CN112812959A

CN112812959A - Nucleic acid extraction, amplification and detection integrated device

Info

Publication number: CN112812959A
Application number: CN201911121012.9A
Authority: CN
Inventors: 梅哲; 贾础豪; 张彤; 王继华
Original assignee: Guangzhou Wondfo Biotech Co Ltd
Current assignee: Guangzhou Wondfo Biotech Co Ltd
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2021-05-18

Abstract

The invention belongs to the field of biological medical treatment, and discloses a nucleic acid extraction, amplification and detection integrated device, which comprises a card box, a lysis solution cavity, a cleaning solution cavity, an eluent cavity, a magnetic bead cavity, a binding solution cavity, a waste solution cavity, a freeze-dried bead cavity, a PCR cavity and a sample processing cavity, wherein the lysis solution cavity, the cleaning solution cavity, the eluent cavity, the magnetic bead cavity, the binding solution cavity, the waste solution cavity, the freeze-dried bead cavity, the: the combination liquid cavity can be communicated with the magnetic bead cavity, the sample processing cavity can be selectively communicated with one of the lysis liquid cavity, the cleaning liquid cavity, the eluent cavity, the magnetic bead cavity, the waste liquid cavity and the freeze-drying bead cavity, and the freeze-drying bead cavity can be communicated with the PCR cavity. According to the invention, through the chambers, automatic processing of nucleic acid extraction, amplification and detection can be realized, the detection result is more accurate, only a few manual operation steps are needed, and the operation is simple, safe and convenient.

Description

Nucleic acid extraction, amplification and detection integrated device

Technical Field

The invention relates to the field of biological medical treatment, in particular to a nucleic acid extraction, amplification and detection integrated device.

Background

Nucleic acid detection plays a very important role in many biochemical analysis fields such as clinical medicine, forensic identification, genetic testing, etc., and has been widely applied in the fields of biological medicine, etc. In the prior art, a centrifugal column method or a magnetic bead method is generally used for extracting nucleic acid, and four steps of cracking, combining, cleaning, eluting and the like are generally required, and subsequent detection steps of nucleic acid molecule hybridization, Polymerase Chain Reaction (PCR), a biochip and the like are added, so that the whole full-automatic instrument from a sample to a result is very difficult to realize. Regarding the transfer of the effective components in each step, the manual transfer mode is mostly adopted in the prior art, the operation is complex, time and labor are wasted, the sample is difficult to be transferred fully and efficiently, the result is unstable easily due to manual operation, and the detection realization difficulty is high.

In addition, the mainstream technology of molecular detection is the fluorescence quantitative PCR technology, and the PCR technology has the characteristic of exponential amplification template, so that the existing open consumables easily cause PCR aerosol pollution in the whole operation process, influence the purity of the extract and become an important factor for limiting the further clinical application of the fluorescence quantitative PCR technology.

In order to solve the above problems, some prior arts have automated processing from sample to result by integrating detection equipment, but the prior art has many technical problems such as complicated structure, poor sensitivity, complicated operation and detection equipment, which are difficult to solve.

Disclosure of Invention

The invention aims to provide an integrated device for extracting, amplifying and detecting nucleic acid, which can realize the automatic processing of extracting, amplifying and detecting nucleic acid, has more accurate detection result, only needs a small number of manual operation steps, and is simple, safe and convenient to operate.

In order to achieve the purpose, the invention adopts the following technical scheme:

the utility model provides a nucleic acid draws, amplifies detection integrated device, include the card box and all set up in the schizolysis sap cavity, washing sap cavity, eluant cavity, magnetic bead cavity, combination sap cavity, waste liquid cavity, freeze-drying bead cavity, PCR chamber and the sample processing chamber of card box, wherein:

the combination liquid cavity can be communicated with the magnetic bead cavity, the sample processing cavity can be selectively communicated with one of the lysis liquid cavity, the cleaning liquid cavity, the eluent cavity, the magnetic bead cavity, the waste liquid cavity and the freeze-drying bead cavity, and the freeze-drying bead cavity can be communicated with the PCR cavity.

Preferably, the cartridge further comprises a first rotary valve rotatably disposed on the cartridge, the first rotary valve is provided with a first rotary valve channel, and the first rotary valve is rotated to selectively communicate the lysis solution chamber, the washing solution chamber, the elution solution chamber and the binding solution chamber.

Preferably, the first rotary valve passage is in communication with a first air pump.

Preferably, the kit further comprises a second rotary valve rotatably arranged on the cartridge, the second rotary valve is provided with a second rotary valve channel, one end of the second rotary valve channel is communicated with the sample processing chamber, and the other end of the second rotary valve channel can be alternatively communicated with the lysis solution chamber, the cleaning solution chamber, the elution solution chamber, the magnetic bead chamber and the waste solution chamber.

Preferably, the top of the sample processing chamber is communicated with a second air pump.

Preferably, the magnetic bead cavity is rotatably arranged on the card box and is rotated to a preset position, and the magnetic bead cavity is simultaneously communicated with the combining liquid cavity and the sample processing cavity.

Preferably, the PCR kit further comprises an air cavity, and the air cavity is communicated with the PCR cavity.

Preferably, a blocking structure is arranged between the PCR cavity and the air cavity and used for blocking the communication between the PCR cavity and the air cavity.

Preferably, a layer of waterproof breathable film is arranged on the blocking structure.

Preferably, the PCR chambers are provided in plurality, and the plurality of PCR chambers are simultaneously communicated with the freeze-dried bead chambers.

Preferably, the kit further comprises a third rotary valve rotatably arranged on the cartridge, the third rotary valve is provided with a third rotary valve channel, a fourth rotary valve channel and fifth rotary valve channels with the same number as that of the PCR chambers, and the third rotary valve can rotate to a first preset position or a second preset position;

the third rotary valve rotates to the first preset position, one end of the third rotary valve channel is communicated with the sample processing chamber, the other end of the third rotary valve channel is communicated with the freeze-dried bead chamber, and the fourth rotary valve channel and the fifth rotary valve channel are in an unconnected state;

the third rotary valve rotates to the second preset position, one end of the fourth rotary valve channel is communicated with the sample processing cavity, the other end of the fourth rotary valve channel is communicated with the freeze-drying bead cavity, one end of the fifth rotary valve channel is communicated with the freeze-drying bead cavity, the other end of the fifth rotary valve channel is communicated with the PCR cavity, and the third rotary valve channel is in an unconnected state.

