A centrifugal fully integrated nucleic acid detection microfluidic chip and nucleic acid detection method thereof

Abstract

The present invention provides a centrifugal fully integrated nucleic acid detection microfluidic chip and a nucleic acid detection method thereof, which relate to the field of in vitro diagnosis. The microfluidic chip of the present invention mainly adds a plurality of cleaning liquid distribution pools. Compared with the conventional technology in which the cleaning liquid is instantly transferred from the nucleic acid capture pool during the centrifugation process, the microfluidic chip of the present invention cleverly utilizes the size of the centrifugal force and is designed for small-scale and multiple graded cleaning, thereby enhancing the cleaning efficiency and avoiding the inhibitory effects of the lysis solution and the sample matrix on amplification. Moreover, the microfluidic chip of the present invention integrates the functions of sample lysis, nucleic acid purification and amplification detection, and only needs to add the sample to the chip to complete the above-mentioned entire analysis process in the fully sealed chip. The microfluidic chip of the present invention has a simple structure and process, can detect multiple samples and multiple indicators in parallel, and is particularly suitable for the field of in vitro diagnosis POCT.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnosis, and in particular to a centrifugal fully integrated nucleic acid detection microfluidic chip and a nucleic acid detection method thereof. Background Art

[0002] Molecular diagnosis is the only in vitro diagnostic method that can diagnose, prevent, and customize treatment plans for diseases at an early stage. Among them, nucleic acid detection based on polymerase chain reaction (PCR) is the gold standard for molecular diagnosis and has important clinical value. Traditional PCR reactions need to be completed in specialized partitioned laboratories to avoid aerosol contamination, and they are highly dependent on professional equipment and personnel, with extremely high construction and maintenance costs.

[0003] Microfluidic technology has the ability to integrate all functions such as sample pre-treatment, reaction, and detection on a centimeter-sized chip. In recent years, molecular POCT (Point-of-Care Testing) chips developed based on microfluidic technology can effectively solve the above-mentioned pain points of PCR.

[0004] However, the nucleic acid detection process involves complex liquid manipulation steps such as sample lysis, nucleic acid extraction and purification, amplification, and detection, and the entire process needs to be completed in a fully sealed chip environment to avoid aerosol contamination. At present, most technical solutions use pressure-driven card box microfluidic chips to achieve this, integrating multiple micro pumps and micro valves to control the fluid in sequence. The chip composition structure is complex, the supporting peripheral equipment is expensive, and the stability is poor.

[0005] Centrifugal-driven microfluidic chips are simpler than pressure-driven ones. The fluid can be manipulated in different areas of the chip by the centrifugal direction and speed, and have the advantage of a simple assembly structure. However, because it is inconvenient to control the mechanical valve synchronously during the centrifugal process, there are fewer valves for fluid control on centrifugal chips, which directly increases the difficulty of applying such chips in nucleic acid testing and becomes a technical difficulty.

[0006] Patent CN118185746A discloses a fully integrated microfluidic nucleic acid analysis chip and a nucleic acid analysis method, which integrates the steps of nucleic acid detection on a centrifugal microfluidic chip, but the sample and lysate need to be mixed manually outside the chip before adding. The liquid reagent is in a liquid pool formed by hollowing out the chip. The reagent pools are interconnected and cannot serve as a storage function. In fact, manual addition is still required before use. It does not really meet the on-site analysis needs of "sample in - result out".

[0007] Patent CN118048231A discloses a centrifugal nucleic acid detection microfluidic chip. The internal design structure of the entire centrifugal microfluidic chip is complex, but the front and rear liquid pools of the detection process are all controlled by siphon valves. The siphon valve itself is affected by the rotation speed, liquid properties and chip surface properties. The patent only relies on the four-stage siphon valve to accurately control all processes, which is essentially difficult to achieve. In addition, the nucleic acid extraction and purification process of the sample has a great impact on the accuracy of the results, but even if the chip structure in the patent is complex, this part of the function is not integrated.

