CN114989971B - Card box device for nucleic acid extraction and automatic liquid separation and analysis method thereof - Google Patents
Card box device for nucleic acid extraction and automatic liquid separation and analysis method thereof Download PDFInfo
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- CN114989971B CN114989971B CN202210941136.7A CN202210941136A CN114989971B CN 114989971 B CN114989971 B CN 114989971B CN 202210941136 A CN202210941136 A CN 202210941136A CN 114989971 B CN114989971 B CN 114989971B
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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
Abstract
The invention provides a card box device for nucleic acid extraction and automatic liquid separation and an analysis method thereof, wherein the card box device comprises a nucleic acid extraction structure and a liquid separation mechanism, wherein the liquid separation mechanism comprises an actuating mechanism and a nucleic acid derivation structure; the nucleic acid extraction structure comprises a lysis liquid tube, a magnetic bead tube, a first cleaning liquid tube, a second cleaning liquid tube, a third cleaning liquid tube, an elution liquid tube and a sample tube which are sequentially and independently connected; a liquid storage tank and a liquid circulation channel are arranged in the nucleic acid leading-out structure, a conical tube is formed at the bottom end of the nucleic acid leading-out structure, the liquid storage tank is communicated with the conical tube through the liquid circulation channel, and the liquid storage tank is communicated with the sample tube through the liquid circulation channel; the actuating mechanism controls the opening and the blocking of the liquid circulation channel through a first valve and a second valve; the liquid storage tank is connected with the liquid pushing rod in a sliding mode, the actuating mechanism controls the liquid pushing rod to move up and down and is matched with the first valve and the second valve to achieve automatic quantitative liquid separation of the reagent, and the work efficiency of checking, extracting and detecting is improved.
Description
Technical Field
The invention relates to a liquid sample preparation and delivery device, in particular to a device for preparing and delivering a nucleic acid sample in a sample and an analysis method thereof.
Background
With the continuous improvement of clinical diagnosis requirements and the sensitivity of nucleic acid detection, nucleic acid substances to be detected in samples become a key part in the clinical diagnosis process, most of the existing pretreatment, extraction and analysis devices for nucleic acid amplification samples rely on manual operation to realize precise liquid transfer, have high requirements on manpower, and can use related equipment and instruments only through professional training. The existing sample quantitative distribution comprises a plurality of complicated steps, and the steps are manually completed or completed by auxiliary equipment, so that the problems of low efficiency, easy error and the like exist. Especially, stable liquid separation and transfer of small-volume liquid are always required by various analysis methods, but a mainstream automatic analysis instrument cannot well solve the technical difficulties and cannot realize miniaturization of equipment.
Therefore, how to realize the automatic distribution of the microliter-scale sample and improve the efficiency of quantitative distribution of the sample and the accuracy of the micro-quantitative distribution are problems to be solved in the field of sample processing and detection of nucleic acid analysis at present. To better address and complement the above-mentioned needs, many companies have been engaged in the development of integrated nucleic acid detection devices, and several types of devices or apparatuses for initially performing integrated nucleic acid extraction and amplification are also available on the market, and the most common cartridges or devices are complicated to assemble and are not suitable for mass production and application.
Disclosure of Invention
The invention provides a card box device for nucleic acid extraction and automatic liquid separation and an analysis method thereof, which are used for solving the problems of low sample distribution efficiency and difficult automatic quantification of trace liquid in the prior art and realizing automatic quantitative distribution of microliter sample reagents.
The invention provides a card box device for nucleic acid extraction and automatic liquid separation, which comprises a nucleic acid extraction structure and a liquid separation mechanism, wherein the liquid separation mechanism comprises an actuating mechanism and a nucleic acid leading-out structure, and the nucleic acid extraction structure comprises a lysis liquid pipe, a magnetic bead pipe, a first cleaning liquid pipe, a second cleaning liquid pipe, a third cleaning liquid pipe, an elution liquid pipe and a sample pipe which are sequentially and independently connected; the nucleic acid leading-out structure is arranged on one side of the nucleic acid extracting structure and is clamped with the nucleic acid extracting structure; a liquid storage tank and a liquid circulation channel are arranged in the nucleic acid leading-out structure, a conical tube is formed at the bottom end of the nucleic acid leading-out structure, the liquid storage tank is communicated with the conical tube through the liquid circulation channel, and the liquid storage tank is communicated with the sample tube through the liquid circulation channel; the actuating mechanism controls the opening and the blocking of the liquid flow channel.