Preferably, a layer of waterproof breathable film is arranged on the third rotary valve.

Preferably, the lysis solution cavity, the cleaning solution cavity, the elution solution cavity and the binding solution cavity are all provided with puncture structures, and the puncture structures are used for puncturing corresponding reagent bags in the lysis solution cavity, the cleaning solution cavity, the elution solution cavity and the binding solution cavity.

Preferably, a rotatable magnetic bead storage chamber is arranged in the magnetic bead cavity, and when the magnetic bead storage chamber rotates to a preset position, the magnetic bead cavity can be communicated with the binding solution cavity and the sample processing cavity.

Preferably, the kit further comprises a pressing module capable of pressing the reagent pack, an ultrasonic module and a magnetic attraction module acting on the sample processing cavity, a thermal cycling module used for digesting the sample in the sample processing cavity, and a detection module used for detecting the liquid in the PCR cavity.

The invention has the beneficial effects that: through each chamber, the automatic processing of nucleic acid extraction, amplification and detection can be realized, only a small number of manual operation steps are needed, and the operation is simple, safe and convenient. In addition, the chambers are arranged on the card box, so that the effective components do not need to be manually transferred, the effective components are prevented from being polluted in the transferring process, and the accuracy of the detection result is improved.

Drawings

FIG. 1 is a schematic perspective view of an integrated nucleic acid extraction, amplification and detection device according to the present invention;

FIG. 2 is a schematic exploded view of the integrated device for nucleic acid isolation, amplification and detection according to the present invention;

FIG. 3 is a schematic diagram of the structure of a reagent pack according to the present invention;

FIG. 4 is a schematic diagram of the construction of an eluate packet according to the present invention;

FIG. 5 is a schematic perspective view of a cartridge according to the present invention;

FIG. 6 is a schematic front view of the cartridge of the present invention;

FIG. 7 is a schematic view of the reverse structure of the cartridge of the present invention;

FIG. 8 is a schematic view showing the communication state of each channel in the cartridge according to the present invention;

FIG. 9 is a schematic view showing a state in which the magnetic bead storage chamber of the present invention is rotated to a predetermined position;

FIG. 10 is a schematic view showing a state where the magnetic bead storage chamber according to the present invention is not rotated to a predetermined position;

FIG. 11 is a schematic diagram of a first rotary valve according to the present invention;

FIG. 12 is a schematic diagram of a second rotary valve according to the present invention;

FIG. 13 is a schematic diagram of a third rotary valve according to the present invention;

FIG. 14 is a schematic view of a third rotary valve of the present invention rotated to a first predetermined position;

FIG. 15 is a schematic view of a third rotary valve of the present invention rotated to a second predetermined position;

FIG. 16 is a schematic diagram of a PCR chamber according to the present invention;

FIG. 17 is a schematic perspective view of a sample processing chamber according to the present invention;

FIG. 18 is a cross-sectional view of a sample processing chamber according to the present invention.

In the figure:

1. a card box; 11. a lysis fluid chamber; 12. a cleaning fluid chamber; 13. an eluate chamber; 14. a magnetic bead cavity; 15. a binding fluid chamber; 16. a waste fluid chamber; 17. a freeze-dried bead chamber; 18. a PCR chamber; 19. an air chamber; 1a, connecting holes; 1b, a blocking structure; 2. a sample processing chamber; 21. a body; 22. a processing chamber; 23. a sample addition port; 24. a communication port; 25. a first seal member; 3. a first rotary valve; 31. a first rotary valve passageway; 32. rotating the valve retainer ring; 4. a second rotary valve; 41. a second rotary valve passageway; 5. a third rotary valve; 51. a third rotary valve passageway; 52. a fourth rotary valve passage; 53. a fifth rotary valve passage; 54. a groove; 6. a magnetic bead storage chamber; 7. a lysis solution bag; 71. a seal member; 8. a cleaning liquid bag; 9. an eluent bag; 10. combining the liquid bag; 20. a first gas passage; 201. a first gas branch passage; 202. a second gas branch channel; 30. a communication channel; 40. a first liquid channel; 401. a first branch channel; 402. a second branch channel; 50. a second gas passage; 501. a third gas branch passage; 502. a fourth gas branch channel; 60. a second liquid passage; 601. a third branch channel; 602. a fourth branch channel; 70. a third gas passage; 701. a fifth gas branch passage; 702. a sixth gas branch passage; 80. a third liquid channel; 801. a fifth branch channel; 802. a sixth branch channel; 90. a fourth gas channel; 901. a seventh gas branch passage; 902. an eighth gas branch passage; 100. a fourth liquid channel; 1001. a seventh branch channel; 1002. an eighth branch channel; 110. a waste liquid channel; 1101. a first waste liquid channel; 1102. a second waste liquid channel; 120. a fifth liquid channel; 1201. a ninth branch channel; 1202. a tenth branch channel; 130. a fifth gas channel; 1301. a ninth gas branch passage; 1302. a tenth gas branch channel; 140. a sixth liquid passage; 150. and (4) end covers.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. It should be further noted that, for the convenience of description, only some of the structures related to the present invention are shown in the drawings, not all of the structures.

In the description of the present invention, unless expressly stated or limited otherwise, the terms "connected," "connected," and "fixed" are to be construed broadly, e.g., as meaning permanently connected, removably connected, or integral to one another; can be mechanically or electrically connected; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

In the present invention, unless otherwise expressly stated or limited, "above" or "below" a first feature means that the first and second features are in direct contact, or that the first and second features are not in direct contact but are in contact with each other via another feature therebetween. Also, the first feature being "on," "above" and "over" the second feature includes the first feature being directly on and obliquely above the second feature, or merely indicating that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature includes the first feature being directly under and obliquely below the second feature, or simply meaning that the first feature is at a lesser elevation than the second feature.

