Nucleic acid extraction device and method

Abstract

The present invention belongs to the field of biological testing, specifically relating to a nucleic acid extraction device and a method for extracting and amplifying target nucleic acids using the device. The device comprises a nucleic acid extraction element, a waste liquid storage chamber, and a reaction chamber. The reaction chamber selectively communicates with the nucleic acid extraction element and the waste liquid storage chamber, and fluid exchange occurs through pressure fluctuations between the waste liquid storage chamber and the reaction chamber. The present invention utilizes an integrated control system that discharges fluid from the reaction chamber into the waste liquid storage chamber under negative pressure, resulting in convenient operation and high efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and in particular relates to a nucleic acid extraction device and a method for extracting nucleic acid and amplifying target nucleic acid using the device. Background Art

[0002] Nucleic acids are the foundation of molecular biology research, and high-quality nucleic acids are a necessary prerequisite for molecular labeling, gene cloning, and gene expression research. Due to the complex composition of biological samples (such as blood, saliva, semen, or other secretions), it is usually necessary to extract, purify, and amplify the target nucleic acids before subsequent research can be carried out. The current nucleic acid extraction and amplification methods have the following main problems: (1) Faced with the huge number and complex sample processing, nucleic acid extraction, purification, and amplification steps, manual operation is prone to errors, and the overall operation steps are complicated, making it impossible to extract and amplify the target nucleic acid efficiently and quickly; (2) Most molecular diagnostics need to be performed in the laboratory. Many grassroots units do not have the conditions to establish standard molecular diagnostic laboratories. In addition, due to the different operating habits and proficiency of operators, nucleic acid is prone to cross-contamination of samples during extraction and amplification; (3) Existing nucleic acid extraction instruments and PCR instruments are often large in size and not suitable for use at the sampling site, which to a certain extent limits the application scope of molecular diagnostics. Making the extraction and amplification of nucleic acids fully automatic, fully enclosed, and integrated can shorten the nucleic acid extraction and amplification process, reduce the impact of human factors, enhance the safety and effectiveness of nucleic acid sample preparation, and achieve the miniaturization and portability of devices that meet the needs of grassroots or on-site rapid testing.

[0003] US9212980 discloses a device for nucleic acid extraction and amplification, which includes a plurality of chambers, a fluid replacement area, and a fluid processing area, wherein the fluid processing area is provided with a fluid processing material such as a filter for cell capture, cell lysis, binding of analytes, etc., and the fluid replacement area is used for temporary storage of fluid, which is connected to the fluid processing area. When in use, the fluid processing area is selectively connected to the plurality of chambers by adjusting the position of the rotary valve, and the fluid is then driven to flow between the fluid processing area, the fluid replacement area, and the chambers by the up and down movement of the piston. In this process, the control of the fluid is through a pair of ports of the valve body, and the two ports are selectively connected to each chamber in turn by rotating the valve body to cause the replacement or movement of the fluid, which may cause cross-contamination of the sample between several pathways, affecting the efficiency of DNA amplification. On the other hand, for example, during DNA extraction, biological cells are fixed in the fluid processing area by a fluid handling material such as a filter. By pushing the piston downward to apply pressure, a cleaning solution and a lysis solution flow sequentially through the fluid processing area, lysing the biological cells and releasing intracellular DNA. However, due to the negative pressure in the fluid displacement area and the waste liquid chamber, as well as clogging of the filter by the broken biological cell tissue, the downward movement of the piston to apply pressure makes it difficult for the fluid to be discharged from the fluid displacement area. This requires a large driving force to apply pressure, which is inconvenient to operate. Furthermore, after the biological cells are lysed, the intracellular DNA mixes with microscopic biological tissue that cannot be adsorbed or filtered by the filter. This lysis solution then directly enters the reagent chamber and mixes with the amplification reagents, resulting in insufficient purity of the extracted template DNA, which may adversely affect the subsequent PCR results. Summary of the Invention

[0004] In response to the shortcomings of the prior art, the present invention aims to provide a nucleic acid extraction device. The present invention uses a microfluidic DNA extraction module to achieve convenient and miniaturized DNA extraction, achieving the goals of functional integration, structural miniaturization, and fully automatic DNA extraction.

[0005] As a first aspect of the present invention, the present invention provides a nucleic acid extraction device, comprising:

[0006] A nucleic acid extraction element, a waste liquid storage chamber and a reaction chamber, wherein the reaction chamber is selectively connected to the nucleic acid extraction element and the waste liquid storage chamber, and is characterized in that the device exchanges fluids through pressure changes between the waste liquid storage chamber and the reaction chamber.

[0007] The term "fluid exchange" as used herein refers to the ability of a fluid to flow from one location to another. Specifically, in the present invention, this means that a fluid can flow from the nucleic acid extraction element or waste liquid storage chamber into the reaction chamber, and vice versa, with the fluid being able to repeatedly switch between these chambers.

[0008] The term "selectively" here means that when the reaction chamber is fluidically connected to the nucleic acid extraction element, there is no fluid communication between the waste liquid storage chamber and the reaction chamber; and when the waste liquid storage chamber and the reaction chamber are fluidically connected, there is no fluid communication between the reaction chamber and the nucleic acid extraction element. That is, the reaction chamber is selectively fluidically connected to the nucleic acid extraction element or to the waste liquid storage chamber at appropriate times.

[0009] Preferably, the device allows the fluid to flow from the reaction chamber into the waste liquid storage chamber by reducing the air pressure in the waste liquid storage chamber.

[0010] Preferably, the device reduces the air pressure in the waste liquid storage chamber via an exhaust element communicated with the waste liquid storage chamber.

[0011] Preferably, the exhaust element is a columnar air intake column.

[0012] Preferably, the exhaust element is a cylindrical air suction column.

[0013] Preferably, a sealing gasket is provided at the connection between the suction column and the waste liquid storage chamber.

[0014] Preferably, a nucleic acid solid phase extraction material is provided in the reaction chamber.

[0015] Preferably, the nucleic acid solid phase extraction material is magnetic beads.

[0016] When the nucleic acid solid phase extraction material is magnetic beads, there is an electromagnet in the reaction chamber, and the magnetic beads are fixed in the reaction chamber. The magnetic beads can specifically bind to free DNA to form a magnetic bead-DNA complex, thereby fixing the DNA in the reaction chamber.

[0017] Preferably, the nucleic acid extraction element comprises:

[0018] a lysis solution chamber, used for adding and storing a mixture of sample and lysis solution; or

[0019] a cleaning liquid chamber for adding and storing cleaning liquid; or

[0020] an eluent chamber, used for adding and storing eluent;

[0021] Wherein, one or more of the lysis liquid cavity, the cleaning liquid cavity or the elution liquid cavity are respectively connected to the reaction chamber fluid.

[0022] Preferably, the cleaning liquid cavity includes a primary cleaning liquid cavity, a secondary cleaning liquid cavity and a tertiary cleaning liquid cavity, and one or more of the primary cleaning liquid cavity, the secondary cleaning liquid cavity or the tertiary cleaning liquid cavity are respectively connected to the reaction chamber fluid.

[0023] It can be understood that the device described in this scheme may include multiple cleaning liquid cavities, such as 2, 3, 4, 5, etc., to meet the needs of using a variety of different cleaning liquids for nucleic acid purification, or using more amounts of the same cleaning liquid for multiple cleanings to improve the purity of the nucleic acid.

[0024] Preferably, the device further comprises a nucleic acid amplification element, which is a PCR reaction liquid cavity in fluid communication with the reaction chamber, and contains reagents required for the PCR reaction.

[0025] Preferably, the nucleic acid amplification element further comprises a PCR reaction tube in fluid communication with the PCR reaction liquid cavity.

[0026] Preferably, the side wall of the PCR reaction liquid cavity is provided with an injection hole and an exhaust hole for communicating with the PCR reaction tube fluid.

