CN117229898A - Rapid nucleic acid detection chip cartridge based on microfluidic technology - Google Patents
Rapid nucleic acid detection chip cartridge based on microfluidic technology Download PDFInfo
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- CN117229898A CN117229898A CN202311026763.9A CN202311026763A CN117229898A CN 117229898 A CN117229898 A CN 117229898A CN 202311026763 A CN202311026763 A CN 202311026763A CN 117229898 A CN117229898 A CN 117229898A
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 75
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 75
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 75
- 238000001514 detection method Methods 0.000 title claims abstract description 62
- 238000005516 engineering process Methods 0.000 title claims abstract description 11
- 239000007788 liquid Substances 0.000 claims abstract description 53
- 239000012188 paraffin wax Substances 0.000 claims abstract description 52
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 47
- 238000006243 chemical reaction Methods 0.000 claims abstract description 43
- 238000005406 washing Methods 0.000 claims abstract description 29
- 239000002699 waste material Substances 0.000 claims abstract description 22
- 238000000605 extraction Methods 0.000 claims abstract description 18
- 238000000746 purification Methods 0.000 claims abstract description 18
- 239000012807 PCR reagent Substances 0.000 claims abstract description 17
- 239000003480 eluent Substances 0.000 claims abstract description 17
- 238000005336 cracking Methods 0.000 claims abstract description 15
- 238000003860 storage Methods 0.000 claims abstract description 15
- 238000012408 PCR amplification Methods 0.000 claims abstract description 10
- 238000010438 heat treatment Methods 0.000 claims description 15
- 239000011324 bead Substances 0.000 claims description 11
- 230000009089 cytolysis Effects 0.000 claims description 11
- 238000013461 design Methods 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 230000000149 penetrating effect Effects 0.000 claims description 3
- 238000004891 communication Methods 0.000 claims 2
- 238000003745 diagnosis Methods 0.000 abstract description 6
- 238000003753 real-time PCR Methods 0.000 abstract description 3
- 238000000034 method Methods 0.000 description 11
- 238000003199 nucleic acid amplification method Methods 0.000 description 10
- 230000003321 amplification Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 5
- 238000010586 diagram Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Abstract
The invention provides a rapid nucleic acid detection chip cartridge based on a microfluidic technology, which comprises: a nucleic acid extraction purification module comprising: the device comprises a sample cracking cavity, a first washing liquid cavity, a second washing liquid cavity, an eluent cavity, a reagent reaction cavity and a waste liquid cavity; the bottoms of the sample cracking cavity, the first washing liquid cavity, the second washing liquid cavity and the eluent cavity are respectively communicated with the reagent reaction cavity through a first paraffin valve body, a second paraffin valve body, a third paraffin valve body and a fourth paraffin valve body, and the bottoms of the reagent reaction cavity are communicated with the waste liquid cavity through a fifth paraffin valve body; a PCR amplification detection module comprising: the bottom of the reagent reaction cavity is communicated with the PCR reagent storage cavity through a sixth paraffin valve body. The invention integrates nucleic acid extraction and purification with real-time fluorescence quantitative PCR detection, greatly reduces the requirements on the professional skills and auxiliary equipment of operators, and has good application prospect in the field of molecular diagnosis.
Description
Technical Field
The invention relates to the field of nucleic acid detection chip cartridges, in particular to a rapid nucleic acid detection chip cartridge based on a microfluidic technology.
Background
The nucleic acid detection technology is a molecular diagnosis technology which is most widely applied in recent years, can rapidly and efficiently amplify the characteristic nucleotide sequence of pathogenic bacteria in the diagnosis of infectious diseases, and has been widely applied in the fields of clinical diagnosis, disease screening and the like.
Conventional nucleic acid detection steps typically include: sample inactivation, nucleic acid extraction and purification, nucleic acid amplification and detection, and the like. The extraction and purification of nucleic acid are key in molecular biology experiments, and relate to whether high-quality nucleic acid can be extracted or not, so that the success or failure of subsequent experiments can be directly influenced. The nucleic acid amplification and detection are to carry out high-efficiency rapid amplification and fluorescence or visual detection on the extracted and purified characteristic nucleotide sequence of the pathogenic bacteria so as to obtain an immediate diagnosis result. The current common method for nucleic acid amplification detection is real-time fluorescent quantitative PCR, which can perform qualitative and quantitative analysis on nucleic acid in a sample, and is a gold standard for current nucleic acid amplification detection. However, in the processes of sample preparation, nucleic acid amplification detection and the like, a specific experimental environment is often required, a complex operation flow is required, and the technical requirements on experimental operators are high; furthermore, the length, sensitivity and specificity of the nucleic acid amplification method will also directly affect the interpretation of the subsequent results.