Preferably, a first locking block is arranged at the right side of the nucleic acid derivation structure, and a groove matched with the first locking block is arranged at the corresponding position of the nucleic acid extraction structure; a second locking block is arranged on the left side of the nucleic acid extraction structure, and a groove matched with the second locking block is arranged at the corresponding position of the nucleic acid derivation structure; the nucleic acid extraction structure and the nucleic acid derivation structure are clamped and fixed through the first locking block and the second locking block.
Preferably, the liquid flow channel comprises a vertical channel in the vertical direction and a horizontal channel in the horizontal direction, the vertical channel is positioned in the middle of the nucleic acid derivation structure, and the horizontal channel is positioned on the right side of the nucleic acid derivation structure; the transverse channel and the vertical channel are intersected and communicated in the nucleic acid leading-out structure to form a T-shaped channel.
Preferably, a first valve slot is arranged on the left side of the nucleic acid derivation structure, and the first valve slot is horizontally arranged and penetrates through the vertical channel; the first valve is in rotating fit with the first valve groove; the first valve is provided with a radial through hole, the through hole is communicated with the vertical channel after the first valve rotates for a certain angle, the actuating mechanism is in transmission connection with the first valve, and the vertical channel is opened/blocked by rotating the first valve.
Preferably, a second valve slot is arranged on the right side of the nucleic acid exporting structure, and the second valve slot is vertically arranged and penetrates through the transverse channel; the second valve is in rotating fit with the second valve groove, a radial fluid channel is formed in the upper portion of the second valve, the fluid channel is communicated with the transverse channel after the second valve rotates for a certain angle, the actuating mechanism is in transmission connection with the second valve, and the transverse channel can be opened/blocked through rotation of the second valve.
Preferably, the liquid pushing rod is connected with the liquid storage tank in a sliding mode, the top of the liquid pushing rod is provided with a connecting part, the middle of the liquid pushing rod is provided with a pushing limiting disc, the bottom of the liquid pushing rod is provided with a lifting limiting disc, the bottom of the lifting limiting disc is fixedly connected with a piston, and the piston is in sealing sliding fit with the liquid storage tank; the upper part of the liquid storage tank is provided with a positioning block, wherein the bottom surface of the pushing limiting disc is matched with the top surface of the positioning block, the top surface of the lifting limiting disc is matched with the bottom surface of the positioning block, and the actuating mechanism is in transmission connection with the connecting part of the liquid pushing rod.
Preferably, a positioning bulge is arranged at the end part of the first valve, a fan-shaped step is arranged at the left end of the first valve groove, the positioning bulge is matched with the fan-shaped step, a first sealing rubber ring is arranged on the periphery of the first valve, and the first sealing rubber ring is an annular sealing structure sealed by a skirt belt; and a second sealing rubber ring is arranged at the periphery of the second valve.
Preferably, the bottom of the pyrolysis liquid pipe is provided with a solid-liquid separation membrane, the reaction liquid is arranged above the solid-liquid separation membrane, and the rehydration liquid or the rehydration reagent is arranged below the solid-liquid separation membrane.
Preferably, the sealing film comprises a glue layer and a sealing film layer, the sealing film layer is adhered to the upper surface of the nucleic acid extraction structure through the glue layer, the sealing film layer is made of a multi-layer composite material, the nucleic acid leading-out structure is matched with the detection reagent tube, and the detection reagent tube is internally preset with detection reagents.
The invention also provides a nucleic acid analysis method, which is implemented based on the card box device for nucleic acid extraction and automatic liquid separation, and mainly comprises the following steps:
s1: the magnetic rod is extended into the magnetic bead tube to adsorb the magnetic beads;
s2: heating to melt the isolating layer in the lysis solution tube, fully mixing the nucleic acid extraction protein lytic enzyme and the lysis solution, adding the biological sample into the lysis solution tube containing the nucleic acid extraction protein lytic enzyme and the lysis solution for lysis and releasing nucleic acid;
s3: extending the magnetic bar adsorbed with the magnetic beads into a lysis liquid tube to pump up and down so as to enable the magnetic beads to adsorb the released nucleic acid;
s4: sequentially extending the magnetic rod adsorbed with the magnetic beads into the first cleaning liquid pipe, the second cleaning liquid pipe and the third cleaning liquid pipe to perform pumping rinsing up and down;
s5, stretching the rinsed magnetic rod into an elution tube for elution, and transferring a reagent in the elution tube to a sample tube after elution is finished;
s6, controlling the second valve to rotate clockwise by the actuating mechanism to open the transverse channel; the actuating mechanism controls the first valve to rotate to block the vertical channel; the actuating mechanism lifts the liquid pushing rod upwards through the connecting part until the top surface of the lifting limiting disc is contacted with the bottom surface of the positioning block, so that the reagent transferring process is completed;
s7: the actuating mechanism controls the second valve to rotate anticlockwise so as to block the transverse channel; the actuating mechanism controls the first valve to rotate to open the vertical channel; the actuating mechanism pushes the liquid pushing rod downwards through the connecting part until the bottom surface of the pushing limiting disc is contacted with the top surface of the positioning block, and the reagent is discharged into the detection reagent tube from the conical tube, so that the quantitative distribution of the reagent is realized.