[0027] Preferably, the PCR reaction liquid chamber includes a primary PCR reaction liquid chamber and a secondary PCR reaction liquid chamber, wherein the primary PCR reaction liquid chamber is in fluid communication with the reaction chamber, wherein an injection port is provided at the bottom of the sidewall of the primary PCR reaction liquid chamber, and an exhaust port is provided at the upper portion of the sidewall of the secondary PCR reaction liquid chamber. A mixture of the PCR reaction liquid and nucleic acid enters the PCR reaction tube through the injection port at the bottom of the sidewall of the primary PCR reaction liquid chamber. As the liquid enters, air in the PCR reaction tube is discharged through the exhaust port at the upper portion of the sidewall of the secondary PCR reaction liquid chamber, thereby allowing the mixed liquid to enter smoothly.

[0028] Preferably, the device comprises a microfluidic channel for fluid communication with the reaction chamber, the waste liquid storage chamber, the nucleic acid extraction element or the nucleic acid amplification element.

[0029] Preferably, the microfluidic channels are radially extended and distributed on a rotating disk, and the reaction chamber, the waste liquid storage chamber, the nucleic acid extraction element or the nucleic acid amplification element are connected or disconnected by the rotation of the rotating disk.

[0030] Preferably, the fluid exchange is achieved through microfluidic channels.

[0031] Preferably, when the reaction chamber is fluidically connected to the nucleic acid extraction element, there is no fluid communication between the waste liquid storage chamber and the reaction chamber; and when the waste liquid storage chamber is fluidically connected to the reaction chamber, there is no fluid communication between the reaction chamber and the nucleic acid extraction element.

[0032] Preferably, the device comprises a piston, and the movement of the piston allows the fluid in the nucleic acid extraction element to enter the reaction chamber.

[0033] Preferably, the movement of the piston is upward along the reaction chamber.

[0034] Preferably, the device further comprises an exhaust element for generating negative pressure in the waste liquid storage chamber, thereby allowing the fluid in the reaction chamber to enter the waste liquid storage chamber.

[0035] Preferably, the negative pressure drives the piston to move downward along the reaction chamber.

[0036] Preferably, the exhaust element for generating negative pressure in the waste liquid storage chamber is an air suction column connected to the waste liquid storage chamber.

[0037] As a second aspect of the present invention, the present invention provides a method for extracting nucleic acid and amplifying specific target nucleic acid using a portable device.

[0038] Preferably, the method comprises the following steps:

[0039] (1) A nucleic acid extraction device is provided, comprising:

[0040] Nucleic acid extraction element, used for extracting nucleic acid;

[0041] The waste liquid storage chamber is used to store waste liquid during the reaction process;

[0042] a reaction chamber selectively communicating with the nucleic acid extraction element and the waste liquid storage chamber;

[0043] (2) Allow the reaction chamber to exchange fluid with the waste liquid storage chamber or nucleic acid extraction element to achieve nucleic acid extraction.

[0044] Preferably, the fluid is exchanged between the reaction chamber, the waste liquid storage chamber or the nucleic acid extraction element by changing the pressure between the waste liquid storage chamber and the reaction chamber.

[0045] Preferably, the fluid is allowed to enter the waste liquid storage chamber from the reaction chamber by reducing the air pressure in the waste liquid storage chamber.

[0046] Preferably, an exhaust element communicated with the waste liquid storage chamber is provided to reduce the air pressure in the waste liquid storage chamber.

[0047] Preferably, the exhaust element is a cylindrical air suction column.

[0048] Preferably, a nucleic acid solid phase extraction material is provided in the reaction chamber, so that the nucleic acid in the sample is bound to the nucleic acid solid phase extraction material.

[0049] Preferably, the nucleic acid solid phase extraction material is magnetic beads.

[0050] Preferably, the nucleic acid extraction element comprises:

[0051] a lysis solution chamber, used for adding and storing a mixture of sample and lysis solution; or

[0052] a cleaning liquid chamber for adding and storing cleaning liquid; or

[0053] an eluent chamber, used for adding and storing eluent;

[0054] One or more of the lysis liquid cavity, the cleaning liquid cavity or the elution liquid cavity are respectively connected to the reaction chamber fluid.

[0055] Preferably, the cleaning liquid cavity includes a primary cleaning liquid cavity, a secondary cleaning liquid cavity and a tertiary cleaning liquid cavity, and one or more of the primary cleaning liquid cavity, the secondary cleaning liquid cavity or the tertiary cleaning liquid cavity are respectively connected to the reaction chamber fluid.

[0056] Preferably, the method further includes a step of nucleic acid amplification, wherein the reaction chamber is fluidically connected to the nucleic acid amplification element to achieve nucleic acid amplification, wherein the nucleic acid amplification element is a PCR reaction liquid cavity fluidically connected to the reaction chamber, and reagents required for the PCR reaction are arranged inside the cavity.

[0057] Preferably, the nucleic acid amplification element further comprises a PCR reaction tube in fluid communication with the PCR reaction liquid cavity, allowing fluid to enter the PCR reaction tube for performing an amplification reaction.

[0058] Preferably, the PCR reaction tube is fluidically connected via an injection hole and an exhaust hole provided on a side wall of the PCR reaction liquid cavity.

[0059] Preferably, the PCR reaction liquid cavity includes a primary PCR reaction liquid cavity and a secondary PCR reaction liquid cavity, and the primary PCR reaction liquid cavity is connected to the reaction cavity fluid, wherein the injection hole is arranged at the bottom of the side wall of the primary PCR reaction liquid cavity, and the exhaust hole is arranged at the upper part of the side wall of the secondary PCR reaction liquid cavity.

[0060] Preferably, fluid communication or exchange is achieved between the reaction chamber, the waste liquid storage chamber, the nucleic acid extraction element or the nucleic acid amplification element through a microfluidic channel provided in the nucleic acid extraction device.

[0061] Preferably, the microfluidic channels are radially extended and distributed on a rotating disk, and the reaction chamber, the waste liquid storage chamber, the nucleic acid extraction element or the nucleic acid amplification element are connected or disconnected by the rotation of the rotating disk.

[0062] Preferably, fluid exchange is performed through microfluidic channels.

[0063] Preferably, when the reaction chamber is fluidically connected to the nucleic acid extraction element, the waste liquid storage chamber and the reaction chamber are not fluidically connected; when the waste liquid storage chamber and the reaction chamber are fluidically connected, the reaction chamber and the nucleic acid extraction element are not fluidically connected.

[0064] Preferably, the fluid in the nucleic acid extraction element enters the reaction chamber through the movement of a piston arranged in the reaction chamber.

[0065] Preferably, the movement of the piston is upward along the reaction chamber.

[0066] Preferably, the fluid in the reaction chamber is allowed to enter the waste liquid storage chamber through an exhaust element that creates a negative pressure in the waste liquid storage chamber.

[0067] Preferably, the exhaust element that generates negative pressure in the waste liquid storage chamber is an air suction column connected to the waste liquid storage chamber, and the negative pressure drives the piston to move downward along the reaction chamber.

[0068] As a third aspect of the present invention, a nucleic acid extraction device is provided. Preferably, the device comprises a nucleic acid extraction element, a waste liquid storage chamber, and a reaction chamber, wherein the reaction chamber is selectively fluidically connected to the nucleic acid extraction element or the waste liquid storage chamber via a microfluidic channel.

[0069] Preferably, the microfluidic channel includes an inlet microfluidic channel for fluidly connecting the reaction chamber with the nucleic acid extraction element, and a plurality of outlet microfluidic channels for fluidly connecting the reaction chamber with the waste liquid storage chamber.

[0070] Preferably, when the reaction chamber is fluidically connected to the nucleic acid extraction element through the liquid inlet microfluidic channel, there is no fluid communication between the waste liquid storage chamber and the reaction chamber; and when the waste liquid storage chamber is fluidically connected to the reaction chamber through the liquid outlet microfluidic channel, there is no fluid communication between the reaction chamber and the nucleic acid extraction element.