[0008] Patent CN118146930A discloses a single-disc full-extraction chip based on centrifugal microfluidics, which pre-embeds the liquid reagents required in the nucleic acid detection process in the form of liquid capsules and integrates the nucleic acid extraction and purification process. However, after the reaction reagents in each step are released from the liquid capsules, they directly pass through the buffer cavity and the silica gel membrane area into the waste liquid pool under the action of centrifugation. On the one hand, the reagents and samples do not have sufficient mixing time. On the other hand, the transfer of liquid during the centrifugation process is completed instantly. In this short period of time, it is difficult to ensure that the cleaning solution or eluent can fully clean the nucleic acid on the silica membrane.

[0009] In summary, it is necessary to develop a new type of centrifugal fully integrated nucleic acid detection microfluidic chip and nucleic acid detection method thereof, with a focus on adding multiple cleaning liquid distribution pools to achieve graded cleaning, further reducing the impact of reagents or impurity residues on the detection results during the extraction process. Summary of the invention

[0010] In view of the problems existing in the prior art, the present invention provides a centrifugal fully integrated nucleic acid detection microfluidic chip and a nucleic acid detection method thereof. The microfluidic chip of the present invention mainly adds a plurality of cleaning liquid distribution pools. Compared with the conventional technology in which the cleaning liquid is instantly transferred from the nucleic acid capture pool during the centrifugation process, the microfluidic chip of the present invention cleverly utilizes the size of the centrifugal force and is designed for small-scale and multiple graded cleaning, thereby enhancing the cleaning efficiency and avoiding the inhibitory effects of the lysis solution and the sample matrix on the amplification. Moreover, the microfluidic chip of the present invention integrates the functions of sample lysis, nucleic acid purification, and amplification detection. It only needs to add the sample to the chip to complete the above-mentioned entire analysis process in a fully sealed chip. The microfluidic chip of the present invention has a simple structure and process, and can detect multiple samples and multiple indicators in parallel, and is particularly suitable for the field of in vitro diagnosis POCT.

[0011] To achieve this object, the present invention adopts the following technical solutions:

[0012] One of the purposes of the present invention is to provide a centrifugal fully integrated nucleic acid detection microfluidic chip, comprising a reagent layer located at an upper layer and a structural layer located at a lower layer, wherein the upper and lower layers are arranged around the same centrifugal axis and are tightly combined;

[0013] The reagent layer includes at least one group of liquid capsule reagent components, and the liquid capsule reagent components include a lysing liquid capsule, a cleaning liquid capsule and an eluent liquid capsule; the reagent layer is also provided with a sample addition tube, and an air hole is provided next to the sample addition tube to ensure smooth entry of fluid during the sample addition process;

[0014] The structural layer includes at least one group of sample lysis function modules, nucleic acid extraction and purification function modules, and amplification function modules distributed in sequence along the radial direction; the sample lysis function module includes a sample pool, a lysis solution pool, and a mixing pool, and the sample pool and the lysis solution pool are independently connected to the mixing pool; the nucleic acid extraction and purification function module includes a nucleic acid capture pool, an eluent pool, a total cleaning solution pool, a sorting pool, a buffer pool, and an extraction waste liquid pool, the mixing pool is connected to the nucleic acid capture pool, and the eluent pool is connected to the nucleic acid capture pool; the total cleaning solution pool is respectively connected to at least two cleaning solution distribution pools, at least two of the cleaning solution distribution pools are distributed and connected in sequence along the radial direction, and the cleaning solution distribution pool with the farthest radial distribution is connected to the nucleic acid capture pool; the nucleic acid capture pool is connected to the sorting pool, and the left and right sides of the sorting pool are respectively connected to the extraction waste liquid pool and the buffer pool; the amplification function module is connected to the buffer pool for performing quantitative and amplification reactions;

[0015] The sample loading tube, lysis liquid capsule, cleaning liquid capsule, and eluent liquid capsule located in the upper reagent layer correspond to the sample pool, lysis liquid pool, total cleaning liquid pool, and eluent pool located in the lower structural layer, respectively, so that the sample liquid in the sample loading tube, the lysis liquid in the lysis liquid capsule, the cleaning liquid in the cleaning liquid capsule, and the eluent in the eluent liquid capsule can enter the corresponding liquid pools respectively.