Compared with the prior art, the invention has the beneficial effects that:
1) The actuator controls the second valve 500 to rotate to open/close the cross passage 205; the actuator controls the first valve 400 to rotate to open/block the vertical channel 208; the actuator realizes the automatic injection and discharge of the reagent by lifting/pushing the plunger rod 300 in cooperation with the movement of the first valve 400 and the second valve.
2) When the top surface of the lifting limiting disc is in contact with the bottom surface of the positioning block 2021, the reagent transferring process is completed, when the bottom surface of the pushing limiting disc is in contact with the top surface of the positioning block 2021, the reagent extracting process is completed, and quantitative distribution of the reagent is realized by arranging the lifting limiting disc, the pushing limiting disc and the positioning block.
3) By arranging the sealing film 109 for sealing the reagent in the nucleic acid extraction structure 100, the sample reagent can be stored for a longer time, and it can be ensured that the liquid does not spill during transportation.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:
FIG. 1 is a schematic diagram of a cartridge device according to an embodiment of the present application.
Fig. 2 is a schematic three-dimensional structure diagram of a cartridge device provided in an embodiment of the present application.
FIG. 3 is a schematic diagram showing the cooperation of a nucleic acid export structure and a detection reagent tube according to the embodiment of the present application.
FIG. 4 is a schematic view of the assembly of the cartridge device according to the embodiment of the present application.
FIG. 5 is a schematic view of a liquid pushing rod and a liquid storage tank provided in the embodiment of the present application.
FIG. 6 is a schematic view of a first valve and a first valve slot in accordance with an embodiment of the present disclosure.
Fig. 7 is a schematic view of fluid control of a liquid separation mechanism according to an embodiment of the present application.
FIG. 8 is a sealing film structure according to an embodiment of the present disclosure.
FIG. 9 shows a partition structure of a lysis liquid tube according to an embodiment of the present application.
In the figure: the nucleic acid extraction structure 100, a lysis liquid tube 101, a magnetic bead tube 102, a first cleaning liquid tube 103, a second cleaning liquid tube 104, a third cleaning liquid tube 105, an elution liquid tube 106, a sample tube 107, a lysis liquid solution 108, a sealing membrane 109, an adhesive layer 1091, a sealing membrane layer 1092, a nucleic acid derivation structure 200, a first valve groove 201, a liquid storage tank 202, a positioning block 2021, a second valve groove 203, a first locking block 204, a transverse channel 205, a tapered tube 206, a second locking block 207, a vertical channel 208, a first valve 400, a first sealing rubber ring 401, a positioning protrusion 402, a second valve 500, a fluid channel 501, a second sealing rubber ring 502, a detection reagent tube 600 and a detection reagent 601.
The drawings are for illustrative purposes only and are not to be construed as limiting the patent; for the purpose of better illustrating the embodiments, certain features of the drawings may be omitted, enlarged or reduced, and do not represent the size of an actual product; it will be understood by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted.
Detailed Description
The principles and features of the present invention are described below in conjunction with the accompanying fig. 1-9, which are provided by way of example only and are not intended to limit the scope of the invention. The invention is described in more detail in the following paragraphs by way of example with reference to the accompanying drawings. Advantages and features of the present invention will become apparent from the following description and from the claims. It is to be noted that the drawings are in a very simplified form and are not to precise scale, which is merely for the purpose of facilitating and distinctly claiming the embodiments of the present invention.