[0071] Preferably, the microfluidic channel is radially extended and distributed on a rotating disk, and the reaction chamber, the waste liquid storage chamber or the nucleic acid extraction element are connected or disconnected by the rotation of the rotating disk.

[0072] Preferably, the lengths of the liquid inlet microfluidic channel and the liquid outlet microfluidic channel are different.

[0073] Preferably, the nucleic acid extraction element includes: a lysis liquid cavity, a cleaning liquid cavity or an elution liquid cavity, wherein one or more of the lysis liquid cavity, the cleaning liquid cavity or the elution liquid cavity are respectively and sequentially connected to the reaction chamber fluid through the liquid inlet microfluidic channel.

[0074] Preferably, the cleaning liquid cavity includes a primary cleaning liquid cavity, a secondary cleaning liquid cavity or a tertiary cleaning liquid cavity, wherein one or more of the primary cleaning liquid cavity, the secondary cleaning liquid cavity or the tertiary cleaning liquid cavity are respectively and sequentially connected to the reaction chamber fluid through the liquid inlet microfluidic channel.

[0075] Preferably, the device further comprises a nucleic acid amplification element, which is a PCR reaction liquid cavity connected to the reaction chamber and contains reagents required for the PCR reaction.

[0076] Preferably, the PCR reaction liquid cavity is in fluid communication with the reaction chamber via a liquid inlet microfluidic channel.

[0077] As a fourth aspect, the present invention provides a nucleic acid extraction device. Preferably, the device comprises a nucleic acid extraction element, a waste liquid storage chamber, a nucleic acid amplification element, and a reaction chamber. The nucleic acid amplification element comprises a PCR reaction liquid chamber and a PCR reaction tube, and the PCR reaction liquid chamber is provided with an injection port and an exhaust port on the sidewall thereof for communicating with the PCR reaction tube.

[0078] Preferably, the PCR reaction liquid cavity includes a primary PCR reaction liquid cavity and a secondary PCR reaction liquid cavity, the injection hole is arranged at the bottom of the side wall of the primary PCR reaction liquid cavity, and the exhaust hole is arranged at the upper part of the side wall of the secondary PCR reaction liquid cavity.

[0079] Preferably, the injection hole is a through hole, which is connected to the first port of the injection channel of the PCR reaction tube and is used to inject fluid into the PCR reaction tube; the exhaust hole is a through hole, which is connected to the second port of the injection channel of the PCR reaction tube and is used to discharge gas in the channel.

[0080] Preferably, the apertures of the injection hole and the exhaust hole are smaller than the apertures of the first port and the second port.

[0081] Preferably, the device is provided with a sealing gasket at the connection between the PCR reaction liquid cavity and the PCR reaction tube.

[0082] Preferably, the sealing gasket has two through holes, wherein the size of the through hole connecting the sealing gasket to the PCR reaction liquid cavity corresponds to the size of the injection hole and the exhaust hole, and the size of the through hole connecting the sealing gasket to the PCR reaction tube corresponds to the size of the first port and the second port of the PCR reaction tube.

[0083] Preferably, the sealing gasket is made of elastic material.

[0084] Preferably, the sealing gasket is made of silicone.

[0085] Preferably, a non-water-absorbing material capable of slowly leaking air is provided on the upper portion of the first-level cavity of the PCR reaction liquid.

[0086] Preferably, the non-water-absorbent material capable of slowly leaking air is high-density hydrophobic cotton.

[0087] Beneficial effects :

[0088] (1) The device of the present invention provides an exhaust element in the waste liquid storage chamber to change the air pressure between the waste liquid storage chamber and the reaction chamber, so that the waste liquid in the reaction chamber can be easily discharged. The exhaust element reduces the air pressure, so that the fluid in the reaction chamber is discharged into the waste liquid storage chamber under the action of negative pressure, without the need to pressurize the reaction chamber to discharge the liquid, thereby facilitating experimental operations.

[0089] (2) The device and method of the present invention avoid the mixing of reagents caused by repeated microflow channels, thereby improving the purity and concentration of the extracted DNA, thereby improving the efficiency of the amplification reaction and making the reaction smoother and faster.

[0090] (3) The device of the present invention achieves gas and liquid communication between the PCR reaction tube and the PCR reaction liquid cavity through the injection hole and the exhaust hole, effectively avoiding the generation of bubbles during the liquid injection process, and can conveniently observe whether the liquid injection is completed through the overflow of the liquid through the exhaust hole.

[0091] (4) The present invention adopts an integrated control system to integrate the traditional manual extraction and purification of nucleic acids into a fully automatic closed processing process, making the operation process convenient and fast, and improving the efficiency of experimental work. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 (A) and (B) are three-dimensional cross-sectional views of the nucleic acid extraction device of the present invention, wherein: Figure 1 (B) is a partial enlarged view of the piston;

[0093] Figure 2 is a perspective view of the nucleic acid extraction device of the present invention;

[0094] Figure 3 yes Figure 2 Exploded view of the device;

[0095] Figure 4 is another perspective view of the nucleic acid extraction device of the present invention;

[0096] Figure 5 is a three-dimensional diagram of the suction column, where Figure 5 (A) is a structural diagram of the suction column with a sealing gasket. Figure 5 (B) is the structure diagram of the suction column without sealing gasket;

[0097] Figure 6 is another perspective view of the nucleic acid extraction device of the present invention;

[0098] Figure 7 is a top view of the nucleic acid extraction device of the present invention;

[0099] Figure 8is another top view of the nucleic acid extraction device of the present invention;

[0100] Figure 9 1. It is a three-dimensional view and a front view of the rotating disk of the nucleic acid extraction device of the present invention;

[0101] Figure 10 is a cross-sectional view of a rotating disk of the nucleic acid extraction device of the present invention;

[0102] Figure 11 is a cross-sectional view of the PCR reaction liquid chamber of the nucleic acid extraction device of the present invention;

[0103] Figure 12 This is a structural diagram of a PCR reaction tube of a nucleic acid extraction device of the present invention;

[0104] Figure 13 is a three-dimensional view of the card slot of the nucleic acid extraction device of the present invention;

[0105] Figure 14 The front view (A) and rear view (B) of the gasket are shown;

[0106] Figure 15 It is a structural diagram of the housing and rotating disk of the nucleic acid extraction device of the present invention.

[0107] Reference numerals: 1 top cover, 2 housing, 3 rotating disk, 4 base, 5 PCR reaction tube, 6 suction column, 7 piston, 8 card slot, 9 sealing gasket, 10 magnetic beads, 11 electromagnet, 201 reaction chamber, 202 waste liquid storage chamber, 203 lysate chamber, 204 cleaning liquid chamber, 2041 cleaning liquid primary chamber, 2042 cleaning liquid secondary chamber, 2043 cleaning liquid tertiary chamber, 205 elution liquid chamber, 206 PCR reaction liquid chamber, 2061 PCR reaction liquid primary chamber, 2062 PCR reaction liquid secondary cavity, 207 through hole, 208 injection hole, 209 exhaust hole, 210 rib position, 211 boss, 301 liquid inlet microfluidic channel, 302 first liquid outlet microfluidic channel, 303 second liquid outlet microfluidic channel, 304 third liquid outlet microfluidic channel, 305 fourth liquid outlet microfluidic channel, 306 opening, 307 boss, 501 first port, 502 second port, 601 sealing gasket, 602 suction column external connection end, 801 card slot opening, 901~904 sealing gasket through holes, 905 groove. DETAILED DESCRIPTION

[0108] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other technical solutions obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0109] In the present invention, unless otherwise specified or limited, the terms "connection" and "fixation" should be understood in a broad sense. For example, "fixation" can refer to fixed connection, detachable connection, or integration; it can refer to direct connection or indirect connection through an intermediate medium; it can refer to internal communication between two elements or interaction between two elements, unless otherwise specified. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0110] Example 1

[0111] Figure 1-4 FIG. 1 shows a specific embodiment of the nucleic acid extraction device of the present invention, which includes a housing 2 having multiple cavities. Figure 2 and Figure 3 As shown, the nucleic acid extraction device comprises a top cover 1 and a base 4 , wherein the top cover 1 has openings for injecting samples or reagents into each cavity.