[0016] The microfluidic chip of the present invention mainly adds multiple cleaning liquid distribution pools. Compared with the conventional technology in which the cleaning liquid is instantly transferred from the nucleic acid capture pool during centrifugation, the microfluidic chip of the present invention cleverly uses the size of the centrifugal force and is designed for small-scale and multiple graded cleaning, thereby enhancing the cleaning efficiency and avoiding the inhibitory effects of the lysis solution and the sample matrix on amplification. Moreover, the microfluidic chip of the present invention integrates the functions of sample lysis, nucleic acid purification, and amplification detection. It only needs to add the sample to the chip to complete the above-mentioned entire analysis process in a fully sealed chip. The microfluidic chip of the present invention has a simple structure and process, and can perform multi-sample and multi-index detection in parallel, and is particularly suitable for the field of in vitro diagnosis POCT.

[0017] It should be noted that in order to achieve parallel multi-sample and multi-index detection, two or more groups of liquid capsule reagent components can be distributed symmetrically along the radial direction on the reagent layer. Accordingly, two or more groups of sample lysis function modules, nucleic acid extraction and purification function modules, and amplification function modules distributed in sequence along the radial direction are opened on the structural layer.

[0018] It should be noted that in order to achieve the upper and lower layers being arranged around the same centrifugal axis and tightly combined, the reagent layer and the structural layer are independently provided with a central rotating ring and a positioning slot; the sample addition tube is often designed close to the liquid capsule reagent component to reduce the reagent flow path; unless otherwise specified in the present invention, the structural layers are all connected through microchannels.

[0019] As a preferred technical solution of the present invention, the amplification function module includes a quantitative pool, an amplification pool, and an amplification waste liquid pool; at least one quantitative pool is arranged along the circumference, and the quantitative pools are interconnected; the quantitative pool farthest from the buffer pool is connected to the amplification waste liquid pool; the quantitative pools correspond to the amplification pools one by one and are distributed in sequence along the radial direction;

[0020] The mixing pool is connected to the nucleic acid capture pool through a first siphon valve; the quantitative pool closest to the buffer pool is connected to the buffer pool through a second siphon valve; the quantitative pool is connected to the amplification pool through a flow resistance valve, the flow resistance valve includes an inlet channel, a breakthrough valve port and an overflow channel, the inlet channel is located at the bottom of the quantitative pool and enables the bottom of the quantitative pool to be connected to the breakthrough valve port, the overflow channel is located at the top of the amplification pool and enables the top of the amplification pool to be connected to the breakthrough valve port, and there is gas in the breakthrough valve port.

[0021] The flow resistance valve structure commonly used in centrifugal chips in the prior art is a flow channel with a small cross-section. However, on the outside of a circle with a larger radius, it is difficult to prevent the liquid from being released prematurely even under low-speed conditions. The present invention designs a gas chamber in the middle of the flow resistance valve, namely a breakthrough valve port, and adopts a bottom-in and top-out form. Only when the fluid breaks through the air resistance of the gas-filled chamber at high speed can it pass through the inlet channel, breakthrough valve port and overflow channel of the flow resistance valve in sequence and enter the corresponding amplification pool. That is, the flow resistance valve described in the present invention can achieve effective blocking.

[0022] As a preferred technical solution of the present invention, the structural layer also includes at least one gas buffer pool, and the gas buffer pool is connected to the extraction waste liquid pool, at least two of the cleaning liquid distribution pools, and the amplification waste liquid pool through gas channels respectively, and the mixing pool is connected to the extraction waste liquid pool through a gas channel, so that gas-liquid circulation is achieved inside the entire chip.

[0023] It should be noted that the structural layer of the present invention also includes at least one gas buffer pool, which is arranged in the area closest to the center of the circle. Through the setting of the gas channel, all liquid pools can be connected to the gas buffer pool, so that during the transfer of liquid between different liquid pools, gas pressure balance can be achieved, preventing the influence of positive or negative pressure on circulation control, and realizing precise control of the fluid in a sealed internal environment.

[0024] As a preferred technical solution of the present invention, a switching valve is provided at the connection between the nucleic acid capture pool and the sorting pool, and is located inside the sorting pool. A switching valve is provided at the connection between the nucleic acid capture pool and the sorting pool, and the switching valve can realize the switching between the following two states:

[0025] State 1: The nucleic acid capture pool is connected to the extraction waste liquid pool through the sorting pool;

[0026] State 2: The nucleic acid capture pool is connected to the buffer pool through the sorting pool.