It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When a component is referred to as being "connected" to another component, it can be directly connected to the other component or intervening components may also be present. When a component is referred to as being "disposed on" another component, it can be directly on the other component or intervening components may also be present. The terms "vertical," "horizontal," "left," "right," and the like as used herein are for illustrative purposes only.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1
With reference to the accompanying drawings 1-4, the invention provides a cartridge device for nucleic acid extraction and automatic liquid separation, which comprises: the nucleic acid extraction device comprises a nucleic acid extraction structure 100 and a liquid separation mechanism, wherein the liquid separation mechanism comprises an execution mechanism and a nucleic acid leading-out structure 200, the nucleic acid extraction structure 100 is arranged on one side of the nucleic acid extraction structure 100, the nucleic acid leading-out structure 200 is clamped with the nucleic acid extraction structure 100, a first locking block 204 is arranged on the right side of the nucleic acid leading-out structure 200, and a groove matched with the first locking block 204 is arranged at the corresponding position of the nucleic acid extraction structure 100; a second locking block 207 is arranged at the left side of the nucleic acid extracting structure 100, and a groove matched with the second locking block 207 is arranged at the corresponding position of the nucleic acid exporting structure 200; the nucleic acid extraction structure 100 and the nucleic acid export structure 200 are clamped and fixed by the first locking block 204 and the second locking block 207.
As shown in fig. 1: the nucleic acid extraction structure 100 comprises 7 reagent tubes, wherein the 7 reagent tubes are a lysis liquid tube 101, a magnetic bead tube 102, a first cleaning liquid tube 103, a second cleaning liquid tube 104, a third cleaning liquid tube 105, an elution liquid tube 106 and a sample tube 107 from right to left in sequence, wherein the lysis liquid tube 101, the magnetic bead tube 102, the first cleaning liquid tube 103, the second cleaning liquid tube 104, the third cleaning liquid tube 105, the elution liquid tube 106 and the sample tube 107 are respectively and independently arranged, lysis liquid is injected into the lysis liquid tube 101, magnetic bead suspension is injected into the magnetic bead tube 102, cleaning liquid 1 is injected into the first cleaning liquid tube 103, cleaning liquid 2 is injected into the second cleaning liquid tube 104, cleaning liquid 3 is injected into the third cleaning liquid tube 105, elution liquid is injected into the elution liquid tube 106, and the sample tube 107 is used for storing a reagent to be detected.
As shown in fig. 1,3,4, the liquid separating mechanism includes an actuator and a nucleic acid discharge structure 200, a liquid storage tank 202 and a liquid flow channel are disposed inside the nucleic acid discharge structure 200, wherein the liquid flow channel includes a vertical channel 208 in a vertical direction and a horizontal channel 205 in a horizontal direction, the vertical channel 208 is located in the middle of the nucleic acid discharge structure 200, and the horizontal channel 205 is located in the right of the nucleic acid discharge structure 200; the transverse channel 205 and the vertical channel 208 are intersected and communicated in the nucleic acid export structure 200 to form a T-shaped channel, and the T-shaped channel is a dead volume part for transferring a reagent sample and cannot be completely discharged, so that the volume of the T-shaped channel is reduced as much as possible; a first valve slot 201 is arranged at the left side of the nucleic acid derivation structure 200, and the first valve slot 201 is horizontally arranged and penetrates through the vertical channel 208; the first valve 400 is rotatably engaged with the first valve slot 201, and the vertical channel 208 is opened/blocked by rotating the first valve 400, so that the reagent in the opening/blocking reservoir 202 flows out of the conical pipe 206; a radial through hole is formed in the first valve 400, and the through hole is communicated with the vertical channel 208 after the first valve 400 rotates for a certain angle;
a second valve groove 203 is formed on the right side of the nucleic acid derivation structure 200, and the second valve groove 203 is vertically arranged and penetrates through the transverse channel 205; the second valve 500 is rotatably engaged with the second valve groove 203, the transverse channel 205 can be opened/blocked by the rotation of the second valve 500, and the reagent can be opened/blocked from flowing from the sample tube 107 to the reservoir 202 by the second valve 500; a radial fluid channel 501 is formed in the upper part of the second valve 500, and after the second valve 500 rotates by a certain angle, the fluid channel 501 is communicated with the transverse channel 205;
the top of the nucleic acid derivation structure 200 is provided with a reservoir 202, and the bottom of the reservoir 202 is communicated with the top of the vertical channel 208 in an intersecting manner.