[0112] Figure 1 The nucleic acid extraction device of the present invention is shown, comprising a nucleic acid extraction element, a waste liquid storage chamber 202, and a reaction chamber 201. The nucleic acid extraction element is used to add or store reagents required for nucleic acid extraction and purification, the waste liquid storage chamber 202 is used to store waste liquid generated during the reaction process, and the reaction chamber 201 is where nucleic acid extraction occurs. The nucleic acid extraction element, waste liquid storage chamber, and reaction chamber are located on the housing 2. In this embodiment, the reaction chamber selectively communicates with the nucleic acid extraction element and the waste liquid storage chamber. The device exchanges fluids through pressure changes between the waste liquid storage chamber and the reaction chamber.

[0113] The "fluid exchange" referred to here means that the fluid can flow from one place to another, and the flow may pass through some physical structures to guide it. The so-called passing through physical structures generally refers to the liquid passing through the surface of these physical structures, or the internal space of these structures to passively or actively flow to another place. Passive flow is generally caused by external forces, such as flow under pressure. In the present invention, it specifically means that the fluid can enter the reaction chamber from the nucleic acid extraction element or the waste liquid storage chamber, and can also enter the nucleic acid extraction element or the waste liquid storage chamber from the reaction chamber. The fluid can be repeatedly switched between the above-mentioned cavities.

[0114] The term "selectively" here means that when the reaction chamber is fluidically connected to the nucleic acid extraction element, there is no fluid communication between the waste liquid storage chamber and the reaction chamber; and when the waste liquid storage chamber and the reaction chamber are fluidically connected, there is no fluid communication between the reaction chamber and the nucleic acid extraction element. That is, the reaction chamber is selectively fluidically connected to the nucleic acid extraction element at an appropriate time, and selectively fluidly connected to the waste liquid storage chamber at another appropriate time.

[0115] Specifically, in one embodiment, a piston 7 is movably placed in the reaction chamber. When the piston moves upward along the reaction chamber, the volume of the reaction chamber expands, the pressure decreases, and the fluid is sucked into the reaction chamber from the nucleic acid extraction element or the waste liquid storage chamber; when the fluid in the reaction chamber needs to be discharged, the piston moves downward along the reaction chamber, the volume of the reaction chamber decreases, the pressure increases, and the fluid is pressed from the reaction chamber into the nucleic acid extraction element or the waste liquid storage chamber.

[0116] In a preferred embodiment, the air pressure in the waste liquid storage chamber is reduced to allow the fluid to enter the waste liquid storage chamber from the reaction chamber under the action of negative pressure, rather than by pressurizing the reaction chamber with a piston. In this embodiment, the negative pressure drives the fluid from the reaction chamber into the waste liquid storage chamber, while the piston moves downward along the reaction chamber under the negative pressure, preparing for the next generation of negative pressure in the reaction chamber.

[0117] In certain specific cases, for example, to further mix the fluid, the waste liquid storage chamber may be depressurized to allow the fluid to flow from the reaction chamber into the waste liquid storage chamber under negative pressure. Then, the reaction chamber may be depressurized to allow the fluid to flow from the waste liquid storage chamber into the reaction chamber under negative pressure. This process is repeated to allow the fluid to be exchanged between the two chambers for thorough mixing. The negative pressure in the reaction chamber is generated by the upward movement of the piston.

[0118] In a preferred embodiment, Figure 1 As shown, the device reduces the air pressure in the waste liquid storage chamber by an exhaust element connected to the waste liquid storage chamber. Preferably, the exhaust element is a columnar air suction column connected to the waste liquid storage chamber, and more preferably a cylindrical air suction column 6. Figure 5As shown, the suction column 6 is a hollow cylinder, one end of which is connected to the waste liquid storage chamber 202. In a preferred embodiment, a sealing gasket 601 is provided at the connection between the suction column and the waste liquid storage chamber 202 to enhance the sealing between the suction column and the waste liquid storage chamber, and the sealing gasket is preferably made of elastic material. The other end of the suction column is exposed outside the nucleic acid extraction device and is used to connect to the exhaust device. Preferably, the external connection end 602 of the suction column is connected to the exhaust device through a hose. The exhaust device described here is preferably a vacuum pump or a negative pressure pump, which reduces the air pressure in the waste liquid storage chamber 202 by evacuating the waste liquid storage chamber 202. As the air pressure in the waste liquid storage chamber 202 decreases, the fluid in the reaction chamber 201 enters the waste liquid storage chamber 202. At this time, the fluid can be discharged from the reaction chamber 201 into the waste liquid storage chamber 202 without pressurizing the reaction chamber 201, which is convenient and labor-saving to operate. In a specific embodiment, the piston 7 connected to the reaction chamber 201 will move downward relative to the reaction chamber as the liquid in the reaction chamber is discharged.

[0119] In a preferred embodiment, a nucleic acid solid phase extraction material is provided in the reaction chamber 201 for capturing target nucleic acids in the sample. In a preferred embodiment, the nucleic acid solid phase extraction material is a magnetic bead 10. When the nucleic acid solid phase extraction material is a magnetic bead, an electromagnet 11 is provided inside the reaction chamber so that the magnetic bead is fixed in the reaction chamber. In a preferred embodiment, the reaction chamber is a cylindrical cavity with a receiving cavity for receiving the electromagnet 11 in the center of the cavity. The electromagnet 11 is placed in the receiving cavity (e.g., Figure 1 As shown in A), the bottom side wall of the piston 7 has a certain groove, and the groove on the bottom side wall of the piston 7 and the side wall of the central accommodating cavity of the reaction chamber form a space for accommodating magnetic beads, as shown in FIG. Figure 1 B. The position indicated by the dotted box. The magnetic beads specifically bind to the free DNA in the sample to form a magnetic bead-DNA complex, thereby fixing the DNA at a specific position in the reaction chamber.

[0120] The sample includes at least one of the following substances: cells, spores, microorganisms, biological tissues, biological fluids or environmental samples.

[0121] In a preferred embodiment, the nucleic acid extraction element includes: a lysis liquid chamber 203 for adding and storing a mixture of a sample and a lysis liquid; a cleaning liquid chamber 204 for adding and storing a cleaning liquid; and an eluent chamber 205 for adding and storing an eluent. By connecting the reaction chamber 201 with the lysis liquid chamber 203, the cleaning liquid chamber 204, and the eluent chamber 205 in sequence, the nucleic acid in the sample is extracted and purified.