[0027] Optionally, the nucleic acid capture pool is a silicon membrane pool provided with a silica gel membrane.

[0028] It should be noted that if no switching valve is provided inside the sorting pool, the sorting pool can be designed as a structure with two left and right branch chambers, and the left and right branch chambers are respectively connected to the extraction waste liquid pool and the buffer pool. By adjusting the forward / reverse centrifugal rotation, the state switching of the nucleic acid capture pool being connected to the extraction waste liquid pool and the buffer pool respectively through the sorting pool can also be achieved.

[0029] As a preferred technical solution of the present invention, the lysis liquid pool, the total cleaning liquid pool, and the eluent pool are respectively provided with liquid capsule puncture needles, so that under the action of pressing, the sealed aluminum foil located in the upper liquid capsule can be punctured and the internal reagent can be released.

[0030] As a preferred technical solution of the present invention, a water-absorbing material, such as absorbent cotton or the like, is arranged in the waste liquid extraction pool to increase the liquid holding volume and prevent the waste liquid from flowing back.

[0031] The second object of the present invention is to provide a nucleic acid detection method for non-medical diagnosis purposes based on the centrifugal fully integrated nucleic acid detection microfluidic chip described in the first object, wherein the lysate capsule contains a lysate, the cleaning liquid capsule contains a cleaning liquid, the eluent capsule contains an eluent, and the buffer pool is pre-embedded with freeze-dried enzyme balls;

[0032] The nucleic acid detection method comprises the following steps:

[0033] Step S1: injecting the sample liquid into the sample pool of the microfluidic chip through the sample tube, and sealing the air hole next to the sample tube, so that the microfluidic chip and the sample tube form a fully enclosed internal environment;

[0034] Step S2: cleavage:

[0035] The microfluidic chip is centrifuged to completely transfer the residual sample liquid in the sample tube to the sample pool; the lysis liquid capsule is pressed to release the lysis liquid into the lysis liquid pool, the microfluidic chip is centrifuged to transfer the sample liquid and the lysis liquid to the mixing pool for full mixing and lysis, and the liquid that has completed lysis enters the nucleic acid capture pool to achieve nucleic acid capture; the microfluidic chip is centrifuged to allow the liquid that has completed nucleic acid capture to enter the extraction waste liquid pool from the nucleic acid capture pool through one side of the sorting pool;

[0036] Step S3: Gradual cleaning:

[0037] The cleaning liquid capsule is pressed to release the cleaning liquid into the total cleaning liquid pool, and under the action of the squeezing pressure of the liquid capsule, the cleaning liquid enters at least two cleaning liquid distribution pools respectively, and the microfluidic chip is centrifuged to achieve the cleaning liquid in at least two cleaning liquid distribution pools to enter the nucleic acid capture pool in order from far to near from the center of the circle, and the graded cleaning is completed. The waste liquid after cleaning enters the extraction waste liquid pool from the nucleic acid capture pool through one side of the sorting pool;

[0038] Step S4 Elution:

[0039] The eluent capsule is pressed to release the eluent into the eluent pool; the microfluidic chip is centrifuged in the opposite direction to the graded cleaning, and the eluent passes through the nucleic acid capture pool to elute the captured nucleic acid, and enters the buffer pool from the nucleic acid capture pool through the other side of the sorting pool, and the eluted nucleic acid is re-dissolved and mixed with the freeze-dried enzyme pellet in the buffer pool;

[0040] Step S5 Amplification:

[0041] The microfluidic chip is centrifuged and rotated in a direction opposite to the graded cleaning, so that the reconstituted and mixed liquid enters the amplification function module for quantitative and amplification reactions.

[0042] The nucleic acid detection method described in the present invention adopts the aforementioned centrifugal fully integrated nucleic acid detection microfluidic chip to perform the nucleic acid detection method, which is for non-medical diagnostic purposes. After the sample to be tested is added, the chip environment is completely closed, and there is no aerosol pollution problem. One chip can detect multiple samples at the same time, and all reagents for nucleic acid extraction and purification are integrated. The entire analysis process is completed only by the drive of centrifugal force; in particular, based on the structural design of multiple cleaning liquid distribution pools, graded cleaning with a small amount of cleaning liquid and multiple times can be achieved, thereby enhancing the cleaning efficiency and avoiding the inhibitory effects of the lysate and sample matrix on amplification.

