 presses the seal top cover 33 to deform the flexible seal top cover 33, so as to change the volume in the pump chamber 31, generate positive pressure or negative pressure in the pump chamber 31, and control the flow direction of the solution by controlling valves on different micro channels.
In an alternative embodiment, the nucleic acid extraction zone comprises a sample lysis chamber 216 arranged in the liquid bag assembly 2 and an adsorption elution bin communicated with the sample lysis chamber 216, wherein a nucleic acid capturing column is arranged in the adsorption elution bin; the sample cracking chamber 216 is communicated with the sample placement area 4, the adsorption elution bin is communicated with the waste liquid storage pool 32, valves are arranged on micro-channels between the sample cracking chamber 216 and the sample placement area 4 and the adsorption elution bin, and valves are arranged on micro-channels between the adsorption elution bin and the waste liquid storage pool 32.
Wherein after the sample tube is arranged in the sample placement area 4, a first sample needle on the puncture assembly is communicated with the pump chamber 31 through a micro-channel between the sample placement area 4 and the pump chamber 31 so as to push the gas in the pump chamber 31 into the sample tube, a second sample needle is communicated with the sample cracking chamber 216 so as to push the sample into the sample cracking chamber 216, the needle head tangential surfaces of the first sample needle and the second sample needle are opposite, and the first sample needle is higher than the second sample needle so as to prevent bubbles from entering the sample cracking chamber 216;
during the injection of the sample into the sample-splitting chamber 216, the pressure head 35 is pressed down, positive pressure is generated in the pump chamber 31 and enters the first sample needle and the sample tube through the micro-channel, the sample-introducing needle 2161 inside the sample-splitting chamber 216 is communicated with the second sample needle, the sample-introducing needle is higher than the liquid level of the sample in the sample-splitting chamber 216, the sample-discharging opening 2160 is arranged in the sample-splitting chamber 216, and the sample-discharging opening 2160 is communicated with the micro-channel and is provided with a valve.
In this embodiment, a nucleic acid trapping column is provided in the adsorption eluting chamber, so that nucleic acid can be attached to the nucleic acid trapping column, and the nucleic acid trapping column is made of a silicon matrix membrane material.
In an alternative embodiment, the nucleic acid amplification area 16 is respectively communicated with the adsorption eluting bin and the waste liquid storage tank 32 through a micro-channel system 11, a valve is arranged on a micro-channel between the nucleic acid amplification area 16 and the waste liquid storage tank 32, and a valve is arranged on a micro-channel between the nucleic acid amplification area 16 and the adsorption eluting bin.
Further, the liquid pack assembly 2 further includes a magnetic particle complex solution chamber 210, a hybridization solution chamber 211, a third cleaning solution chamber 212, an eluent chamber 213, a second cleaning solution chamber 214, and a first cleaning solution chamber 215; the eluent chamber 213, the second cleaning liquid chamber 214 and the first cleaning liquid chamber 215 are all communicated with the adsorption elution cartridge through the microchannel system 11, and valves are provided in the microchannels for respectively communicating the eluent chamber 213, the second cleaning liquid chamber 214 and the first cleaning liquid chamber 215 with the adsorption elution cartridge; the magnetic particle multiplexing solution chamber 210, the hybridization solution chamber 211, and the third washing solution chamber 212 are communicated with the nucleic acid detection region through the microchannel system 11, and valves are provided in the microchannels for communicating the magnetic particle multiplexing solution chamber 210, the hybridization solution chamber 211, and the third washing solution chamber 212 with the nucleic acid detection region.
In this embodiment, after the sample is cracked in the sample cracking chamber 216, the pressure head 35 of the pump chamber 31 is lifted up, the cracked sample enters the waste liquid storage pool 32 through the adsorption eluting bin, then the first cleaning liquid in the first cleaning liquid chamber 215 and the second cleaning liquid in the second cleaning liquid chamber 214 are respectively sucked to repeatedly clean the nucleic acid capturing column, the waste liquid is sucked into the waste liquid storage pool 32, and finally the eluent is sucked from the eluent chamber 213 and injected into the adsorption chamber to elute the nucleic acid.