[0122] The lysate comprises at least one of the following: guanidine hydrochloride, chaotropic salt, red blood cell lysis reagent, chelating agent, sodium hydroxide, DNA enzyme inhibitor, RNA enzyme inhibitor, anticoagulant, coagulant, protease, surfactant, etc.; the cleaning solution comprises at least one of the following: ethanol, sodium chloride, Tris hydrochloric acid, etc.; the eluent is sterile deionized water or Tris hydrochloric acid. The specific process is as follows: (1) the lysis solution chamber 203 and the reaction chamber 201 are connected, and the sample and the lysis solution enter the reaction chamber 201 for a lysis reaction. In this process, on the one hand, the sample can be lysed by connecting an additional device such as an acoustic vibrator to the device of the present invention. On the other hand, after the mixture of the lysis solution and the sample enters the reaction chamber 201, the reaction chamber 201 is connected to the waste liquid storage chamber 202. By changing the pressure between the reaction chamber and the waste liquid storage chamber, the mixture of the lysis solution and the sample is repeatedly exchanged between the reaction chamber and the waste liquid storage chamber to promote complete sample lysis; (2) After the lysis reaction is completed, the nucleic acid in the sample is bound to the magnetic beads 10 and fixed in the reaction chamber 201, and the lysis waste enters the waste liquid storage chamber 202. ; (3) connecting the cleaning liquid chamber 204 and the reaction chamber 201, the cleaning liquid enters the reaction chamber 201, and the magnetic beads that have been bound to the nucleic acid are immersed in the cleaning liquid, and the impurities are cleaned and removed. In this process, mixing can be promoted by acoustic vibration or the like; (4) connecting the reaction chamber 201 and the waste liquid storage chamber 202, the waste liquid storage chamber 202 is depressurized, and the waste liquid is discharged into the waste liquid storage chamber 202. The nucleic acid is bound to the magnetic beads and fixed in the reaction chamber 201. The cleaning process can be repeated several times according to actual conditions; (5) connecting the elution liquid chamber 205 and the reaction chamber 201, the elution liquid enters the reaction chamber 201, and the magnetic beads that have been bound to the nucleic acid are immersed in the elution liquid. The nucleic acid is separated from the magnetic beads and dissolved in the elution liquid. In this process, mixing can also be promoted and elution can be accelerated by acoustic vibration or the like.

[0123] In a preferred embodiment, after the nucleic acid is separated from the magnetic beads, the extraction of the nucleic acid in the sample is completed. At this time, the magnetic beads can be cleaned and used for the extraction of nucleic acid from the next sample. That is, the device of the present invention can be reused multiple times, saving experimental costs.

[0124] In a preferred embodiment, Figure 4 As shown, the cleaning liquid chamber 204 includes a primary cleaning liquid chamber 2041, a secondary cleaning liquid chamber 2042, and a tertiary cleaning liquid chamber 2043. The primary cleaning liquid chamber 2041, the secondary cleaning liquid chamber 2042, and the tertiary cleaning liquid chamber 2043 are respectively connected to the reaction chamber 201 in sequence. It will be understood that the device described in this embodiment can include multiple cleaning liquid chambers, for example, 2, 3, or 4, to meet the needs of using multiple different cleaning liquids for nucleic acid purification, or using larger amounts of the same cleaning liquid for multiple cleanings to improve nucleic acid purity.

[0125] In a preferred embodiment, the lysis liquid chamber 203, the cleaning liquid chamber 204, the eluent chamber 205 and the waste liquid storage chamber 202 are connected to the reaction chamber 201 in sequence through a plurality of independent microfluidic channels. Figure 3 、 Figure 4 and Figure 6 As shown, the lysis liquid chamber 203, the cleaning liquid chamber 204, the eluent chamber 205, the waste liquid storage chamber 202 and the reaction chamber 201 are located in a cylindrical shell 2, wherein the reaction chamber 201 is located in the center of the inner ring of the cylindrical shell, and the lysis liquid chamber 203, the cleaning liquid chamber 204, the eluent chamber 205 and the waste liquid storage chamber 202 are distributed on the outer ring of the cylindrical shell and distributed along the circumference of the reaction chamber 201, and are radially connected to the reaction chamber 201; the multiple independent microfluidic channels are arranged on a rotating disk 3, and the rotating disk is constructed by two parts; the shell 2 containing the above-mentioned multiple chambers is connected to the rotating disk 3, and the rotation of the rotating disk 3 can enable the reaction chamber 201 to be fluidically connected to the lysis liquid chamber 203, the cleaning liquid chamber 204, the eluent chamber 205 or the waste liquid storage chamber 202 in sequence through the multiple microfluidic channels.

[0126] from Figure 4 、 Figure 6 and Figure 7 It can be seen that the bottom of the lysate chamber 203, the cleaning liquid chamber 204, the eluent chamber 205, and the waste liquid storage chamber 202 each have a through hole 207, and the bottom of the reaction chamber 201 has multiple through holes 207 that correspond one-to-one to the through holes at the bottom of the above chambers. Through the two corresponding through holes 207, the lysate chamber 203, the cleaning liquid chamber 204, the eluent chamber 205, the waste liquid storage chamber 202 and the reaction chamber 201 can be connected. Figure 7 As can be seen, the distances between the two through-holes connecting the lysing liquid chamber 203 and the reaction chamber 201, the primary cleaning liquid chamber 2041 and the reaction chamber 201, the secondary cleaning liquid chamber 2042 and the reaction chamber 201, the tertiary cleaning liquid chamber 2043 and the reaction chamber 201, and the eluent chamber 205 and the reaction chamber 201 are equal and equidistantly spaced about the center of the reaction chamber. However, the distance between the two through-holes connecting the waste liquid storage chamber 202 and the reaction chamber 201 is different from the distance between the two through-holes connecting the aforementioned chambers and the reaction chamber.

[0127] Figure 9The rotating disk is shown to be distributed with multiple radially extending independent microfluidic channels, wherein each microfluidic channel has two openings 306 on the outer surface of the rotating disk, and the two openings 306 correspond to the two through holes 207 connecting the lysis liquid cavity and the reaction chamber, the cleaning liquid cavity and the reaction chamber, the eluent cavity and the reaction chamber, and the waste liquid storage chamber and the reaction chamber, respectively. When the rotating disk is rotated horizontally by a certain angle, the two openings 306 of a certain microfluidic channel match with the corresponding two through holes 207 on the shell, thereby achieving fluid communication between the above-mentioned cavity and the reaction chamber. The microfluidic channel includes an inlet microfluidic channel 301 and a plurality of outlet microfluidic channels. In a preferred embodiment, the number of the outlet microfluidic channels is 4, which are respectively named as the first outlet microfluidic channel 302, the second outlet microfluidic channel 303, the third outlet microfluidic channel 304, and the fourth outlet microfluidic channel 305. The lengths of the outlet microfluidic channels 302, 303, 304 and 305 are the same, but different from the length of the inlet microfluidic channel 301. It is understood that the number of outlet microfluidic channels can be set according to the number of times waste liquid needs to be discharged.

[0128] In a preferred embodiment, the length of the liquid inlet microfluidic channel 301 is equal to the distance between the two through holes connecting the lysate cavity 203 and the reaction chamber 201, the cleaning liquid primary cavity 2041 and the reaction chamber 201, the cleaning liquid secondary cavity 2042 and the reaction chamber 201, the cleaning liquid tertiary cavity 2043 and the reaction chamber 201, and the eluent cavity 205 and the reaction chamber 201. The length of the liquid outlet microfluidic channels 302, 303, 304, and 305 is equal to the distance between the two through holes connecting the waste liquid storage cavity 202 and the reaction chamber 201. Through the rotation of the rotating disk, the liquid inlet microfluidic channel 301 connects the lysate chamber 203, the cleaning liquid primary chamber 2041, the cleaning liquid secondary chamber 2042, the cleaning liquid tertiary chamber 2043, and the eluent chamber 205 to the reaction chamber 201 in sequence, while the waste liquid storage chamber 202 connects to the reaction chamber 201 through the liquid outlet microfluidic channels 302, 303, 304, and 305 in sequence.

[0129] In a preferred embodiment, the five groups of through holes connecting the lysate cavity 203 and the reaction chamber 201, the cleaning liquid primary cavity 2041 and the reaction chamber 201, the cleaning liquid secondary cavity 2042 and the reaction chamber 201, the cleaning liquid tertiary cavity 2043 and the reaction chamber 201, and the eluent cavity 205 and the reaction chamber 201 are equidistantly distributed with the center of the reaction chamber 201 as the center of the circle, that is, the radial extension lines of the above five groups of through holes intersect at the center of the reaction chamber, and the angles formed by the radial extension lines of the five groups of through holes are the same, preferably 36 degrees (such as Figure 7 shown).