[0043] As a preferred technical solution of the present invention, in step S2, after the lysis is completed, the centrifugal rotation of the microfluidic chip is stopped, and under the capillary action, the lysed liquid breaks through the first siphon valve and enters the nucleic acid capture pool from the mixing pool;

[0044] In step S5, after the re-dissolving and mixing is completed, the centrifugal rotation of the microfluidic chip is stopped, and under the capillary action, the re-dissolving and mixing liquid breaks through the second siphon valve and enters the quantitative pool of the amplification function module from the buffer pool;

[0045] The microfluidic chip is centrifuged in a direction opposite to the graded cleaning, and the eluted samples are distributed to at least one quantitative pool, and the excess eluted samples enter the amplification waste liquid pool; the gas resistance of the breakthrough valve port of the flow resistance valve is broken through by high-speed centrifugation, so that the eluted samples in each quantitative pool pass through the inlet channel, the breakthrough valve port and the overflow channel of the flow resistance valve in turn, and enter the corresponding amplification pool to complete the quantitative and amplification reactions.

[0046] As a preferred technical solution of the present invention, in step S2, the sample solution and the lysate are transferred to a mixing pool for full mixing and lysis through an instrument ultrasonic module and / or forward and reverse reciprocating centrifugation.

[0047] As a preferred technical solution of the present invention, in step S4, the eluted nucleic acid is reconstituted and mixed with the freeze-dried enzyme pellets in the buffer pool by reciprocating forward and reverse centrifugation.

[0048] Compared with the prior art solutions, the present invention has at least the following beneficial effects:

[0049] (1) The microfluidic chip of the present invention integrates sample lysis, nucleic acid purification, and amplification detection functions. It only needs to add the sample to the chip to complete the above-mentioned entire analysis process in a fully sealed chip. The microfluidic chip of the present invention has a simple structure and process, and can perform multi-sample and multi-index detection in parallel, and is particularly suitable for the field of in vitro diagnostic POCT.

[0050] (2) Cleaning design with step-by-step release of cleaning solution: The microfluidic chip of the present invention mainly adds multiple cleaning solution distribution pools. Compared with the conventional technology in which the cleaning solution is instantly transferred from the nucleic acid capture pool during centrifugation, the microfluidic chip of the present invention cleverly utilizes the centrifugal force and is designed to perform graded cleaning in small amounts and multiple times, thereby enhancing the cleaning efficiency and avoiding the inhibitory effects of the lysate and sample matrix on amplification.

[0051] (3) Internal gas-liquid circulation design of the chip: The microfluidic chip of the present invention is designed with at least one gas buffer pool in the structural layer, and is arranged in the area closest to the center of the circle. Through the arrangement of the gas channel, all liquid pools can be connected to the gas buffer pool, so that during the transfer of liquid between different liquid pools, gas pressure balance can be achieved, preventing the influence of positive or negative pressure on the circulation control, and realizing precise control of the fluid in a sealed internal environment;

[0052] (4) High-efficiency flow resistance valve design: The flow resistance valve structure commonly used in centrifugal chips in the prior art is a flow channel with a small cross-section. However, on the outside of a circle with a large radius, it is difficult to prevent the liquid from being released prematurely even under low-speed conditions. The present invention designs a gas chamber in the middle of the flow resistance valve, namely, a breakthrough valve port, and adopts a bottom-in and top-out form. Only when the fluid breaks through the air resistance of the gas chamber at high speed can it pass through the inlet channel, breakthrough valve port and overflow channel of the flow resistance valve in sequence and enter the corresponding amplification pool. That is, the flow resistance valve described in the present invention can achieve effective isolation. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 1 is a schematic diagram of the structure of the centrifugal fully integrated nucleic acid detection microfluidic chip described in Example 1 of the present invention;

[0054] Figure 2 yes Figure 1 Exploded diagram of the structure;