In the description of the present embodiment, the terms "upper", "lower", "right", etc. are used in an orientation or positional relationship based on that shown in the drawings only for convenience of description and simplicity of operation, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used only for descriptive purposes and are not intended to have a special meaning.

The invention provides a nucleic acid extraction, amplification and detection integrated device, which comprises a card box 1, a lysis solution cavity 11, a cleaning solution cavity 12, an eluent cavity 13, a magnetic bead cavity 14, a binding solution cavity 15, a waste solution cavity 16, a freeze-drying bead cavity 17, a PCR cavity 18 and a sample processing cavity 2, wherein the lysis solution cavity 11, the cleaning solution cavity 12, the eluent cavity 13, the elution cavity 9, the magnetic bead cavity 14, the binding solution cavity 15 and the waste solution cavity 16 are all arranged in the card box 1, the lysis solution cavity 11 can be used for placing a lysis solution bag 7, the cleaning solution cavity 12 is used for placing a cleaning solution bag 8, the eluent cavity 13 is used for placing an eluent bag 9, the magnetic bead cavity 14 is used for placing a magnetic bead storage chamber 6, the binding solution cavity 15 is used for placing a binding solution bag 10, and the waste. The sample processing chamber 2 is used for placing a sample and performing operations such as cracking, combining, cleaning, eluting and the like. The freeze-dried bead chamber 17 is used for placing freeze-dried beads, and the PCR chamber 18 is used for placing reaction liquid mixed with the freeze-dried beads.

In this embodiment, the bottoms of the lysis solution cavity 11, the cleaning solution cavity 12, the elution solution cavity 13, and the binding solution cavity 15 are provided with a puncturing structure (not shown in the drawings), and when the corresponding reagent bags (i.e., the respective lysis solution bag 7, the cleaning solution bag 8, the elution solution bag 9, and the binding solution bag 10, which are collectively referred to as reagent bags in this embodiment) are placed in the respective reagent cavities (i.e., the respective lysis solution cavity 11, the cleaning solution cavity 12, the elution solution cavity 13, and the binding solution cavity 15, which are collectively referred to as reagent cavities), the reagent bags (i.e., the respective lysis solution bag 7, the cleaning solution bag 8, the elution solution bag 9, and the binding solution bag 10, which are collectively referred to as reagent bags in this embodiment) may be pressed, and at this time, the puncturing structure may puncture the reagent bags, so that the reagents in the reagent bags flow into. If the lysate is needed, the lysate bag 7 in the lysate cavity 11 is pressed, and the puncture structure of the lysate cavity 11 punctures the lysate bag 7, so that the lysate flows into the lysate cavity 11.

As shown in fig. 3, the lysis solution bag 7, the cleaning solution bag 8, and the binding solution bag 10 have the same structure, and are all cylindrical structures with upper ends closed and lower ends provided with the packaging films, and a sealing member 71 is disposed at one end of the cylindrical structure provided with the packaging films, and the cylindrical structures can be hermetically mounted in the reagent chambers through the sealing member 71, so that when the reagents in the lysis solution bag 7, the cleaning solution bag 8, and the binding solution bag 10 flow into the corresponding reagent chambers, the reagents are in a sealed environment, and the occurrence of pollution caused by contact with the outside is avoided. The sealing films of the lysis buffer pack 7, the washing buffer pack 8, and the binding buffer pack 10 can be punctured by the puncturing mechanism, in addition to sealing the reagent.

As shown in fig. 4, the eluent bag 9 is a cylindrical structure with an upper end closed and a lower end provided with an encapsulation film, and the two ends of the cylindrical structure are both provided with a sealing member 71, the eluent bag 9 can be placed in the eluent cavity 13 in a sealing manner through the sealing members 71, and when the encapsulation film of the eluent bag 9 is punctured by the puncturing structure, eluent flows into the eluent cavity 13 and is in a closed state, so that the eluent cannot contact with the outside, and pollution is avoided.

As shown in fig. 5 and fig. 6, in this embodiment, the lysis solution chamber 11, the cleaning solution chamber 12, the elution solution chamber 13, the magnetic bead chamber 14, the binding solution chamber 15, the waste solution chamber 16, the freeze-drying bead chamber 17, and the PCR chamber 18 are all disposed on the card box 1, and the lysis solution chamber 11, the cleaning solution chamber 12, the elution solution chamber 13, the magnetic bead chamber 14, the binding solution chamber 15, and the waste solution chamber 16 are located at one end of the card box 1, the PCR chamber 18 is disposed at the other end of the card box 1, a connecting hole 1a is disposed in the middle of the card box 1 for connecting and installing the sample processing chamber 2, and the freeze-drying bead chamber 17 is disposed between the sample processing chamber 2 and the PCR chamber 18.

Further, in this embodiment, the binding liquid cavity 15 can be communicated with the magnetic bead cavity 14, as shown in fig. 6 to 8, the binding liquid cavity 15 is communicated with a first gas channel 20 and a communicating channel 30, wherein the first gas channel 20 is used for gas to enter, the communicating channel 30 is used for communicating the binding liquid cavity 15 with the magnetic bead cavity 14, and the binding liquid (the binding liquid packet 10 flows into the binding liquid cavity 15 after being punctured) in the binding liquid cavity 15 can be conveyed to the magnetic bead cavity 14 by the gas entering the first gas channel 20, so that the binding liquid and the magnetic beads in the magnetic bead cavity 14 are uniformly mixed. Optionally, the first gas channel 20 includes a first gas branch channel 201 opened in one side surface (generally referred to as the first side surface in this embodiment) of the cartridge 1 and a second gas branch channel 202 opened in the other side surface (generally referred to as the second side surface in this embodiment) of the cartridge 1, two ends of the first gas branch channel 201 are respectively communicated with the second gas branch channel 202 and the bonding liquid cavity 15, and the second gas branch channel 202 is used for gas to enter.