[0130] The microfluidic channels are radially distributed around the center of the rotating disk 3. The radial extensions of the outlet microfluidic channels 302, 303, 304, and 305 intersect at the center of the rotating disk 3, and the radial extensions form the same angle, preferably 36 degrees. The center of the reaction chamber of the device coincides with the center of the rotating disk.

[0131] When the nucleic acid extraction device is in a fluid disconnected state ( Figure 8 The angle between the radial extension of the liquid inlet microfluidic channel 301 and the radial extension of the two through-holes connecting the lysate chamber 203 and the reaction chamber 201 is 18 degrees. By rotating the rotating disk 18 degrees clockwise relative to the lysate chamber 203, the lysate chamber 203 and the reaction chamber 201 can be connected via the liquid inlet microfluidic channel 301. Further clockwise rotation of the rotating disk can sequentially connect the waste liquid storage chamber 202, the cleaning liquid chamber 204, or the eluent chamber 205 to the reaction chamber 201.

[0132] In a preferred embodiment, the nucleic acid extraction device further comprises a nucleic acid amplification element, which comprises: a PCR reaction liquid chamber 206 connected to the reaction chamber, wherein reagents required for the PCR reaction are arranged inside the chamber.

[0133] The PCR reaction reagent includes at least one of the following: Bst polymerase, Taq polymerase, reverse transcriptase, dNTPs, primers, and probes. The PCR reaction reagent is preferably liquid.

[0134] In a preferred embodiment, Figure 6 As shown, the PCR reaction liquid cavity 206 includes a PCR reaction liquid primary cavity 2061 and a PCR reaction liquid secondary cavity 2062, and the two cavities are independent of each other. The PCR reaction liquid primary cavity 2061 is connected to the reaction chamber 201 through the liquid inlet microfluidic channel 301, and the nucleic acid is dissolved in the eluent after elution. At this time, the reaction chamber 201 and the PCR reaction liquid primary cavity 2061 are connected, and the piston is further moved upward, and the PCR reaction liquid enters the reaction chamber 201 and mixes with the nucleic acid. Furthermore, the PCR reaction liquid and the nucleic acid can be fully mixed by acoustic vibration. In this process, it is necessary to ensure that some air in the PCR reaction liquid is inhaled. The purpose of inhaling air is to ensure that all PCR reaction liquids enter the reaction chamber 201.

[0135] In a preferred embodiment, the nucleic acid amplification component further includes a PCR reaction tube 5 connected to the PCR reaction liquid chamber 206. The entire PCR reaction and corresponding optical result detection are completed in the PCR reaction tube 5. The PCR reaction tube 5 can be directly placed in a temperature-controlled instrument for PCR amplification or can be removed from the nucleic acid amplification device and placed in a temperature-controlled instrument for amplification.

[0136] In a preferred embodiment, an injection hole 208 is provided at the bottom of the side wall of the first-level PCR reaction liquid chamber 2061. The injection hole 208 is provided on the side wall with a certain height difference from the bottom of the first-level PCR reaction liquid chamber 2061. An exhaust hole 209 is provided at the upper part of the side wall of the second-level PCR reaction liquid chamber. Figure 11 ), the height of the vent 209 from the bottom of the cavity is higher than the height of the injection hole 208 from the bottom of the cavity. The injection hole 208 and the vent 209 are both through holes, and the injection hole 208 and the vent 209 correspond to the first port 501 and the second port 502 of the PCR reaction tube injection channel respectively ( Figure 12 The PCR reaction tube is in fluid and gas communication with the PCR reaction liquid cavity through the injection hole 208 , the exhaust hole 209 , the first port 501 and the second port 502 .

[0137] The piston 7 moves downward to pressurize the reaction chamber 201. The mixed solution of PCR reaction solution and nucleic acid enters the PCR reaction solution cavity 206 from the reaction chamber 201 through the liquid inlet microfluidic channel 301. When the liquid level of the mixed solution in the PCR reaction solution cavity 206 exceeds the injection hole 208, the air above the PCR reaction solution cavity 206 begins to compress, prompting the mixed solution to enter the PCR reaction tube 5 through the injection hole 208. As the liquid enters, the air in the PCR reaction tube 5 is discharged from the exhaust hole 209, so that the liquid can smoothly enter the PCR reaction tube.

[0138] The vent hole plays two roles: (1) to exhaust the air in the PCR reaction tube 5 to facilitate the entry of liquid into the PCR reaction tube 5; (2) to indicate whether the liquid has fully filled the PCR reaction tube 5. When liquid overflows from the vent hole 209, it indicates that the mixed liquid has been completely filled in the PCR reaction tube 5.

[0139] In a preferred embodiment, injection hole 208 and exhaust hole 209 are two small holes with diameters smaller than those of the first port 501 and second port 502 of the PCR reaction tube. Pressurization of reaction chamber 201 forces the mixed solution into the PCR reaction tube through injection hole 208. This structure effectively prevents the formation of bubbles in the mixed solution injected into the PCR reaction tube or facilitates the exhaust of bubbles, thereby preventing the introduction of bubbles into the mixed solution and interfering with PCR reaction results.

[0140] In a preferred embodiment, a sealing gasket 9 is provided at the connection between the PCR reaction tube 5 and the PCR reaction liquid cavity 206. Preferably, the sealing gasket 9 is made of an elastic material, such as silicone. Figure 14As shown, the sealing gasket 9 is a rectangular columnar structure having upper and lower through holes extending through the column. One side of the sealing gasket 9 is used to connect to the PCR reaction tube 5, and the other side is used to connect to the PCR reaction liquid cavity 206. The two through holes 901 and 902 on the surface connecting to the PCR reaction tube 5 correspond to the first port 501 and the second port 502 of the PCR reaction tube 5, respectively, and their aperture sizes correspond to the first port 501 and the second port 502 of the PCR reaction tube. The first port 501 and the second port 502 of the PCR reaction tube 5 are inserted into the through holes 901 and 902, respectively, and have an interference fit with the sealing gasket 9. The two through holes 903 and 904 on the surface connecting to the PCR reaction liquid cavity 206 correspond to the injection hole 208 and the exhaust hole 209 of the PCR reaction liquid cavity 206, respectively, and their aperture sizes correspond to the sizes of the injection hole 208 and the exhaust hole 209. Furthermore, a groove 905 is formed at the junction of through-holes 901 and 903, or 902 and 904. Groove 905 aligns injection hole 208 and first port 501, or vent 209 and second port 502. Fluid enters through through-hole 903, flows through groove 905, and enters the injection channel of PCR reaction tube 5 through first port 501, thereby allowing the fluid to enter PCR reaction tube 5.

[0141] In a preferred embodiment, a sponge-like, non-absorbent material capable of slow air leakage is disposed above the primary PCR reaction liquid chamber 2061. Preferably, the material is high-density hydrophobic cotton. The high-density hydrophobic cotton serves to slowly exhaust air, maintaining consistent air pressure in the primary PCR reaction liquid chamber 2061 and the secondary PCR reaction liquid chamber 2062. This prevents the mixed solution from flowing within the primary PCR reaction liquid chamber 2061 and avoids the formation of bubbles in the fluid. Specifically, the downward movement of piston 7 pressurizes reaction chamber 201. The mixture of nucleic acid and PCR reaction solution enters primary PCR reaction liquid chamber 2061 from reaction chamber 201 through liquid inlet microfluidic channel 302. The liquid level in primary PCR reaction liquid chamber 2061 continuously rises. When the liquid level exceeds the height of the injection hole, primary PCR reaction liquid chamber 2061 begins to be pressurized. At this point, the pressure in secondary PCR reaction liquid chamber 2062 reaches atmospheric pressure. By placing high-density hydrophobic cotton above primary PCR reaction liquid chamber 2061, the primary PCR reaction liquid chamber 2061 can be slowly vented, thereby aligning the pressure in primary PCR reaction liquid chamber 2061 with that in secondary PCR reaction liquid chamber 2062, making it easier for liquid to enter PCR reaction tube 5. In a preferred embodiment, the venting time of the high-density hydrophobic cotton is set to 5-10 seconds. Preferably, the high-density hydrophobic cotton is filled at 1 / 3 of the height of primary PCR reaction liquid chamber 2061, for example, in the form of a plunger fixedly placed above the primary PCR reaction liquid chamber. High-density hydrophobic cotton does not absorb water and will not absorb PCR reaction liquid, thus not having any impact on the nucleic acid extraction process.