As shown in fig. 5, the liquid pushing rod 300 is slidably connected with the liquid storage tank 202, the liquid pushing rod 300 is a fluid control push rod, the top of the liquid pushing rod 300 is provided with a connecting part, the middle part is provided with a pushing limiting disc, the bottom is provided with a lifting limiting disc, the bottom of the lifting limiting disc is fixedly connected with a piston, and the piston is in sealing sliding fit with the liquid storage tank 202; a positioning block 2021 is disposed at the upper portion of the liquid storage tank 202, wherein the bottom surface of the push-in limiting disc is matched with the top surface of the positioning block 2021, and the top surface of the lift-up limiting disc is matched with the bottom surface of the positioning block 2021. The bottom end of the nucleic acid export structure 200 forms a tapered tube 206, and a vertical channel 208 extends from the reservoir 202 to the orifice of the tapered tube 206. In operation, the plunger 300 is pulled up through the connecting portion, and the reagent is injected from the sample tube 107 into the reservoir 202 through cooperation with the first valve 400 and the second valve 500; pushing the plunger 300 downward through the connection portion, so that the reagent flows downward from the reservoir 202 into the tapered tube 206 and is discharged through the tapered tube 206; when the liquid pushing rod 300 is pulled up through the connecting part, the reagent transferring process is completed when the top surface of the lifting limiting disc is contacted with the bottom surface of the positioning block 2021; the liquid pushing rod 300 is pushed downwards through the connecting part until the bottom surface of the pushing limiting disc is contacted with the top surface of the positioning block 2021, so that the extraction process of the reagent is completed; the injection and the discharge of the reagent are realized by pushing and pulling the liquid pushing rod 300, and the quantitative distribution of the reagent is realized by arranging the structure arrangement of pushing the limit disc, lifting the limit disc and the positioning block 2021.
As shown in fig. 6, the first valve 400 is rotatably connected to the first valve slot 201, a positioning protrusion 402 is disposed at an end of the first valve 400, a fan-shaped step is formed at a left end of the first valve slot 201, the positioning protrusion 402 is engaged with the fan-shaped step, and the positioning protrusion 402 contacts with a side wall of the fan-shaped step in a process of rotating the first valve 400 to define a rotation angle of the first valve 400. A first sealing rubber ring 401 is further arranged on the periphery of the first valve 400, and the first sealing rubber ring 401 is an annular sealing structure with a skirt seal and can prevent liquid from flowing in an unexpected direction. The first valve 400 may also be in the form of a push rod, or other means for achieving rotational or sliding retraction, connected to the first valve slot 201 to open or close the vertical channel 208;
the second valve 500 is a reagent inlet valve, a radial fluid passage 501 is formed at an upper portion of the second valve 500, and a second sealing rubber ring 502 is disposed at an outer circumference of the second valve 500.
FIG. 7 shows a schematic diagram of fluid control of the liquid separation mechanism, the reagent after nucleic acid extraction is put into the sample tube 107, the external driving mechanism is connected to the connecting portion of the liquid pushing rod, when the liquid pushing rod 300 is pulled up through the connecting portion, the external control system controls the second valve 500 to rotate clockwise to open the transverse channel 205, when the top surface of the lifting limiting disc contacts the bottom surface of the positioning block 2021, the external control system controls the second valve 500 to rotate counterclockwise to block the transverse channel 205, completing the reagent transfer process, in which the first valve 400 is in a state of blocking the vertical channel 208; before the connecting part starts to push the liquid pushing rod 300 downwards, the external control system rotates the first valve 400 to open the vertical channel 208, when the bottom surface of the push limiting disc is contacted with the top surface of the positioning block 2021, the external control system rotates the first valve 400 to block the vertical channel 208, the extraction process of the reagent is completed, and in the process, the second valve 500 is in a state of blocking the transverse channel 205; the injection and the discharge of the reagent are realized by pushing and pulling the liquid pushing rod 300, and the quantitative distribution of the reagent is realized by arranging the structure arrangement of pushing the limit disc, lifting the limit disc and the positioning block 2021.
The actuating mechanism is in transmission connection with the connecting parts of the first valve 400, the second valve 500 and the liquid pushing rod 300, and the actuating mechanism automatically controls the movement of the first valve 400, the second valve 500 and the liquid pushing rod 300 to realize automatic liquid distribution.