In view of the above, there have been studies on integrating nucleic acid extraction and purification with automated detection with higher sensitivity in a cartridge, and simplifying the detection flow based on magnetic bead transfer or driven reagent flow distribution. The existing chip card box detection mode is insufficient in that an automatic process from sample input to result output cannot be realized, human intervention is often required in the transfer process, and the risk of pollution to experimental environment exists. Therefore, the application of the high-sensitivity and simple operation device integrating nucleic acid extraction, purification and automatic detection in the field of molecular diagnosis still has the problem to be solved.
Disclosure of Invention
The invention aims to provide a rapid nucleic acid detection chip cartridge based on a microfluidic technology, so as to solve the problem that the nucleic acid detection chip cartridge in the prior art needs human intervention and has risk of pollution to experimental environment.
In order to solve the problems, the invention adopts the following technical scheme:
provided is a rapid nucleic acid detection chip cartridge based on a microfluidic technology, comprising: a nucleic acid extraction purification module comprising: the device comprises a sample cracking cavity, a first washing liquid cavity, a second washing liquid cavity, an eluent cavity, a reagent reaction cavity and a waste liquid cavity; the bottoms of the sample cracking cavity, the first washing liquid cavity, the second washing liquid cavity and the eluent cavity are respectively communicated with the reagent reaction cavity through a first paraffin valve body, a second paraffin valve body, a third paraffin valve body and a fourth paraffin valve body, and the bottoms of the reagent reaction cavity are communicated with the waste liquid cavity through a fifth paraffin valve body; a PCR amplification detection module, comprising: the PCR reagent storage cavity, the microfluidic chip and the lead screw push rod are arranged in the reagent reaction cavity in a penetrating way, wherein the bottom of the reagent reaction cavity is communicated with the PCR reagent storage cavity through a sixth paraffin valve body.
Preferably, the sample cracking cavity is arranged at the uppermost part, and the first washing liquid cavity, the second washing liquid cavity and the eluent cavity are at the same height and are arranged below the sample cracking cavity.
Preferably, the bottom of the sample cracking cavity is of an inclined design, and the first paraffin valve body is arranged at the lowest position of the bottom of the sample cracking cavity.
Preferably, the screw rod is driven by a motor and is used for providing negative pressure for the reagent reaction cavity so that nucleic acid molecules in the reagent reaction cavity enter the PCR reagent storage cavity below through the opened sixth paraffin valve body.
Preferably, the first, second, third, fourth, fifth and sixth paraffin valve bodies realize liquid sealing and liquid conducting functions through respective heating modules.
Preferably, a sample injection hole closed by a removable sealing plug is arranged at the top of the sample cracking cavity for sample injection.
Preferably, the top of the rapid nucleic acid detection chip cartridge is further provided with a first vent hole and a second vent hole, the first vent hole is communicated with the reagent reaction cavity, and the second vent hole is communicated with the waste liquid cavity.
Preferably, a magnet for adsorbing magnetic beads and a motor for driving the magnet to move are also arranged in the rapid nucleic acid detection chip box.
According to the invention, nucleic acid extraction and purification are integrated with real-time fluorescence quantitative PCR detection, and the reagent required by the reaction is pre-buried in the reagent storage cavity in advance based on the encapsulation of plastic materials; the paraffin microfluidic pump valve design ensures that the reactions of the reagents are independently carried out in the operation process without mutual interference; the adsorption, enrichment, washing and elution of the nucleic acid molecules are completed by the adsorption of the magnetic beads by the permanent magnets. Based on the preparation of the sample in the nucleic acid extraction and purification part, the special fluid design is combined, and in the nucleic acid amplification and detection part, the accurate detection can be realized through limiting dilution and poisson distribution statistics.