The magnetic bead chamber 14 can be communicated with the sample processing chamber 2 through the first liquid channel 40, so that the binding solution carrying the magnetic beads is uniformly mixed and delivered to the sample processing chamber 2. In this embodiment, the first liquid channel 40 may be a channel directly opened in one side of the cartridge 1, or as shown in fig. 6 to 8, the first liquid channel 40 includes a first branch channel 401 opened in a first side of the cartridge 1 and a second branch channel 402 opened in a second side of the cartridge 1, the first branch channel 401 is connected to the magnetic bead chamber 14, and two ends of the second branch channel 402 are respectively connected to the first branch channel 401 and the sample processing chamber 2.

In this embodiment, the magnetic bead storage chamber 6 is rotatably installed in the magnetic bead chamber 14, and when the magnetic bead storage chamber 6 rotates to a preset position, the magnetic bead storage chamber 6 enables the magnetic bead chamber 14 to communicate with the binding solution chamber 15 and the sample processing chamber 2, specifically, the communication channel 30 and the first branch channel 401, so as to communicate with the binding solution chamber 15 and the sample processing chamber 2 (shown in fig. 9). When the magnetic bead storage chamber 6 is not rotated to the predetermined position, the magnetic bead chamber 14 is sealed by the magnetic bead storage chamber 6, and the magnetic bead chamber 14 cannot communicate with the binding solution chamber 15 and the sample processing chamber 2, that is, the magnetic bead chamber 14 is not communicated with the communication channel 30 and the first branch channel 401 (shown in fig. 10).

In this embodiment, the lysis solution chamber 11 is connected to a second gas channel 50 and a second liquid channel 60, wherein the second gas channel 50 is used for gas to enter, and the second liquid channel 60 is used for connecting the lysis solution chamber 11 to the sample processing chamber 2. Optionally, the second gas channel 50 includes a third gas branch channel 501 opened in the first side of the cartridge 1 and a fourth gas branch channel 502 opened in the second side of the cartridge 1, two ends of the third gas branch channel 501 are respectively communicated with the fourth gas branch channel 502 and the lysis solution cavity 11, and the fourth gas branch channel 502 is used for gas to enter.

Alternatively, the second liquid channel 60 may be a channel directly opened in one side surface of the cartridge 1, or as shown in fig. 6-8, the second liquid channel 60 includes a third branch channel 601 opened in the first side surface of the cartridge 1 and a fourth branch channel 602 opened in the second side surface of the cartridge 1, the third branch channel 601 is connected to the lysis solution chamber 11, and two ends of the fourth branch channel 602 are respectively connected to the third branch channel 601 and the sample processing chamber 2. The lysate in the lysate chamber 11 (the lysate bag 7 is punctured and flows into the lysate chamber 11) can enter the sample processing chamber 2 through the third branch channel 601 and the fourth branch channel 602 under the action of the gas pressure in the second gas channel 50.

It is understood that the cleaning solution chamber 12 is connected to a third gas channel 70 and a third liquid channel 80, wherein the third gas channel 70 is used for gas to enter, and the third liquid channel 80 is used for connecting the cleaning solution chamber 12 to the sample processing chamber 2. Optionally, the third gas channel 70 includes a fifth gas branch channel 701 opened in the first side of the cartridge 1 and a sixth gas branch channel 702 opened in the second side of the cartridge 1, two ends of the fifth gas branch channel 701 are respectively communicated with the sixth gas branch channel 702 and the cleaning liquid chamber 12, and the sixth gas branch channel 702 is used for gas to enter.

Alternatively, the third liquid channel 80 may be a channel directly formed in one side surface of the cartridge 1, or as shown in fig. 6 to 8, the third liquid channel 80 includes a fifth branch channel 801 formed in the first side surface of the cartridge 1 and a sixth branch channel 802 formed in the second side surface of the cartridge 1, the fifth branch channel 801 is communicated with the wash liquid chamber 12, and both ends of the sixth branch channel 802 are respectively communicated with the fifth branch channel 801 and the sample processing chamber 2. The cleaning liquid in the cleaning liquid chamber 12 (which flows into the cleaning liquid chamber 12 after the cleaning liquid pack 8 is punctured) can enter the sample processing chamber 2 through the fifth branch channel 801 and the sixth branch channel 802 under the action of the gas pressure in the third gas channel 70. In this embodiment, the number of the cleaning solution chambers 12 is preferably two, and more may be selected according to the need of cleaning.

The eluent chamber 13 is communicated with a fourth gas channel 90 and a fourth liquid channel 100, wherein the fourth gas channel 90 is used for gas to enter, the fourth liquid channel 100 is used for communicating the eluent chamber 13 with the sample processing chamber 2, optionally, the fourth gas channel 90 comprises a seventh gas branch channel 901 arranged in the first side surface of the cartridge 1 and an eighth gas branch channel 902 arranged in the second side surface of the cartridge 1, two ends of the seventh gas branch channel 901 are respectively communicated with the eighth gas branch channel 902 and the eluent chamber 13, and the eighth gas branch channel 902 is used for gas to enter.

Alternatively, the fourth liquid channel 100 may be a channel directly opened in one side surface of the cartridge 1, or as shown in fig. 6 to 8, the fourth liquid channel 100 includes a seventh branch channel 1001 opened in the first side surface of the cartridge 1 and an eighth branch channel 1002 opened in the second side surface of the cartridge 1, the seventh branch channel 1001 is communicated with the eluent chamber 13, and both ends of the eighth branch channel 1002 are respectively communicated with the seventh branch channel 1001 and the sample processing chamber 2. The eluent in the eluent chamber 13 (the eluent packet 9 flows into the eluent chamber 13 after being punctured) can enter the sample processing chamber 2 through the seventh branch channel 1001 and the eighth branch channel 1002 under the action of the gas pressure in the fourth gas channel 90.