[0142] In a preferred embodiment, Figure 6 As shown, ribs 210 are provided on the wall of the eluent cavity 205 to reduce the amount of eluent used.

[0143] In a preferred embodiment, the PCR reaction tube 5 is fixedly connected to the PCR reaction liquid chamber 206 through a slot 8. Figure 13 The 3D diagram of the card slot 8 is shown. The card slot 8 is connected to the PCR reaction liquid chamber 206 , and the PCR reaction tube 5 is inserted and fixed in the opening 801 of the card slot 8 .

[0144] In a preferred embodiment, Figure 15 As shown, a boss 211 is provided at the connection between the bottom of the shell 2 and the rotating disk 3 to enhance the sealing effect between the shell 2 and the rotating disk 3; a boss 307 is provided at the connection between the rotating disk 3 and the base 4 to reduce the friction force when the rotating disk rotates.

[0145] Example 2

[0146] This embodiment is a method for extracting nucleic acid using the nucleic acid extraction device described in Example 1.

[0147] Step 1: Open the lysis solution chamber 203, add the liquid sample, and mix the sample with the lysis solution;

[0148] Step 2: Rotate the rotating disk 3 clockwise 18 degrees horizontally, so that the lysate chamber 203 is connected to the reaction chamber 201 through the liquid inlet microfluidic channel 301. External force pushes the reaction chamber piston 7 upward, allowing the mixture of lysate and sample to enter the reaction chamber 201. The nucleic acids released after sample lysis bind to the magnetic beads 10 in the reaction chamber.

[0149] Step 3: Rotate the rotating disk 3 clockwise 18 degrees horizontally, so that the waste liquid storage chamber 202 is connected to the reaction chamber 201 through the first liquid outlet microfluidic channel 302. The vacuum pump is connected to the suction column 6 to extract air, and the lysed waste liquid enters the waste liquid storage chamber 202. The magnetic beads bound to the nucleic acid are fixed to the reaction chamber 201.

[0150] Step 4: Rotate the rotating disk 3 clockwise horizontally by 18 degrees, so that the cleaning liquid primary chamber 2041 is connected to the reaction chamber 201 through the liquid inlet microfluidic channel 301. External force pushes the reaction chamber piston 7 upward, and the cleaning liquid enters the reaction chamber 201, immersing the magnetic beads bound to the nucleic acid in the cleaning liquid to wash and remove impurities;

[0151] Step 5: Rotate the rotating disk 3 clockwise 18 degrees horizontally, so that the waste liquid storage chamber 202 is connected to the reaction chamber 201 through the second liquid outlet microfluidic channel 303. The vacuum pump is connected to the suction column 6 to extract air, and the cleaning waste liquid enters the waste liquid storage chamber 202. The magnetic beads bound to the nucleic acid are fixed to the reaction chamber 201.

[0152] Step 6: Rotate the rotating disk 3 clockwise horizontally by 18 degrees, so that the cleaning liquid secondary chamber 2042 is connected to the reaction chamber 201 through the liquid inlet microfluidic channel 301. External force pushes the reaction chamber piston 7 upward, and the cleaning liquid enters the reaction chamber 201, immersing the magnetic beads bound to the nucleic acid in the cleaning liquid to wash and remove impurities;

[0153] Step 7: Rotate the rotating disk 3 clockwise 18 degrees horizontally, so that the waste liquid storage chamber 202 is connected to the reaction chamber 201 through the third liquid outlet microfluidic channel 304. The vacuum pump is connected to the suction column 6 to extract air, and the cleaning waste liquid enters the waste liquid storage chamber 202. The magnetic beads bound to the nucleic acid are fixed to the reaction chamber 201.

[0154] Step 8: Rotate the rotating disk 3 clockwise 18 degrees horizontally, so that the cleaning liquid tertiary cavity 2043 is connected to the reaction chamber 201 through the liquid inlet microfluidic channel 301. External force pushes the reaction chamber piston 7 upward, and the cleaning liquid enters the reaction chamber 201, immersing the magnetic beads bound to the nucleic acid in the cleaning liquid to wash and remove impurities;

[0155] Step 9: Rotate the rotating disk 3 clockwise 18 degrees horizontally, so that the waste liquid storage chamber 202 is connected to the reaction chamber 201 through the fourth liquid outlet microfluidic channel 305. The vacuum pump is connected to the suction column 6 to extract air, and the cleaning waste liquid enters the waste liquid storage chamber 202. The magnetic beads bound to the nucleic acid are fixed to the reaction chamber 201.

[0156] Step 10: The rotating disk 3 rotates horizontally clockwise by 18 degrees, so that the eluent chamber 205 is connected to the reaction chamber 201 through the liquid inlet microfluidic channel 301. External force pushes the reaction chamber piston 7 upward, and the eluent enters the reaction chamber 201. The magnetic beads bound to the nucleic acid are immersed in the eluent, and the nucleic acid is separated from the magnetic beads and dissolved in the eluent.

[0157] Step 11: Rotate the rotating disk 3 clockwise 36 degrees horizontally, so that the PCR reaction liquid primary chamber 2061 is connected to the reaction chamber 201 through the liquid inlet microfluidic channel 302. External force continues to push the reaction chamber piston 7 upward, and the PCR reaction liquid enters the reaction chamber 201, so that the nucleic acid dissolved in the eluent is mixed with the PCR reaction reagents;

[0158] Step 12: External force pushes the reaction chamber piston 7 downward, allowing the mixed liquid in the reaction chamber 201 to enter the PCR reaction liquid primary chamber 2061 through the liquid inlet microfluidic channel 301. When the liquid level of the mixed liquid in the PCR reaction liquid primary chamber 2061 is higher than the injection hole 208 of the PCR reaction liquid primary chamber 2061, the mixed liquid enters the PCR reaction tube 5 through the injection hole 208 until the mixed liquid overflows from the vent 209 of the PCR reaction liquid secondary chamber 2062, completing the liquid filling of the PCR reaction tube 5.

[0159] Step 13: Place the PCR reaction tube 5 in a temperature control device and perform nucleic acid fluorescence PCR amplification or fluorescence constant temperature amplification according to the set amplification program.

[0160] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A nucleic acid extraction device, comprising: A nucleic acid extraction element, a waste liquid storage chamber, and a reaction chamber, wherein the reaction chamber selectively communicates with the nucleic acid extraction element and the waste liquid storage chamber. The device is characterized in that the device exchanges fluids through pressure changes between the waste liquid storage chamber and the reaction chamber; the device allows fluid to enter the waste liquid storage chamber from the reaction chamber by reducing the air pressure in the waste liquid storage chamber; and the device reduces the air pressure in the waste liquid storage chamber via an exhaust element in communication with the waste liquid storage chamber, one end of the exhaust element being exposed outside the nucleic acid extraction device for connection to an exhaust device. The device also includes a nucleic acid amplification element, which is a PCR reaction liquid cavity connected to the reaction chamber fluid, and the reagents required for the PCR reaction are arranged inside the nucleic acid amplification element; the nucleic acid amplification element also includes a PCR reaction tube connected to the PCR reaction liquid cavity fluid; the side wall of the PCR reaction liquid cavity is provided with an injection hole and an exhaust hole for connecting to the PCR reaction tube fluid; the PCR reaction liquid cavity includes a primary PCR reaction liquid cavity and a secondary PCR reaction liquid cavity, the primary PCR reaction liquid cavity is connected to the reaction chamber fluid, the injection hole is arranged at the bottom of the side wall of the primary PCR reaction liquid cavity, and the exhaust hole is arranged at the upper part of the side wall of the secondary PCR reaction liquid cavity.