In an alternative embodiment, the reagent card body 1 includes a magnetic particle lyophilization zone 12, a hybridization solution lyophilization zone 13, a magnetic particle reconstitution buffer channel 14, and a hybridization solution reconstitution buffer channel 15;
the hybridization solution re-dissolution buffer channel 15 is sequentially communicated with the hybridization solution freeze-drying area 13 and the hybridization solution chamber 211, and the magnetic particle re-dissolution buffer channel 14 is sequentially communicated with the magnetic particle freeze-drying area 12 and the magnetic particle re-dissolution chamber 210;
the waste liquid storage tank 32 can be respectively communicated with the magnetic particle re-dissolution buffer channel 14 and the hybridization solution re-dissolution buffer channel 15 through the micro-channel system 11, and a valve is arranged in a micro-channel for respectively communicating the waste liquid storage tank 32 with the magnetic particle re-dissolution buffer channel 14 and the hybridization solution re-dissolution buffer channel 15;
the nucleic acid detection area is respectively communicated with the magnetic particle re-dissolution buffer channel 14 and the hybridization solution re-dissolution buffer channel 15 through the micro-channel system 11, and a valve is arranged in a micro-channel for respectively communicating the nucleic acid detection area with the magnetic particle re-dissolution buffer channel 14 and the hybridization solution re-dissolution buffer channel 15.
In an alternative embodiment, the nucleic acid detection zone comprises chip 71; the chip 71 has a plurality of probes on its surface for detecting amplified target nucleic acids and hybridizing.
In this embodiment, the amplified solution is driven to enter the nucleic acid detection region by the negative pressure generated by the pump chamber 31, and the probe on the chip 71 in the nucleic acid detection region hybridizes with the amplified target nucleic acid of the biotin group to be bonded to the surface of the chip 71. Then, magnetic nanoparticles are injected into a detection area of the chip 71, the magnetic nanoparticles are combined on the surface of the chip 71 through biotin-avidin reaction, and the giant magnetoresistance chip 71 detects nucleic acid by detecting magnetic signals generated by the magnetic nanoparticles fixed on the surface of the chip 71.
In an alternative embodiment, the magnetic particle complex solution chamber 210, the hybridization solution chamber 211, the third washing solution chamber 212, the eluent chamber 213, the second washing solution chamber 214, the first washing solution chamber 215, and the sample lysis chamber 216 are sequentially spaced apart.
In an alternative embodiment, the liquid pack assembly 2 includes a plurality of liquid pack substrates 21, a semipermeable membrane 22 and a tearable sealing membrane 23 disposed above the liquid pack substrates 21, a bottom sealing membrane 24 disposed below the liquid pack substrates 21, and a cavity formed between two adjacent liquid pack substrates 21.
In this embodiment, the tearable sealing film 23 is configured to be torn manually during use, the bottom sealing film 24 is used to keep the liquid package substrate 21 sealed, and the liquid package is pierced by the opening needle during use, and the opening needle can continue to pierce the bottom sealing film 24 without being stained with the solution in the cavity of the liquid package assembly because the bottom microchannel sealing film is a soft film; when the tearable sealing membrane 23 is torn off and the bottom sealing membrane 24 is pierced, the liquid in the chamber in the liquid pack assembly 2 is sucked into the corresponding micro-channel through the corresponding micro-channel inlet on the reagent card main body 1 by means of negative pressure, and the semipermeable membrane 22 plays roles of balancing air pressure and preventing leakage of the solution, preferably a breathable and liquid-impermeable semipermeable membrane material.
In an alternative embodiment, the nucleic acid extraction, amplification and detection integrated reagent card further comprises a microchannel sealing top die 5, a mounting groove, a bottom microchannel sealing film 6, a circuit board 7, a liquid package sealing film 9 and a bottom cover 8; the mounting groove is fixed on the reagent card main body 1, the mounting groove is used for mounting the liquid package assembly 2, the liquid package sealing film 9 is arranged between the mounting groove and the liquid package assembly 2, the micro-channel sealing top die 5 and the bottom micro-channel sealing film 6 are arranged at the upper end and the lower end of the reagent card, the bottom cover 8 is arranged below the reagent card main body 1, the chip 71 is arranged on the circuit board 7, and the circuit board 7 is arranged between the bottom micro-channel sealing film 6 and the bottom cover 8.