Example 2
Based on this application provides a cartridge device of nucleic acid extraction and automatic minute liquid carries out the process that nucleic acid extracted and is:
s1: magnetic beads are adsorbed by extending a magnetic rod into the magnetic bead tube 102;
s2: heating to melt the isolating layer in the lysis solution tube 101, fully mixing the nucleic acid extraction protein lytic enzyme and the lysis solution, adding the biological sample into the lysis solution tube 101 containing the nucleic acid extraction protein lytic enzyme and the lysis solution for lysis and releasing nucleic acid;
s3: extending the magnetic rod with the magnetic beads into the lysis liquid tube 101 to be pumped up and down so that the magnetic beads adsorb the released nucleic acid;
s4: sequentially extending the magnetic rod adsorbed with the magnetic beads into the first cleaning liquid pipe 103, the second cleaning liquid pipe 104 and the third cleaning liquid pipe 105 for up-and-down pumping rinsing;
s5, the rinsed magnetic rod extends into an elution liquid pipe 106 for elution, and after elution is finished, a reagent in the elution liquid pipe 106 is transferred to a sample pipe 107;
s6, the actuating mechanism controls the second valve 500 to rotate clockwise to open the transverse channel 205; the actuator controls the first valve 400 to rotate to block the vertical channel 208; the actuating mechanism lifts the liquid pushing rod 300 upwards through the connecting part until the top surface of the lifting limiting disc is contacted with the bottom surface of the positioning block 2021, so that the reagent transferring process is completed;
s7: the actuator controls the second valve 500 to rotate counterclockwise, blocking the cross passage 205; the actuator controls the first valve 400 to rotate to open the vertical channel 208; the actuator pushes the liquid pushing rod 300 downwards through the connecting part until the bottom surface of the pushing limiting disc is contacted with the top surface of the positioning block 2021, and the reagent is discharged into the detection reagent tube 600 from the conical tube 206, so that the quantitative distribution of the reagent is realized;
wherein the volume of the lysis solution is 100-600 mul, the volume of the magnetic bead suspension is 50-600 mul, the volumes of the cleaning solution 1, the cleaning solution 2 and the cleaning solution 3 are 100-1000 mul, and the volume of the eluent is 5-200 mul.
Further, an external automation device is in transmission connection with the magnetic rod, and the movement of the magnetic rod is controlled by the external automation device so as to automatically complete the nucleic acid extraction process of the steps S1-S5.
Example 3
Based on embodiments 1 and 2, the present embodiment also provides a sealing membrane 109 for encapsulating reagents within the nucleic acid extraction structure 100; on one hand, the sealing film can store the sample reagent for a longer time; on the other hand, the sealing film 109 can also ensure that liquid does not spill during transportation; before the sample reagent needs to be detected, the sealing film 109 is manually torn, so that each reagent tube of the nucleic acid extraction structure 100 is exposed for subsequent nucleic acid extraction and analysis.
The nucleic acid derivation structure 200 is matched with the detection reagent tube 600, the detection reagent tube 600 is pre-provided with a detection reagent 601, the detection reagent 601 is usually a nucleic acid detection reagent for carrying out a nucleic acid amplification reaction, the detection reagent 601 usually contains a fluorescence detection or color development related reagent for judging the end point of the nucleic acid amplification reaction, and the detection reagent 601 can be a reagent dry powder, a gel or a liquid reagent.
As shown in FIG. 8, the sealing film 109 comprises a glue layer 1091 and a sealing film layer 1092, the sealing film layer 1092 is adhered to the upper surface of the nucleic acid extracting structure 100 by the glue layer 1091, and the glue layer 1091 is a special adhesive material and needs to withstand a freezing temperature of-20 ℃ at the lowest, and can maintain long-term adhesion stability at 4 ℃. The sealing film layer 1092 is typically a multi-layer composite material, in which the supporting material is an aluminum foil, and the top and bottom of the aluminum foil are attached with surface coating structures.
Example 4
Based on embodiments 1 and 2, the present embodiment provides a separation structure of the lysis liquid tube 101, as shown in fig. 9, wherein a solid-liquid separation membrane 1012 is disposed at the bottom of the lysis liquid tube 101, a reaction liquid 1011 is disposed above the solid-liquid separation membrane 1012, and a rehydration liquid or rehydration agent 1013 is disposed below the solid-liquid separation membrane 1012; in use, the solid-liquid separation membrane 1012 is opened by movement of a mechanical lever (not shown) above for mixing the reconstituting liquid or reconstituting reagent 1013 with the reaction liquid 1011. In addition, a separation structure may be provided on the magnetic bead tube 102, the first cleaning solution tube 103, the second cleaning solution tube 104, the third cleaning solution tube 105, and the elution tube 106 as needed.
According to the cartridge device for nucleic acid extraction and automatic liquid separation and the analysis method thereof, the actuating mechanism controls the second valve 500 to rotate to open/close the transverse channel 205; the actuator controls the first valve 400 to rotate to open/block the vertical channel 208; the actuator realizes the automatic injection and discharge of the reagent by lifting/pushing the plunger rod 300 in cooperation with the movement of the first valve 400 and the second valve. When the top surface of the lifting limiting disc is in contact with the bottom surface of the positioning block 2021, the reagent transferring process is completed, when the bottom surface of the pushing limiting disc is in contact with the top surface of the positioning block 2021, the reagent extracting process is completed, and quantitative distribution of the reagent is realized by arranging the lifting limiting disc, the pushing limiting disc and the positioning block. By arranging the sealing film 109 for sealing the reagent in the nucleic acid extraction structure 100, the sample reagent can be stored for a longer time, and it can be ensured that the liquid does not spill during transportation.