The waste liquid chamber 16 is communicated with a waste liquid channel 110, and is communicated with the sample processing chamber 2 through the waste liquid channel 110, specifically, waste liquid in the sample processing chamber 2 can flow into the waste liquid chamber 16 through the waste liquid channel 110, so that the waste liquid can be cleaned conveniently, and subsequent processes can be carried out. Alternatively, as shown in fig. 6 to 8, the waste channel 110 comprises a first waste channel 1101 opened in the first side of the cartridge 1, and a second waste channel 1102 opened in the second side of the cartridge 1, wherein the first waste channel 1101 communicates with the waste chamber 16, and two ends of the second waste channel 1102 respectively communicate with the first waste channel 1101 and the sample processing chamber 2.

In this embodiment, further, a first rotary valve 3 may be installed at a first side of the cartridge 1, the first rotary valve 3 is installed on the cartridge 1 by a rotary valve snap ring 32 and the first rotary valve 3 can be rotated with respect to the cartridge 1. As shown in fig. 11, the first rotary valve 3 is provided with a first rotary valve port 31, and the first rotary valve port 31 is capable of communicating with the first gas line 20, the second gas line 50, the third gas line 70, or the fourth gas line 90 in accordance with the rotation of the first rotary valve 3. It is understood that the first rotary valve channel 31 of the present embodiment is connected to a first air pump (not shown), through which air can be pumped into the first rotary valve channel 31, and then the air can enter the first air channel 20, the second air channel 50, the third air channel 70 or the fourth air channel 90 which are communicated with the first rotary valve channel 31, so as to deliver the corresponding reagent into the sample processing chamber 2.

Further, the present embodiment may also be provided with a second rotary valve 4 at the first side of the cartridge 1, the second rotary valve 4 being mounted to the cartridge 1 together with the first rotary valve 3 by means of a rotary valve retaining ring 32 and the second rotary valve 4 being rotatable relative to the cartridge 1. Preferably, the second rotary valve 4 of the present embodiment is installed below the sample processing chamber 2. As shown in fig. 12, the second rotary valve 4 is provided with a second rotary valve channel 41, one end of the second rotary valve channel 41 is connected to the bottom of the sample processing chamber 2 (specifically, the bottom of the sample processing chamber 2 is opened, and the hole is connected to the second rotary valve channel 41), and the other end is connected to the second branch channel 402, the fourth branch channel 602, the sixth branch channel 802, the eighth branch channel 1002, and the second waste liquid channel 1102 along with the rotation of the second rotary valve 4, thereby receiving the reagent and discharging the waste liquid.

In this embodiment, preferably, the surfaces of the first rotary valve 3 and the second rotary valve 4, which are attached to the first side surface of the cartridge 1, are made of soft rubber, so as to ensure good sealing between the first rotary valve 3, the second rotary valve 4 and the first side surface.

Optionally, the freeze-dried bead chamber 17 of the present embodiment is connected to a fifth liquid channel 120, a fifth gas channel 130, and a plurality of sixth liquid channels 140, wherein the fifth liquid channel 120 and the fifth gas channel 130 can connect the sample processing chamber 2 and the freeze-dried bead chamber 17, and the sixth liquid channels 140 are used for connecting the freeze-dried bead chamber 17 to the PCR chamber 18. When the fifth liquid channel 120 connects the sample processing chamber 2 to the bead freeze-drying chamber 17, the fifth gas channel 130 cannot connect the sample processing chamber 2 to the bead freeze-drying chamber 17, and the bead freeze-drying chamber 17 cannot connect to the PCR chamber 18. Conversely, when the fifth gas channel 130 communicates the sample processing chamber 2 with the freeze-dried bead chamber 17 and the freeze-dried bead chamber 17 communicates with the PCR chamber 18, the fifth liquid channel 120 cannot communicate the sample processing chamber 2 with the freeze-dried bead chamber 17.

As shown in fig. 8, the fifth liquid channel 120 is used to connect the sample processing chamber 2 to the freeze-dried bead chamber 17, alternatively, the fifth liquid channel 120 may be a channel directly opened in one side surface of the cartridge 1, and both ends of the channel are respectively connected to the second rotary valve channel 41 and the freeze-dried bead chamber 17, or as shown in fig. 6-8, the fifth liquid channel 120 includes a ninth branch channel 1201 opened in the first side surface of the cartridge 1 and a tenth branch channel 1202 opened in the second side surface of the cartridge 1, the ninth branch channel 1201 is connected to the freeze-dried bead chamber 17, and both ends of the tenth branch channel 1202 are respectively connected to the ninth branch channel 1201 and the second rotary valve channel 41, so as to connect the freeze-dried bead chamber 17 to the sample processing chamber 2.

The liquid in the sample processing chamber 2 (specifically, the liquid after lysis, washing and elution) can enter the freeze-dried bead chamber 17 through the second rotary valve channel 41, the tenth branch channel 1202 and the ninth branch channel 1201 in sequence under the action of the gas pressure delivered by the second gas pump.

The fifth gas channel 130 may be a channel directly formed in one side surface of the cartridge 1, and both ends of the channel are respectively connected to the second rotary valve channel 41 and the freeze-dried bead chamber 17, or as shown in fig. 6-8, the fifth gas channel 130 includes a ninth gas branch channel 1301 formed in the first side surface of the cartridge 1 and a tenth gas branch channel 1302 formed in the second side surface of the cartridge 1, wherein the ninth gas branch channel 1301 is connected to the freeze-dried bead chamber 17, and both ends of the tenth gas branch channel 1302 are respectively connected to the ninth gas branch channel 1301 and the second rotary valve channel 41, so as to achieve the connection between the freeze-dried bead chamber 17 and the sample processing chamber 2. A gas inlet 21 is formed at the top of the sample processing chamber 2, and the gas inlet 21 can be connected to a second gas pump, so that gas can be delivered into the fifth gas channel 130 by the second gas pump. It will be appreciated that in this embodiment, it is preferred that an end cap 150 (shown in FIG. 1) be snapped directly onto the top of the sample processing chamber 2, the end cap 150 being capable of closing the sample processing chamber 2, if the use of a second air pump is not required.

The sixth liquid channel 140 is formed in the first side of the cartridge 1, and both ends thereof are respectively connected to the freeze-dried bead chamber 17 and the PCR chamber 18. Through the fifth air channel 130 and the sixth liquid channel 140, the liquid in the freeze-dried bead chamber 17 can be transferred to the PCR chamber 18 through the sixth liquid channel 140 by the second air pump under the action of the air pressure in the fifth air channel 130.