According to the rapid nucleic acid detection chip cartridge based on the microfluidic technology, compared with the prior art, the rapid nucleic acid detection chip cartridge based on the microfluidic technology has the following beneficial effects:
1) The sample preparation and the nucleic acid detection are integrated together and are applied to the rapid detection of pathogens;
2) The specially designed paraffin valve body not only has the function of separating reagents in the nucleic acid extraction and purification part, but also plays a role in sealing air when the nucleic acid amplification detection is carried out on the subsequent plastic material;
3) Through the arrangement of the sample cracking cavity, the first washing liquid cavity, the second washing liquid cavity, the eluent cavity, the reagent reaction cavity and the waste liquid cavity in the chip card box from top to bottom along the vertical direction, the micro-flow liquid moves from top to bottom under the action of gravity in the whole process of nucleic acid extraction and purification, and manual operation of operators is not needed;
4) The driving motor in the instrument is used for driving the screw rod push rod to move, so that the pressure required by the mixed reagent to enter the PCR amplification detection module from the nucleic acid extraction and purification module is provided, and the whole process of sample inlet and outlet can be realized basically without driving other processes;
5) In the detection process, the nucleic acid molecules can be sent into the PCR instrument to finish target sample detection only by simple manual operation without injection of reagents and other operations, so that the requirements on the professional skills and auxiliary equipment of operators are greatly reduced, and the method has potential applicability in the field of molecular diagnosis.
Drawings
FIG. 1 is a schematic diagram showing a rapid nucleic acid detecting chip cartridge according to a preferred embodiment of the present invention;
FIG. 2 is a schematic diagram of the PCR amplification detection module in the rapid nucleic acid detection cartridge;
FIG. 3 is a schematic diagram of a flow chart of a rapid nucleic acid detection using the rapid nucleic acid detection cartridge;
wherein the reference numerals have the following meanings:
100: a sample lysis chamber; 101: a first wash liquid chamber; 102: a second wash liquid chamber; 103: an eluent chamber; 104: a reagent reaction chamber; 105: a waste liquid chamber; 106: a first paraffin valve body; 107: a second paraffin valve body; 108: a third paraffin valve body; 109: a fourth paraffin valve body; 110: a fifth paraffin valve body; 111: a sixth paraffin valve body; 112: a PCR reagent storage chamber; 113: a microfluidic chip; 114: a lead screw push rod; 115: a sample inlet; 116: a first exhaust hole; 117: a second exhaust hole; 119: a sample inlet; 120: a PCR amplification reaction region; 121: and a sample outlet.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The technical means used in the examples are, unless specified otherwise, conventional in the art or are in accordance with the experimental methods recommended by the manufacturers of the kits and instruments. Reagents and materials used in the examples were obtained commercially, unless otherwise specified.
As shown in fig. 1, the present invention provides a rapid nucleic acid detection cartridge integrating nucleic acid extraction, purification and automatic detection, which is based on a microfluidic pump valve for controlling a liquid flow path, and simultaneously uses a special flow path design and efficient heat transfer efficiency to achieve rapid nucleic acid amplification and detection. The rapid nucleic acid detection chip cartridge uses plastic material as a base material and mainly comprises: a nucleic acid extraction and purification module and a PCR amplification detection module.
As shown in FIG. 1, the nucleic acid extraction purification module comprises: a sample lysis chamber 100, a first washing solution chamber 101, a second washing solution chamber 102, an eluent chamber 103, a reagent reaction chamber 104, and a waste solution chamber 105. The bottoms of the sample cracking chamber 100, the first washing liquid chamber 101, the second washing liquid chamber 102 and the eluent chamber 103 are respectively communicated with the reagent reaction chamber 104 below through a first paraffin valve body 106, a second paraffin valve body 107, a third paraffin valve body 108 and a fourth paraffin valve body 109, and the bottom of the reagent reaction chamber 104 is communicated with the waste liquid chamber 105 below through a fifth paraffin valve body 110.
The PCR amplification detection module comprises: the bottom of the reagent reaction chamber 104 is also communicated with the PCR reagent storage chamber 112 through a sixth paraffin valve body 111, the PCR reagent storage chamber is used for storing freeze-dried PCR reagents, and the screw rod push rod 114 is arranged in the reagent reaction chamber 104 in a penetrating way.
The sample splitting chamber 100 is disposed at the uppermost position, the first washing liquid chamber 101, the second washing liquid chamber 102, and the eluent chamber 103 are at the same height, and disposed below the sample splitting chamber 100, and the waste liquid chamber 105 is disposed below the reagent reaction chamber 104. Therefore, in the whole nucleic acid extraction and purification process, each reactant flows from top to bottom under the action of gravity, and the extraction and purification of the nucleic acid can be completed without driving.