It should be noted that, in this document, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Those not described in detail in this specification are within the skill of the art.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner; the present invention may be readily implemented by those of ordinary skill in the art as illustrated in the accompanying drawings and described above; however, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the scope of the invention as defined by the appended claims; meanwhile, any equivalent changes, modifications and evolutions made to the above embodiments according to the substantial technology of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (10)
1. The utility model provides a card box device of nucleic acid extraction and automatic minute liquid, includes nucleic acid extraction structure and divides liquid mechanism, and wherein divide liquid mechanism to include actuating mechanism and nucleic acid and derive structure, its characterized in that: the nucleic acid extraction structure comprises a lysis liquid tube (101), a magnetic bead tube (102), a first cleaning liquid tube (103), a second cleaning liquid tube (104), a third cleaning liquid tube (105), an elution liquid tube (106) and a sample tube (107) which are sequentially and independently connected;
the nucleic acid exporting structure (200) is arranged on one side of the nucleic acid extracting structure (100) and is clamped with the nucleic acid extracting structure (100);
a liquid storage tank (202) and a liquid flow channel are arranged in the nucleic acid leading-out structure (200), a conical tube (206) is formed at the bottom end of the nucleic acid leading-out structure (200), the liquid storage tank (202) is communicated with the conical tube (206) through the liquid flow channel, and the liquid storage tank (202) is communicated with a sample tube through the liquid flow channel; the actuating mechanism controls the opening and the blocking of the liquid circulation channel.
2. The cartridge device for nucleic acid isolation and automatic liquid distribution according to claim 1, wherein: a first locking block (204) is arranged on the right side of the nucleic acid derivation structure (200), and a groove matched with the first locking block (204) is arranged at the corresponding position of the nucleic acid extraction structure (100); a second locking block (207) is arranged on the left side of the nucleic acid extraction structure (100), and a groove matched with the second locking block (207) is arranged at the corresponding position of the nucleic acid leading-out structure (200); the nucleic acid extraction structure (100) and the nucleic acid derivation structure (200) are clamped and fixed by the first locking block (204) and the second locking block (207).
3. The cartridge device for nucleic acid isolation and automatic liquid distribution according to claim 2, wherein: the liquid flow channel comprises a vertical channel (208) in a vertical direction and a horizontal channel (205) in a horizontal direction, wherein the vertical channel (208) is positioned in the middle of the nucleic acid derivation structure (200), and the horizontal channel (205) is positioned at the right side of the nucleic acid derivation structure (200); the transverse channel (205) and the vertical channel (208) are intersected and communicated in the nucleic acid exporting structure (200) and form a T-shaped channel.
4. The cartridge device for nucleic acid isolation and automatic liquid distribution according to claim 3, wherein: a first valve slot (201) is formed in the left side of the nucleic acid derivation structure (200), and the first valve slot (201) is horizontally arranged and penetrates through the vertical channel (208); the first valve (400) is in rotating fit with the first valve groove (201); the first valve (400) is provided with a radial through hole, the through hole is communicated with the vertical channel (208) after the first valve (400) rotates for a certain angle, the actuating mechanism is in transmission connection with the first valve (400), and the vertical channel (208) is opened/blocked by rotating the first valve (400).
5. The cartridge device for nucleic acid isolation and automatic liquid distribution according to claim 4, wherein: a second valve groove (203) is formed on the right side of the nucleic acid derivation structure (200), and the second valve groove (203) is vertically arranged and penetrates through the transverse channel (205); the second valve (500) is in rotating fit with the second valve groove (203), a radial fluid channel (501) is formed in the upper portion of the second valve (500), after the second valve (500) rotates for a certain angle, the fluid channel (501) is communicated with the transverse channel (205), the actuating mechanism is in transmission connection with the second valve (500), and the transverse channel (205) can be opened/closed through the rotation of the second valve (500).
6. The cartridge device for nucleic acid isolation and automatic liquid distribution according to claim 1, wherein: the liquid pushing rod (300) is connected with the liquid storage tank (202) in a sliding mode, a connecting portion is arranged at the top of the liquid pushing rod (300), a pushing limiting disc is arranged in the middle of the liquid pushing rod, a lifting limiting disc is arranged at the bottom of the liquid pushing rod, a piston is fixedly connected to the bottom of the lifting limiting disc, and the piston is in sealing sliding fit with the liquid storage tank (202); a positioning block (2021) is arranged at the upper part of the liquid storage tank (202), wherein the bottom surface of the push-in limiting disc is matched with the top surface of the positioning block (2021), the top surface of the push-up limiting disc is matched with the bottom surface of the positioning block (2021), and the actuating mechanism is in transmission connection with the connecting part of the liquid pushing rod (300).