[0055] Figure 3 yes Figure 1 Schematic diagram of the reagent layer;

[0056] Figure 4 yes Figure 1 Schematic diagram of the middle structural layer;

[0057] Figure 5 is a schematic structural diagram of the flow resistance valve described in Example 1 of the present invention;

[0058] Figure 6 This is a schematic diagram of the structure of the centrifugal fully integrated nucleic acid detection microfluidic chip with a switching valve as described in Example 2 of the present invention;

[0059] Figure 7 It is a schematic diagram of the structural layers in the centrifugal fully integrated nucleic acid detection microfluidic chip described in Example 3 of the present invention;

[0060] Figure 8 This is a comparison diagram of the cleaning effect represented by the pigment dry dots on the silicon membrane in the nucleic acid capture pool of the present invention;

[0061] Fig. 9 It is a comparison diagram of the interception effect of the quantitative pool liquid according to the present invention;

[0062] In the figure: 1-reagent layer; 2-structural layer; 11-rotating ring; 12-positioning card slot; 13-sample tube; 14-air hole; 15-lysate liquid capsule; 16-elution liquid capsule; 17-cleaning liquid capsule; 21-gas buffer pool; 22-sample pool; 23-lysate pool; 24-total cleaning liquid pool; 25-capsule puncture needle; 26-elution liquid pool; 27-mixing pool; 28-extraction waste liquid pool; 29-cleaning liquid separation pool Distribution pool; 30-first siphon valve; 31-second siphon valve; 32-nucleic acid capture pool; 33-sorting pool; 34-buffer pool; 35-quantification pool; 36-flow resistance valve; 37-amplification pool; 38-switching valve; 39-amplification waste liquid pool; 291-first distribution pool flow channel; 292-second distribution pool flow channel; 293-third distribution pool flow channel; 361-entrance channel; 362-breakthrough valve port; 363-overflow channel. DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below with reference to the accompanying drawings and through specific implementation methods.

[0064] To better illustrate the present invention and facilitate understanding of the technical solution of the present invention, typical but non-limiting embodiments of the present invention are as follows:

[0065] Example 1

[0066] This embodiment provides a centrifugal fully integrated nucleic acid detection microfluidic chip, such as Figure 1-Figure 4 As shown, it includes a reagent layer 1 located at the upper layer and a structural layer 2 located at the lower layer, and the upper and lower layers are arranged around the same centrifugal shaft and are tightly combined; the sample loading layer 1 is composed of a circular plastic disc and an assembly part thereon, with a rotating ring 11 distributed in the center, and two positioning slots 12 are symmetrically distributed on both sides of the rotating ring 11, which are matched and fixed with the centrifugal shaft of the peripheral device; the structural layer 2 includes all fluid functional structures in the nucleic acid detection process, and a rotating ring 11 of the same size as the rotating ring 11 in the reagent layer 1 is distributed at the center of the circle, and two positioning slots 12 are symmetrically distributed on both sides of the rotating ring 11, which are matched and fixed with the centrifugal shaft of the peripheral device;

[0067] The reagent layer 1 includes two groups of symmetrically distributed liquid capsule reagent components, and the liquid capsule reagent components include a lysing liquid capsule 15, a cleaning liquid capsule 17 and an eluent liquid capsule 16; the reagent layer 1 is also provided with a sample addition tube 13 near the center of the circle, and an air hole 14 is provided next to the sample addition tube 13 to ensure smooth entry of fluid during the sample addition process;