According to the preferred embodiment, the bottom of the sample cracking chamber 100 is of an inclined design, the first paraffin valve 106 is disposed at the lowest position of the bottom of the sample cracking chamber 100, and when the sample is cracked, all of the sample flows downwards from the first paraffin valve 106, so that liquid residues are avoided.
According to the preferred embodiment, the screw rod 114 is provided to penetrate the reagent reaction chamber 104, and is driven by a motor in the cartridge, so that a negative pressure is supplied to the reagent reaction chamber 104, and nucleic acid molecules in the reagent reaction chamber 104 are allowed to enter the PCR reagent storage chamber 112 below through the opened sixth paraffin valve body 111.
It should be appreciated that the first, second, third, fourth, fifth, and sixth paraffin valve bodies 106-111 are warmed and cooled by the respective heating modules to thereby achieve the liquid conduction and liquid sealing functions.
According to the preferred embodiment, the top of the sample-lysing chamber 100 is provided with a sample-feeding aperture 115 closed by a removable sealing plug for feeding. The top of the rapid nucleic acid detecting chip cartridge is further provided with a first vent hole 116 and a second vent hole 117, wherein the first vent hole 116 is communicated with the reagent reaction chamber 104, and the second vent hole 117 is communicated with the waste liquid chamber 105. The quick nucleic acid detection chip card box is also internally provided with a magnet for adsorbing magnetic beads and a motor for driving the magnet to move.
As shown in fig. 2, the microfluidic chip 113 includes a sample inlet 119, a PCR amplification reaction zone 120, and a sample outlet 121.
According to a preferred embodiment of the present invention, there is provided a method for rapid nucleic acid detection using the rapid nucleic acid detection cartridge, as follows:
firstly, as shown in step A in FIG. 3, sample lysate is added into a sample lysis chamber 100 in advance, washing solution A and washing solution B are respectively added into a first washing solution chamber 101 and a second washing solution chamber 102, eluent is added into an eluent chamber 103, magnetic beads are placed in a reagent reaction chamber 104 in advance, and then a swab sample is added into the sample lysis chamber 100 through a sample inlet 115;
then, as shown in steps B-C in fig. 3, when cells or viruses in the sample lysis chamber 100 are lysed, the first paraffin valve 106 is heated by the TEC heating module inside the cartridge to be opened, and the first exhaust hole 116 is opened, and the nucleic acid molecules and the sample lysis solution flow into the reagent reaction chamber 104 below together, and the released nucleic acid molecules are adsorbed by the magnetic beads;
then, as shown in step D of fig. 3, the magnet motor is raised, the fifth paraffin valve body 110 is heated by the TEC heating module inside the cartridge, and the second exhaust hole 117 is opened at the same time, so that the waste liquid is discharged into the waste liquid chamber 105;
then, as shown in steps E-F in fig. 3, the second exhaust hole 117 is closed, the second paraffin valve body 107 is heated by the TEC heating module inside the cartridge, the washing liquid a is released into the reagent reaction chamber 104, the magnetic beads are ensured to be completely dispersed, so as to clean impurities on nucleic acid molecules, the magnet motor is lifted, the fifth paraffin valve body 110 is heated by the TEC heating module inside the cartridge, the second exhaust hole 117 is opened, and the waste liquid is discharged into the waste liquid chamber 105;
repeating steps E-F, specifically, closing the second exhaust hole 117, heating the third paraffin valve body 108 by the TEC heating module in the cartridge, releasing the washing liquid B into the reagent reaction cavity 104, ensuring that the magnetic beads are completely dispersed again to further clean impurities on the nucleic acid molecules, heating the fifth paraffin valve body 110 by the TEC heating module in the cartridge, opening the second exhaust hole 117, and discharging the waste liquid into the waste liquid cavity 105;
then, as shown in step G in fig. 3, the second exhaust hole 117 is closed, the fourth paraffin valve body 109 is heated by the TEC heating module inside the cartridge, the eluent is released into the reagent reaction chamber 104, the magnetic beads are ensured to be completely dispersed again, the nucleic acid molecules are eluted from the magnetic beads, the fifth paraffin valve body 110 is heated by the TEC heating module inside the cartridge, the second exhaust hole 117 is opened, and the waste liquid is discharged into the waste liquid chamber 105;
finally, as shown in step H in fig. 3, the second vent hole 117 is closed, the first vent hole 116 is opened, the sixth paraffin valve body 111 is heated by the TEC heating module inside the cartridge, the motor controls the screw rod 114 to move, so that negative pressure is generated in the reagent reaction chamber 104, the eluent and the nucleic acid molecules enter the PCR reagent storage chamber 112 together, the freeze-dried PCR reagent pre-stored in the PCR reagent storage chamber is melted, the screw rod 114 continues to move, the mixed reagent is injected into the microfluidic chip 113, the first vent hole 116 is closed, the sixth paraffin valve body 111 at the inlet is sealed after cooling, the PCR amplification detection module is sealed, finally, related programs set by the PCR detection analyzer are completed by the heating cycle inside the cartridge, and a detection result report is output after the reaction is completed.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications can be made to the above-described embodiment of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of the present application fall within the scope of the patent claims. The present invention is not described in detail in the conventional art.