In this embodiment, it is also preferable that a third rotary valve 5 is further installed at the first side of the cartridge 1, the third rotary valve 5 being installed on the cartridge 1 and the third rotary valve 5 being rotatable with respect to the cartridge 1. Preferably, the third rotary valve 5 of the present embodiment is installed below the freeze-dried bead chamber 17. As shown in FIG. 13, a third rotary valve pathway 51, a fourth rotary valve pathway 52 and the same number of fifth rotary valve pathways 53 as the number of PCR chambers 18 are provided on the third rotary valve 5, and the third rotary valve 5 can be rotated to the first predetermined position or the second predetermined position.

When the third rotary valve 5 is rotated to the first predetermined position, as shown in fig. 14, one end of the third rotary valve channel 51 is connected to the ninth branch channel 1201, and the other end is connected to the bead chamber 17, so as to connect the bead chamber 17 to the sample processing chamber 2. And the fourth rotary valve passage 52 and the fifth rotary valve passage 53 are both in an unconnected state.

When the third rotary valve 5 is rotated to the second predetermined position, as shown in fig. 15, one end of the fourth rotary valve channel 52 is connected to the ninth gas branch channel 1301, and the other end is connected to the bead freezing chamber 17, so as to connect the bead freezing chamber 17 to the sample processing chamber 2. Meanwhile, one end of the fifth rotary valve channel 53 is communicated with the sixth liquid channel 140, and the other end is communicated with the freeze-dried bead chamber 17, so as to realize the communication between the freeze-dried bead chamber 17 and the PCR chamber 18. And the third rotary valve passage 51 is now in an unconnected state, i.e. the third rotary valve passage 51 is not connected to the ninth branch passage 1201.

In this embodiment, referring to fig. 13, a groove 54 for communicating the freeze-dried bead chamber 17 may be disposed on the third rotary valve 5, and the third rotary valve channel 51, the fourth rotary valve channel 52 and the fifth rotary valve channel 53 are all communicated with the groove 54, so as to realize the communication between the freeze-dried bead chamber 17 and the sample processing chamber 2 and the PCR chamber 18.

It is understood that in this embodiment, the number of the PCR chambers 18 may be plural, and accordingly, the number of the fifth rotary valve passages 53 is plural, and each fifth rotary valve passage 53 communicates with one PCR chamber 18.

As shown in fig. 6 and 16, the PCR chamber 18 is further connected to an air chamber 19, and the air chamber 19 can collect air discharged from the PCR chamber 18 when the liquid in the freeze-dried bead chamber 17 is transferred to the PCR chamber 18 through the sixth liquid channel 140. A blocking structure 1b is also provided between the PCR chamber 18 and the air chamber 19, which blocking structure 1b can be connected to a top pressure valve of an external instrument, which can block the flow of liquid by means of the top pressure valve. It will be appreciated that a one-way valve may also be provided directly at the blocking structure 1b to block the flow of liquid.

In this embodiment, it is preferable that a waterproof and air-permeable membrane is disposed on the blocking structure 1b, which can remove the excess air bubbles in the liquid, so that the excess air can permeate into the air cavity 19, and the liquid can be retained in the PCR cavity 18 by the excess air bubbles.

In this embodiment, the structure of the sample processing chamber 2 is as shown in fig. 17 and 18, the sample processing chamber 2 includes a body 21, a processing chamber 22 disposed in the body 21, a sample inlet 23 disposed at the top of the body 21 and communicated with the processing chamber 22, and a communication port 24 disposed at the bottom of the body 21 and communicated with the second rotary valve channel 41, and the processing chamber 22 is used for performing steps of lysing a sample (virus and bacteria), mixing the sample with magnetic beads, washing, eluting, and the like. A first seal 25 is provided at the communication port 24 to achieve a tight connection and sealing action between the sample processing chamber 2 and the cartridge 1.

In this embodiment, the integrated device for nucleic acid extraction, amplification and detection further includes a pressing module, an ultrasonic module, a magnetic attraction module, a thermal cycle module and a detection module, wherein the pressing module is used for pressing the reagent pack, so that the reagent pack can be punctured by the puncturing structure. The ultrasonic module can act on the sample processing cavity 2, and can realize the steps of sample cracking, sample and magnetic bead mixing, cleaning, elution and the like in the sample processing cavity 2. The magnetic attraction module also acts on the sample processing cavity 2, and can adsorb magnetic beads in the sample processing cavity 2. The thermal cycling module is used for digesting the sample in the sample processing cavity 2 and controlling the temperature of the liquid in the PCR cavity 18. The detection module is used for detecting the liquid in the PCR cavity 18 to obtain a detection result.

The integrated device for nucleic acid extraction, amplification and detection of the present embodiment is used as follows:

1) loading: i.e. the sample to be tested, which may be a patient sample, is introduced into the sample processing chamber 2.

2) The sample in the sample processing cavity 2 is uniformly mixed and digested through the ultrasonic module and the thermal cycle module.

3) The lysis solution bag 7 is pushed downwards by the pressing module, the packaging film at the bottom of the lysis solution bag 7 is pierced by the puncture structure of the lysis solution cavity 11, and the lysis solution flows out of the lysis solution bag 7 and is placed in the lysis solution cavity 11. In the case where the first rotary valve 3 and the second rotary valve 4 are not rotated to a specific position and the first air pump is not activated, the lysate stays in the lysate chamber 11.

4) By rotating the first rotary valve 3 to the a1 position (shown in fig. 8) and rotating the second rotary valve 4 to the B1 position, the lysate is pushed into the sample processing chamber 2 through the second gas channel 50 and the second liquid channel 60 by the first gas pump.

5) The sample is digested or lysed by the action of a thermal cycling module or an ultrasound module outside the sample processing chamber 2.