2. The device according to claim 1, characterized in that The exhaust element is a columnar air suction column.

3. The device according to claim 2, characterized in that The exhaust element is a cylindrical air suction column.

4. The device according to claim 3, characterized in that A sealing gasket is provided at the connection between the air suction column and the waste liquid storage cavity.

5. The device according to claim 1, characterized in that Nucleic acid solid phase extraction material is arranged in the reaction chamber.

6. The device according to claim 5, characterized in that The nucleic acid solid phase extraction material is magnetic beads.

7. The device according to claim 1, characterized in that The nucleic acid extraction element includes: a lysis solution chamber for adding and storing a mixture of a sample and a lysis solution; or a cleaning liquid chamber for adding and storing cleaning liquid; or an eluent chamber, used for adding and storing eluent; Wherein, one or more of the lysis liquid cavity, the cleaning liquid cavity or the elution liquid cavity are respectively connected to the reaction chamber fluid.

8. The device according to claim 7, characterized in that The cleaning liquid cavity includes a primary cleaning liquid cavity, a secondary cleaning liquid cavity and a tertiary cleaning liquid cavity. One or more of the primary cleaning liquid cavity, the secondary cleaning liquid cavity or the tertiary cleaning liquid cavity are respectively connected to the reaction chamber fluid.

9. The device according to any one of claims 1 to 8, characterized in that: The device comprises a microfluidic channel for fluid communication with a reaction chamber, a waste liquid storage chamber, a nucleic acid extraction element or a nucleic acid amplification element.

10. The device according to claim 9, characterized in that The microfluidic channels are radially extended and distributed on a rotating disk, and the reaction chamber, the waste liquid storage chamber, the nucleic acid extraction element or the nucleic acid amplification element are connected or disconnected by the rotation of the rotating disk.

11. The device according to claim 1, characterized in that The fluid exchange is achieved through microfluidic channels.

12. The device according to claim 1, wherein When the reaction chamber is in fluid communication with the nucleic acid extraction element, the waste liquid storage chamber and the reaction chamber are not in fluid communication; when the waste liquid storage chamber and the reaction chamber are in fluid communication, the reaction chamber and the nucleic acid extraction element are not in fluid communication.

13. The device according to claim 12, wherein The device comprises a piston, and the movement of the piston allows the fluid in the nucleic acid extraction element to enter the reaction chamber.

14. The device according to claim 13, wherein The movement of the piston is upward along the reaction chamber.

15. The device according to claim 14, wherein The device also includes an exhaust element for generating negative pressure in the waste liquid storage chamber, thereby allowing the fluid in the reaction chamber to enter the waste liquid storage chamber.

16. The device according to claim 15, wherein The negative pressure drives the piston to move downward along the reaction chamber.

17. The device according to claim 16, wherein The exhaust element for generating negative pressure in the waste liquid storage chamber is an air suction column connected to the waste liquid storage chamber.

18. A method for extracting nucleic acid, comprising the following steps: (1) A nucleic acid extraction device is provided, comprising: Nucleic acid extraction element, used for extracting nucleic acid; The waste liquid storage chamber is used to store waste liquid during the reaction process; a reaction chamber selectively communicating with the nucleic acid extraction element and the waste liquid storage chamber; The nucleic acid extraction device allows fluid to enter the waste liquid storage chamber from the reaction chamber by reducing the air pressure in the waste liquid storage chamber. The nucleic acid extraction device reduces the air pressure in the waste liquid storage chamber by an exhaust element connected to the waste liquid storage chamber, and one end of the exhaust element is exposed outside the nucleic acid extraction device and is used to connect to an exhaust device. (2) exchanging fluid between the reaction chamber and the waste liquid storage chamber or the nucleic acid extraction element to extract nucleic acid; The method also includes a step of nucleic acid amplification, wherein the reaction chamber is fluidically connected to a nucleic acid amplification element to achieve nucleic acid amplification, wherein the nucleic acid amplification element is a PCR reaction liquid cavity fluidically connected to the reaction chamber, and reagents required for the PCR reaction are arranged therein; the nucleic acid amplification element also includes a PCR reaction tube fluidically connected to the PCR reaction liquid cavity, allowing fluid to enter the PCR reaction tube for an amplification reaction; the PCR reaction tube is fluidically connected via an injection hole and an exhaust hole arranged on the side wall of the PCR reaction liquid cavity; the PCR reaction liquid cavity includes a primary PCR reaction liquid cavity and a secondary PCR reaction liquid cavity, wherein the primary PCR reaction liquid cavity is fluidically connected to the reaction chamber, wherein the injection hole is arranged at the bottom of the side wall of the primary PCR reaction liquid cavity, and the exhaust hole is arranged at the upper part of the side wall of the secondary PCR reaction liquid cavity.

19. The method according to claim 18, characterized in that Fluid is exchanged between the reaction chamber, the waste liquid storage chamber or the nucleic acid extraction element through pressure changes between the waste liquid storage chamber and the reaction chamber.

20. The method according to claim 18, wherein The exhaust element is a cylindrical air suction column.

21. The method according to claim 18, wherein A nucleic acid solid phase extraction material is arranged in the reaction chamber, and the nucleic acid in the sample is bound to the nucleic acid solid phase extraction material.

22. The method according to claim 21, characterized in that The nucleic acid solid phase extraction material is magnetic beads.

23. The method according to claim 18, wherein The nucleic acid extraction element comprises: a lysis solution chamber, used for adding and storing a mixture of sample and lysis solution; or a cleaning liquid chamber for adding and storing cleaning liquid; or an eluent chamber, used for adding and storing eluent; One or more of the lysis liquid cavity, the cleaning liquid cavity or the elution liquid cavity are respectively connected to the reaction chamber fluid.

24. The method according to claim 23, wherein The cleaning liquid cavity includes a primary cleaning liquid cavity, a secondary cleaning liquid cavity and a tertiary cleaning liquid cavity, and one or more of the primary cleaning liquid cavity, the secondary cleaning liquid cavity or the tertiary cleaning liquid cavity are respectively connected to the reaction chamber fluid.

25. The method according to any one of claims 18 to 24, characterized in that The microfluidic channel arranged in the nucleic acid extraction device enables fluid communication or exchange between the reaction chamber, the waste liquid storage chamber, the nucleic acid extraction element or the nucleic acid amplification element.

26. The method according to claim 25, characterized in that The microfluidic channels are radially extended and distributed on a rotating disk, and the reaction chamber, the waste liquid storage chamber, the nucleic acid extraction element or the nucleic acid amplification element are connected or disconnected through the rotation of the rotating disk.

27. The method according to claim 18, wherein Fluid exchange is achieved through microfluidic channels.

28. The method according to claim 18, wherein When the reaction chamber is in fluid communication with the nucleic acid extraction element, the waste liquid storage chamber and the reaction chamber are not in fluid communication; when the waste liquid storage chamber and the reaction chamber are in fluid communication, the reaction chamber and the nucleic acid extraction element are not in fluid communication.

29. The method according to claim 28, wherein The fluid in the nucleic acid extraction element enters the reaction chamber through the movement of a piston arranged in the reaction chamber.

30. The method according to claim 29, wherein The movement of the piston is upward along the reaction chamber.

31. The method according to claim 30, wherein The fluid in the reaction chamber is allowed to enter the waste liquid storage chamber through an exhaust element which generates negative pressure in the waste liquid storage chamber.

32. The method according to claim 31, wherein The exhaust element for generating negative pressure in the waste liquid storage chamber is an air suction column connected to the waste liquid storage chamber, and the negative pressure drives the piston to move downward along the reaction chamber.

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