7. The cartridge device for nucleic acid isolation and automatic liquid distribution according to claim 5, wherein: a positioning bulge (402) is arranged at the end part of the first valve (400), a fan-shaped step is arranged at the left end of the first valve groove (201), the positioning bulge (402) is matched with the fan-shaped step, a first sealing rubber ring (401) is arranged on the periphery of the first valve (400), and the first sealing rubber ring (401) is an annular sealing structure with a skirt seal; and a second sealing rubber ring (502) is arranged on the periphery of the second valve (500).
8. The cartridge device for nucleic acid isolation and automatic liquid distribution according to any one of claims 1 to 7, wherein: wherein the bottom of the pyrolysis liquid pipe (101) is provided with a solid-liquid separation membrane (1012), the reaction liquid (1011) is arranged above the solid-liquid separation membrane (1012), and the rehydration liquid or the rehydration reagent (1013) is arranged below the solid-liquid separation membrane (1012).
9. The cartridge device for nucleic acid isolation and automatic liquid distribution according to any one of claims 1 to 7, wherein: the sealing film (109) comprises an adhesive layer (1091) and a sealing film layer (1092), the sealing film layer (1092) is adhered to the upper surface of the nucleic acid extraction structure (100) through the adhesive layer (1091), the sealing film layer (1092) is made of a multi-layer composite material, the nucleic acid derivation structure (200) is matched with the detection reagent tube (600), and the detection reagent tube (600) is internally preset with the detection reagent (601).
10. A method for analyzing nucleic acid, which is implemented based on the cartridge device for nucleic acid extraction and automatic liquid separation according to any one of claims 1 to 7, and which mainly comprises the following steps:
s1: the magnetic rod is extended into the magnetic bead tube (102) to adsorb the magnetic beads;
s2: heating to melt the isolating layer in the lysis solution tube (101), fully mixing the nucleic acid extraction protein lytic enzyme and the lysis solution, adding the biological sample into the lysis solution tube (101) containing the nucleic acid extraction protein lytic enzyme and the lysis solution for lysis and releasing nucleic acid;
s3: extending the magnetic bar adsorbed with the magnetic beads into a lysis liquid tube (101) to pump up and down so as to make the magnetic beads adsorb the released nucleic acid;
s4: sequentially extending the magnetic rod adsorbed with the magnetic beads into a first cleaning liquid pipe (103), a second cleaning liquid pipe (104) and a third cleaning liquid pipe (105) for up-and-down pumping rinsing;
s5, the rinsed magnetic rod extends into an elution liquid pipe (106) for elution, and after the elution is finished, the reagent in the elution liquid pipe (106) is transferred to a sample pipe (107);
s6, controlling the second valve (500) to rotate clockwise by the actuating mechanism, and opening the transverse channel (205); the actuator controls the first valve (400) to rotate to block the vertical channel (208); the actuating mechanism lifts the liquid pushing rod (300) upwards through the connecting part until the top surface of the lifting limiting disc is contacted with the bottom surface of the positioning block (2021), so that the reagent transferring process is completed;
s7: the actuator controls the second valve (500) to rotate anticlockwise so as to block the transverse channel (205); the actuator controls the first valve (400) to rotate to open the vertical channel (208); the actuating mechanism pushes the liquid pushing rod (300) downwards through the connecting part until the bottom surface of the pushing limiting disc is contacted with the top surface of the positioning block (2021), and the reagent is discharged into the detection reagent tube (600) from the conical tube (206), so that the quantitative distribution of the reagent is realized.
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| CN111004797A (en) * | 2019-12-06 | 2020-04-14 | 深圳市宝安区沙井人民医院 | Nucleic acid extraction device and nucleic acid extraction method |
| CN111909835A (en) * | 2020-08-25 | 2020-11-10 | 中国科学院合肥物质科学研究院 | Closed micro-fluidic nucleic acid detection card box |
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| CN111004797A (en) * | 2019-12-06 | 2020-04-14 | 深圳市宝安区沙井人民医院 | Nucleic acid extraction device and nucleic acid extraction method |
| CN111909835A (en) * | 2020-08-25 | 2020-11-10 | 中国科学院合肥物质科学研究院 | Closed micro-fluidic nucleic acid detection card box |
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