6) By pushing the bonding pad 10 downward by the pressing module, the piercing structure of the bonding pad 15 pierces the sealing film at the bottom of the bonding pad 10, and the bonding solution flows out of the bonding pad 10. The first rotary valve 3 is rotated to the a2 position, the second rotary valve 4 is rotated to the B2 position, and the magnetic bead storage chamber 6 is rotated to communicate the magnetic bead chamber 14 with the first gas channel 20 and the first branch channel 401 (see fig. 8 and 9). And starting a first air pump, pushing the binding solution into the magnetic bead cavity 14, uniformly mixing to carry magnetic beads, extracting biomacromolecule extracts such as nucleic acid and the like by adopting magnetic particles including the magnetic beads, and finally reaching the sample processing cavity 2.

7) Under the ultrasonic action of the ultrasonic module outside the sample processing cavity 2, the magnetic beads in the sample processing cavity 2 are fully mixed with the liquid.

8) Under the magnetic attraction effect of the magnetic attraction module outside the sample processing cavity 2, magnetic beads (or magnetic particles) are adsorbed on the inner wall of the sample processing cavity 2. The second rotary valve 4 is rotated to the position B3, and the reaction liquid in the sample processing chamber 2 is pushed into the waste liquid chamber 16 through the waste liquid channel 110 by the second air pump.

9) The cleaning liquid bag 8 is pushed downwards by the pressing module, the puncture structure of the cleaning liquid cavity 12 pierces the packaging film at the bottom of the cleaning liquid bag 8, and the cleaning liquid flows out of the cleaning liquid bag 8. The first rotary valve 3 is rotated to the a3 position and the second rotary valve 4 is rotated to the B4 position. The cleaning solution is pushed into the sample processing chamber 2 through the third gas passage 70 and the third liquid passage 80 by the first gas pump.

10) The magnetic attraction of the magnetic attraction module is controlled to disappear, and the magnetic beads (or magnetic particles) adsorbed with biomacromolecule extracts such as nucleic acid and the like are fully washed under the ultrasonic action of the ultrasonic module. Then, under the action of the magnetic attraction module, the magnetic beads (or magnetic particles) are adsorbed on the inner wall of the sample processing cavity 2. Subsequently, the second rotary valve 4 is rotated to the position B3, and the reaction liquid in the sample processing chamber 2 is pushed into the waste liquid chamber 16 through the waste liquid channel 110 by the second air pump.

11) By pushing another cleaning solution bag 8 downwards (two cleaning solution bags 8 are provided in the embodiment) through the pressing module, the puncture structure of the cleaning solution cavity 12 pierces the packaging film at the bottom of the cleaning solution bag 8, and the cleaning solution flows out of the cleaning solution bag 8. The first rotary valve 3 is rotated to the a4 position and the second rotary valve 4 is rotated to the B5 position. The cleaning solution is pushed into the sample processing chamber 2 through the third gas passage 70 and the third liquid passage 80 by the first gas pump.

12) The magnetic attraction of the magnetic attraction module is controlled to disappear, and the magnetic beads (magnetic particles) adsorbed with biomacromolecule extracts such as nucleic acid are fully washed under the ultrasonic action of the ultrasonic module. Then, under the action of the magnetic attraction module, the magnetic beads (or magnetic particles) are adsorbed on the inner wall of the sample processing cavity 2. Subsequently, the second rotary valve 4 is rotated to the position B3, and the reaction liquid in the sample processing chamber 2 is pushed into the waste liquid chamber 16 through the waste liquid channel 110 by the second air pump.

13) By pushing the eluent packet 9 downwards by the pressing module, the puncturing structure of the eluent chamber 13 pierces the encapsulation film at the bottom of the eluent packet 9, and the liquid flows out of the eluent packet 9. The first rotary valve 3 is rotated to the a5 position and the second rotary valve 4 is rotated to the B6 position. The eluent is pushed into the sample processing chamber 2 through the fourth gas channel 90 and the fourth liquid channel 100 by means of the first gas pump.

14) The magnetic attraction of the magnetic attraction module is controlled to disappear, and the nucleic acid on the magnetic beads is sufficiently eluted under the ultrasonic action of the ultrasonic module. Under the magnetic attraction effect of magnetic attraction module, magnetic bead (magnetic particle) adsorbs on the inner wall of sample processing chamber 2. The second rotary valve 4 is then rotated to the B7 position and the third rotary valve 5 is rotated to the C1 position. The reaction liquid in the sample processing chamber 2 is pushed into the freeze-dried bead chamber 17 through the fifth liquid passage 120 by the second air pump.

15) The gas is continuously delivered to the freeze-dried bead cavity 17 by the second gas pump, so that the reaction liquid in the freeze-dried bead cavity 17 is fully mixed with the freeze-dried beads. This embodiment is preferably covered with a waterproof gas permeable membrane above the third rotary valve 5, from which excess gas can be discharged. Through this waterproof ventilated membrane, can guarantee atmospheric pressure balance on the one hand, consequently liquid can smoothly push to freeze-drying pearl intracavity 17 in, on the other hand it can get rid of unnecessary bubble in the liquid. But also helps to remove excess air bubbles before the next step of liquid enters the PCR chamber 18.

16) The second rotary valve 4 is rotated to the position B8, the third rotary valve 5 is rotated to the position C2, and the reaction liquid in the freeze-dried bead chamber 17 is pushed into the four PCR chambers 18 through the sixth liquid channels 140 (in the present embodiment, four sixth liquid channels 140 are provided, i.e., the channels D1, D2, D3, and D4) by the second air pump via the fifth air channel 130.

17) When the reaction liquid is filled up to the 4 PCR chambers 18, the top pressure valve of the external instrument (or the one-way valve on the cartridge 1) acts on the blocking structure 1b, so that the 4 PCR chambers 18 are isolated from the air chamber 19 and the outside. At this time, the PCR chamber 18 is subjected to temperature rise and decrease cycles, and the amplification result is detected by the detection module.