Claims (9)
1. A microfluidic technology-based rapid nucleic acid detection chip cartridge, comprising:
a nucleic acid extraction purification module comprising: the device comprises a sample cracking cavity, a first washing liquid cavity, a second washing liquid cavity, an eluent cavity, a reagent reaction cavity and a waste liquid cavity; the bottoms of the sample cracking cavity, the first washing liquid cavity, the second washing liquid cavity and the eluent cavity are respectively communicated with the reagent reaction cavity through a first paraffin valve body, a second paraffin valve body, a third paraffin valve body and a fourth paraffin valve body, and the bottoms of the reagent reaction cavity are communicated with the waste liquid cavity through a fifth paraffin valve body; and
a PCR amplification detection module, comprising: the PCR reagent storage cavity, the microfluidic chip and the lead screw push rod are arranged in the reagent reaction cavity in a penetrating way, wherein the bottom of the reagent reaction cavity is communicated with the PCR reagent storage cavity through a sixth paraffin valve body.
2. The rapid nucleic acid detection cartridge of claim 1, wherein the sample lysis chamber is disposed uppermost, the first wash chamber, the second wash chamber, and the eluent chamber are at the same height, and are disposed below the sample lysis chamber.
3. The rapid nucleic acid detection cartridge of claim 1, wherein the bottom of the sample lysis chamber is of an inclined design, and the first paraffin valve body is disposed at a lowest position of the bottom of the sample lysis chamber.
4. The rapid nucleic acid testing chip cartridge of claim 1, wherein the screw rod is driven by a motor for providing negative pressure to the reagent reaction chamber to allow nucleic acid molecules in the reagent reaction chamber to enter the underlying PCR reagent storage chamber through the open sixth paraffin valve body.
5. The rapid nucleic acid testing chip cartridge of claim 1, wherein the first, second, third, fourth, fifth, and sixth paraffin valve bodies are liquid sealed and liquid conductive by respective heating modules.
6. The rapid nucleic acid detection cartridge of claim 1, wherein the top of the sample lysis chamber is provided with a sample introduction aperture closed by a removable sealing plug for sample introduction.
7. The rapid nucleic acid detection chip cartridge of claim 1, wherein the top of the rapid nucleic acid detection chip cartridge is further provided with a first vent and a second vent, the first vent being in communication with the reagent reaction chamber, the second vent being in communication with the waste chamber.
8. The rapid nucleic acid detection cartridge of claim 1, wherein a magnet for attracting magnetic beads and a motor for driving the magnet to move are further provided in the rapid nucleic acid detection cartridge.
9. The rapid nucleic acid detection cartridge of claim 1, wherein the PCR reagent storage chamber is configured to store lyophilized PCR reagents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311026763.9A CN117229898A (en) | 2023-08-15 | 2023-08-15 | Rapid nucleic acid detection chip cartridge based on microfluidic technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311026763.9A CN117229898A (en) | 2023-08-15 | 2023-08-15 | Rapid nucleic acid detection chip cartridge based on microfluidic technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117229898A true CN117229898A (en) | 2023-12-15 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311026763.9A Pending CN117229898A (en) | 2023-08-15 | 2023-08-15 | Rapid nucleic acid detection chip cartridge based on microfluidic technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117229898A (en) |
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2023
- 2023-08-15 CN CN202311026763.9A patent/CN117229898A/en active Pending
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