The liquid package sealing film is used for ensuring good sealing between the reagent card and the liquid package assembly and isolating each cavity on the unsealed liquid package assembly 2 to prevent liquid channeling.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (8)
1. A nucleic acid extraction, amplification and detection integrated reagent card, comprising: the kit comprises a reagent card main body, a liquid package component, a sample placement area, a driving component, a nucleic acid extraction area, a nucleic acid amplification area, a nucleic acid detection area and a micro-channel system;
the extending direction of the liquid pack assembly is the same as the length direction of the reagent card main body, the driving assembly and the liquid pack assembly are arranged in parallel, and the sample placing area is arranged between the liquid pack assembly and the driving assembly;
the sample tube is arranged in the sample placing area, and the sample placing area is also provided with a puncturing assembly for puncturing the sample tube; the liquid package component, the sample placement region, the driving component, the nucleic acid extraction region, the nucleic acid amplification region and the nucleic acid detection region are all arranged on the reagent card main body, the liquid package component is respectively communicated with the nucleic acid extraction region, the nucleic acid amplification region and the nucleic acid detection region, the micro-channel system is used for communicating the driving component with the nucleic acid extraction region, the nucleic acid amplification region, the nucleic acid detection region and the liquid package component, and valves are arranged on all micro-channels between the driving component and the nucleic acid extraction region, the nucleic acid amplification region, the nucleic acid detection region and the liquid package component, so that the driving component can be independently communicated with any one of the nucleic acid extraction region, the nucleic acid amplification region and the nucleic acid detection region;
the drive assembly is for liquid flow within the microchannel system;
the driving assembly comprises a pump chamber, a waste liquid storage tank, a sealing top cover, a flexible sealing cover and a pressure head;
the pump chamber is communicated with the waste liquid storage tank, the flexible sealing cover and the sealing top cover are sequentially arranged above the pump chamber and the waste liquid storage tank, the pressure head is arranged above the sealing top cover and can squeeze the sealing top cover, the pump chamber and the waste liquid storage tank are respectively communicated with the nucleic acid extraction area, the nucleic acid amplification area, the nucleic acid detection area and the liquid package component through the micro-channel system, and valves are arranged in micro-channels between the pump chamber and the waste liquid storage tank and the nucleic acid extraction area, between the pump chamber and the waste liquid storage tank and the nucleic acid amplification area, and between the pump chamber and the waste liquid storage tank and the nucleic acid detection area and between the pump chamber and the waste liquid storage tank and the liquid package component;
the nucleic acid extraction area comprises a sample cracking chamber and an adsorption elution bin, wherein the sample cracking chamber is arranged in the liquid bag component, the adsorption elution bin is communicated with the sample cracking chamber, and a nucleic acid capturing column is arranged in the adsorption elution bin;
the sample cracking chamber is communicated with the sample placing area, the adsorption eluting bin is communicated with the waste liquid storage pool, a valve is arranged on a micro-channel between the sample cracking chamber and the sample placing area and the adsorption eluting bin respectively, and a valve is arranged on a micro-channel between the adsorption eluting bin and the waste liquid storage pool;
the pump chamber is communicated with the waste liquid storage tank through a passage port, when the pressure head moves downwards or upwards, the pressure head extrudes the sealing top cover so as to deform the flexible sealing top cover, so that the volume in the pump chamber is changed, positive pressure or negative pressure is generated in the pump chamber, and the flow direction of the solution is controlled by controlling valves on different micro-channels.
2. The integrated nucleic acid extraction, amplification and detection reagent card of claim 1, wherein the nucleic acid amplification zone is respectively communicated with the adsorption elution bin and the waste liquid storage tank through a micro-channel system, a valve is arranged on a micro-channel between the nucleic acid amplification zone and the waste liquid storage tank, and a valve is arranged on the micro-channel between the nucleic acid amplification zone and the adsorption elution bin.
3. The integrated nucleic acid extraction, amplification and detection reagent card of claim 2, wherein the liquid pack assembly further comprises a magnetic particle complex solution chamber, a hybridization solution chamber, a third wash solution chamber, an eluent chamber, a second wash solution chamber, and a first wash solution chamber;
the eluent chamber, the second cleaning liquid chamber and the first cleaning liquid chamber are communicated with the adsorption elution bin through a micro-channel system, and valves are arranged in micro-channels for respectively communicating the eluent chamber, the second cleaning liquid chamber and the first cleaning liquid chamber with the adsorption elution bin;
the magnetic particle complex solution chamber, the hybridization solution chamber and the third cleaning solution chamber are communicated with the nucleic acid detection area through a micro-channel system.