The integrated device for extracting, amplifying and detecting nucleic acid can realize the automatic treatment of nucleic acid extraction, amplification and detection, only needs a few manual operation steps, and is simple to operate, safe and convenient. The detection time is short, and is less than 1 hour. In addition, the chambers are all arranged on the card box 1 and isolated from the outside, so that the cross contamination among samples is avoided, and the probability of the occurrence of false positive structures is reduced. And the effective components do not need to be manually transferred, so that the effective components are prevented from being polluted in the transfer process, and the accuracy of the detection result is improved. In addition, the card box 1 is arranged separately from each reagent bag, so that the problem of reagent storage is effectively solved.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Numerous obvious variations, adaptations and substitutions will occur to those skilled in the art without departing from the scope of the invention. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The utility model provides a nucleic acid extraction, amplification and detection integrated device, characterized in that, include card box (1) and all install in lysis liquid chamber (11), washing liquid chamber (12), eluant chamber (13), magnetic bead chamber (14), combination sap chamber (15), waste liquid chamber (16), freeze-drying bead chamber (17), PCR chamber (18) and sample processing chamber (2) of card box (1), wherein:

the binding liquid cavity (15) can be communicated with the magnetic bead cavity (14), the sample processing cavity (2) can be selectively communicated with one of the lysis liquid cavity (11), the cleaning liquid cavity (12), the eluent cavity (13), the magnetic bead cavity (14), the waste liquid cavity (16) and the freeze-drying bead cavity (17), and the freeze-drying bead cavity (17) can be communicated with the PCR cavity (18).

2. The integrated nucleic acid extraction, amplification and detection device according to claim 1, further comprising a first rotary valve (3) rotatably disposed on the cartridge (1), wherein the first rotary valve (3) is provided with a first rotary valve channel (31), and the first rotary valve (3) is rotated, and the first rotary valve channel (31) can alternatively communicate with the lysis solution chamber (11), the washing solution chamber (12), the elution solution chamber (13) and the binding solution chamber (15).

3. The integrated nucleic acid extraction, amplification and detection device of claim 2, wherein the first rotary valve channel (31) is connected to a first air pump.

4. The integrated nucleic acid extraction, amplification and detection device according to claim 1, further comprising a second rotary valve (4) rotatably disposed on the cartridge (1), wherein the second rotary valve (4) is provided with a second rotary valve channel (41), one end of the second rotary valve channel (41) is connected to the sample processing chamber (2), and the other end of the second rotary valve channel can alternatively connect to the lysis solution chamber (11), the washing solution chamber (12), the elution solution chamber (13), the magnetic bead chamber (14) and the waste solution chamber (16).

5. The integrated nucleic acid extraction, amplification and detection device according to claim 4, wherein a second air pump is connected to the top of the sample processing chamber (2).

6. The integrated nucleic acid extraction, amplification and detection device according to any one of claims 1 to 5, wherein the magnetic bead chamber (14) is rotatably disposed on the cartridge (1), and the magnetic bead chamber (14) is rotated to a predetermined position, and the magnetic bead chamber (14) is simultaneously communicated with the binding solution chamber (15) and the sample processing chamber (2).

7. The integrated nucleic acid extraction, amplification and detection device of any one of claims 1 to 5, further comprising an air chamber (19), wherein the air chamber (19) is in communication with the PCR chamber (18).

8. The integrated nucleic acid extraction, amplification and detection device according to claim 7, wherein a blocking structure (1b) is disposed between the PCR cavity (18) and the air cavity (19), and the blocking structure (1b) is used for blocking the communication between the PCR cavity (18) and the air cavity (19).

9. The integrated nucleic acid extraction, amplification and detection device according to claim 8, wherein the blocking structure (1b) is provided with a waterproof and breathable membrane.

10. The integrated nucleic acid extraction, amplification and detection device according to claim 6, wherein a plurality of PCR chambers (18) are provided, and the plurality of PCR chambers (18) are simultaneously communicated with the freeze-dried bead chambers (17).

11. The integrated nucleic acid extraction, amplification and detection device according to claim 10, further comprising a third rotary valve (5) rotatably disposed on the cartridge (1), wherein the third rotary valve (5) has a third rotary valve channel (51), a fourth rotary valve channel (52) and the same number of fifth rotary valve channels (53) as the number of the PCR chambers (18), and the third rotary valve (5) can be rotated to a first predetermined position or a second predetermined position;

the third rotary valve (5) rotates to the first preset position, one end of the third rotary valve channel (51) is communicated with the sample processing chamber (2), the other end is communicated with the freeze-dried bead chamber (17), and the fourth rotary valve channel (52) and the fifth rotary valve channel (53) are in an unconnected state;

the third rotary valve (5) rotates to the second preset position, one end of the fourth rotary valve channel (52) is communicated with the sample processing chamber (2), the other end of the fourth rotary valve channel is communicated with the freeze-dried bead chamber (17), one end of the fifth rotary valve channel (53) is communicated with the freeze-dried bead chamber (17), the other end of the fifth rotary valve channel is communicated with the PCR chamber (18), and the third rotary valve channel (51) is in an unconnected state.

12. The integrated nucleic acid extraction, amplification and detection device of claim 11, wherein the third rotary valve (5) is provided with a waterproof and breathable membrane.

13. The integrated nucleic acid extraction, amplification and detection device according to claim 1, wherein the lysis solution chamber (11), the washing solution chamber (12), the elution solution chamber (13) and the binding solution chamber (15) are provided with puncture structures for puncturing reagent packs in the corresponding lysis solution chamber (11), the washing solution chamber (12), the elution solution chamber (13) and the binding solution chamber (15).

14. The integrated nucleic acid extraction, amplification and detection device according to claim 1, wherein a rotatable magnetic bead storage chamber (6) is disposed in the magnetic bead chamber (14), and when the magnetic bead storage chamber (6) rotates to a preset position, the magnetic bead chamber (14) is capable of communicating the binding solution chamber (15) and the sample processing chamber (2).

15. The integrated device for nucleic acid extraction, amplification and detection according to claim 1, further comprising a pressing module capable of pressing a reagent pack, an ultrasonic module and a magnetic attraction module acting on the sample processing chamber (2), a thermal cycling module for digesting the sample in the sample processing chamber (2), and a detection module for detecting the liquid in the PCR chamber (18).