4. The integrated nucleic acid extraction, amplification and detection reagent card of claim 3, wherein the reagent card body comprises a magnetic particle lyophilization zone, a hybridization solution lyophilization zone, a magnetic particle reconstitution buffer channel, and a hybridization solution reconstitution buffer channel;
the hybridization solution re-dissolution buffer channel is sequentially communicated with the hybridization solution freeze-drying area and the hybridization solution chamber, and the magnetic particle re-dissolution buffer channel is sequentially communicated with the magnetic particle freeze-drying area and the magnetic particle re-solution chamber;
the waste liquid storage pool can be respectively communicated with the magnetic particle re-dissolution buffer channel and the hybridization solution re-dissolution buffer channel through a micro-channel system, and a valve is arranged in the micro-channel for respectively communicating the waste liquid storage pool with the magnetic particle re-dissolution buffer channel and the hybridization solution re-dissolution buffer channel;
the nucleic acid detection area is respectively communicated with the magnetic particle re-dissolution buffer channel and the hybridization solution re-dissolution buffer channel through a micro-channel system, and a valve is arranged in the micro-channel for respectively communicating the nucleic acid detection area with the magnetic particle re-dissolution buffer channel and the hybridization solution re-dissolution buffer channel.
5. The integrated nucleic acid extraction, amplification and detection reagent card of claim 3, wherein the nucleic acid detection zone comprises a chip;
the surface of the chip is provided with a plurality of probes, and the probes are used for detecting amplified target nucleic acid for hybridization.
6. The integrated nucleic acid extraction, amplification and detection reagent card of claim 4, wherein the magnetic particle complex solution chamber, the hybridization solution chamber, the third washing solution chamber, the eluent chamber, the second washing solution chamber, the first washing solution chamber and the sample lysis chamber are sequentially arranged at intervals.
7. The integrated nucleic acid extraction, amplification and detection reagent card of claim 3, wherein the liquid pack assembly comprises a plurality of liquid pack substrates, a semipermeable membrane and a tearable sealing membrane arranged above the liquid pack substrates, a bottom sealing membrane arranged below the liquid pack substrates, and a cavity formed between two adjacent liquid pack substrates.
8. The integrated nucleic acid extraction, amplification and detection reagent card of claim 5, further comprising a microchannel sealing top mold, a mounting groove, a bottom microchannel sealing membrane, a circuit board, a liquid bag sealing membrane, and a bottom cover;
the mounting groove is fixed on the reagent card main body, the mounting groove is used for mounting the liquid package assembly, the liquid package sealing film is arranged between the mounting groove and the liquid package assembly, the micro-channel sealing top die and the bottom micro-channel sealing film are arranged at the upper end and the lower end of the reagent card, the bottom cover is arranged below the reagent card main body, the chip is arranged on the circuit board, and the circuit board is arranged between the bottom micro-channel sealing film and the bottom cover.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110964716A (en) * | 2019-12-05 | 2020-04-07 | 广州万孚生物技术股份有限公司 | In-vitro diagnosis and analysis device and reagent card |
| CN110964715B (en) * | 2019-12-05 | 2021-11-26 | 广州万孚生物技术股份有限公司 | In-vitro diagnosis and analysis device and reagent card |
| CN111321056B (en) * | 2020-02-27 | 2023-11-21 | 浙江大学 | Cartridge device and method for nucleic acid extraction and analysis |
| CN112221546B (en) * | 2020-08-27 | 2021-11-26 | 中国科学院苏州生物医学工程技术研究所 | Sample transfer device, transfer system and method |
| CN112501007A (en) * | 2020-12-01 | 2021-03-16 | 杭州康金来技术有限公司 | Rotation type structure of nucleic acid extraction element |
| CN112680340A (en) * | 2020-12-23 | 2021-04-20 | 广州和实生物技术有限公司 | Full-automatic nucleic acid detection device and detection method thereof |
| CN113846014A (en) * | 2021-10-29 | 2021-12-28 | 青岛全诊生物技术有限公司 | Nucleic acid extraction and amplification device |
| CN114164091A (en) * | 2021-12-07 | 2022-03-11 | 广东润鹏生物技术有限公司 | Molecular diagnostic sample processing system and control method |
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