[0068] The structural layer 2 includes two groups of sample lysis functional modules, nucleic acid extraction and purification functional modules, and amplification functional modules distributed in sequence along the radial direction; the sample lysis functional module includes a sample pool 22, a lysis liquid pool 23, and a mixing pool 27, and the sample pool 22 and the lysis liquid pool 23 are independently connected to the mixing pool 27; the nucleic acid extraction and purification functional module includes a nucleic acid capture pool 32, an eluent pool 26, a total cleaning liquid pool 24, a sorting pool 33, a buffer pool 34, and an extraction waste liquid pool 28, the mixing pool 27 is connected to the nucleic acid capture pool 32, and the eluent pool 26 is connected to the nucleic acid capture pool 32; the total cleaning liquid pool 24 is connected to three cleaning liquid distribution pools 29, respectively, and the three cleaning liquid distribution pools 29 are distributed and connected in sequence along the radial direction, and the cleaning liquid distribution pool 29 farthest in the radial distribution is connected to the nucleic acid capture pool 32 ; The nucleic acid capture pool 32 is connected to the sorting pool 33, and the sorting pool 33 is designed to have a structure with two left and right branch chambers, and the left and right branch chambers are respectively connected to the extraction waste liquid pool 28 and the buffer pool 34; the amplification function module is located at the outermost periphery of the chip, including eight quantitative pools 35, eight amplification pools 37, and one amplification waste liquid pool 39; the eight quantitative pools 35 are arranged along the circumference, and the quantitative pools 35 are interconnected; the quantitative pool 35 farthest from the buffer pool 34 is connected to the amplification waste liquid pool 39; the quantitative pools 35 correspond to the amplification pools 37 one by one and are distributed in sequence along the radial direction; the amplification pool 37 is pre-embedded with probe primers of different indicators, and combined with the multi-color fluorescence channels of peripheral instruments, multi-indicator parallel detection can be realized; and according to the detection requirements, the quantitative pools 35 and the amplification pools 37 can be designed in different numbers;

[0069] The mixing pool 27 is connected to the nucleic acid capture pool 32 through a first siphon valve 30; the quantitative pool 35 closest to the buffer pool 34 is connected to the buffer pool 34 through a second siphon valve 31; the quantitative pool 35 is connected to the amplification pool 37 through a flow resistance valve 36. Figure 5 As shown, the flow resistance valve 36 includes an inlet channel 361, a breakthrough valve port 362 and an overflow channel 363. The inlet channel 361 is located at the bottom of the quantitative pool 35 and makes the bottom of the quantitative pool 35 communicate with the breakthrough valve port 362. The overflow channel 363 is located at the top of the amplification pool 37 and makes the top of the amplification pool 37 communicate with the breakthrough valve port 362. There is gas in the breakthrough valve port 362.

[0070] The structural layer 2 has two gas buffer pools 21 arranged close to the center of the circle. The gas buffer pool 21 is connected to the extraction waste liquid pool 28, the three cleaning liquid distribution pools 29, and the amplification waste liquid pool 39 through gas channels, respectively. The mixing pool 27 is connected to the extraction waste liquid pool 28 through a gas channel, so that gas-liquid circulation is achieved inside the entire chip to form an internal gas-liquid balance.

[0071] Among them, the sample loading tube 13, lysis liquid capsule 15, cleaning liquid capsule 17, and eluent capsule 16 located in the upper reagent layer 1 correspond to the sample pool 22, lysis liquid pool 23, total cleaning liquid pool 24, and eluent pool 26 located in the lower structural layer 2, respectively. Three liquid capsule puncture needles 25 are respectively arranged in the lysis liquid pool 23, the total cleaning liquid pool 24, and the eluent pool 26, so that under the action of pressing, they can pierce the sealed aluminum foil located in the upper liquid capsule and release the internal reagent; absorbent cotton is arranged in the extraction waste liquid pool 28 to increase the liquid holding volume and prevent the waste liquid from flowing back; the nucleic acid capture pool 32 is a silicon membrane pool provided with a silica gel membrane.

[0072] Application Example 1

[0073] This application example provides a nucleic acid detection method for non-medical diagnosis purposes based on the centrifugal fully integrated nucleic acid detection microfluidic chip described in Example 1, wherein the lysing liquid capsule 15 is encapsulated with a lysing liquid, the cleaning liquid capsule 17 is encapsulated with a cleaning liquid, the eluent liquid capsule 16 is encapsulated with an eluent, and the buffer pool 34 is pre-embedded with freeze-dried enzyme balls;

[0074] The nucleic acid detection method comprises the following steps:

[0075] Insert the sample tube 13 with the sample liquid into the microfluidic chip, squeeze the liquid sample in the sample tube 13 into the sample pool 22, and seal the air hole 14 next to the sample tube 13, so that the microfluidic chip and the sample tube 13 form a fully enclosed internal environment; then put the whole into the supporting instrument, set the parameters and start the operation with one button, and the following actions are automatically completed on the instrument:

[0076] Lysis: The microfluidic chip is centrifuged and rotated, and the residual sample liquid in the sample tube 13 is completely transferred to the sample pool 22 under the action of counterclockwise low-speed centrifugation; the instrument pressure rod presses the lysis liquid capsule 15 to release the lysis liquid into the lysis liquid pool 23, and the microfluidic chip is centrifuged and transferred to the mixing pool 27 under the action of counterclockwise medium-speed centrifugation to fully mix and lyse, and the sample reagent is fully mixed and lysed through the instrument ultrasonic module or forward and reverse reciprocating centrifugation; after the lysis is completed, the centrifugal rotation of the microfluidic chip is stopped, and under the action of capillary action, the liquid that has completed the lysis breaks through the first siphon valve 30 and enters the nucleic acid capture pool 32 from the mixing pool 27 to achieve nucleic acid capture; the microfluidic chip is centrifuged and, under the action of counterclockwise high-speed centrifugation, the liquid that has completed the nucleic acid capture enters the extraction waste liquid pool 28 from the nucleic acid capture pool 32 through the left side of the sorting pool 33;

[0077] Gradual cleaning: Press the cleaning liquid capsule 17 to release the cleaning liquid into the total cleaning liquid pool 24. At the same time, under the action of the capsule squeezing pressure, the cleaning liquid enters three radially connected cleaning liquid distribution pools 29 respectively. Then, the microfluidic chip is centrifuged. Under the counterclockwise medium-speed centrifugation, because the centrifugal force is related to the radius of the liquid pool from the center of the circle and the centrifugal speed, the liquids in the three cleaning liquid distribution pools 29 from far to near the center of the circle are transferred to the nucleic acid capture pool 32 in sequence according to the centrifugal speed from small to large, completing small and multiple graded cleaning to enhance the cleaning efficiency. The waste liquid after cleaning enters the extraction waste liquid pool 28 from the nucleic acid capture pool 32 through the left side of the sorting pool 33;

[0078] Elution: Press the eluent capsule 16 to release the eluent into the eluent pool 26, and rotate the microfluidic chip centrifugally. Under the action of clockwise high-speed centrifugation, the eluent passes through the nucleic acid capture pool 32 to elute the captured nucleic acid, and enters the buffer pool 34 from the nucleic acid capture pool 32 through the right side of the sorting pool 33; when all the eluents are transferred, the eluent is reconstituted and mixed with the freeze-dried enzyme balls in the buffer pool 34 by reciprocating forward and reverse low-speed rotation; after the reconstitution and mixing are completed, the centrifugal rotation of the microfluidic chip is stopped, and under the action of capillary action, the reconstituted and mixed liquid breaks through the second siphon valve 31, and enters the eight quantitative pools 35 of the amplification function module from the buffer pool 34, and the excess eluted sample enters the amplification waste liquid pool 39;

[0079] Amplification: The microfluidic chip is centrifuged and rotated in a clockwise high-speed centrifugal force to break through the gas resistance of the breakthrough valve port 362 of the flow resistance valve 36, so that the eluted samples in each quantitative pool 35 pass through the inlet channel 361, the breakthrough valve port 362 and the overflow channel 363 of the flow resistance valve 36 in turn, and enter the corresponding amplification pool 37. After being mixed with the probe primer dry powder reagent in the amplification pool 37, the amplification is completed under the circulating temperature control conditions provided by the instrument.

[0080] The centrifugal fully integrated nucleic acid detection microfluidic chip described in Example 1 is used. In the nucleic acid detection method for non-medical diagnosis purposes performed in this application example, on the one hand, the volume of the total cleaning solution is 300 μL. Based on the depth of the color of the residual pigment, it can be judged that the efficiency of the graded cleaning is excellent. On the other hand, during the amplification process, the chip rotates synchronously at 2000 rpm for 60 s, and the flow resistance valve effectively intercepts the liquid in the quantitative pool and enters the amplification pool in advance.

[0081] Example 2

[0082] This embodiment provides a centrifugal fully integrated nucleic acid detection microfluidic chip. Compared with Embodiment 1, the only difference is that: a switching valve 38 is arranged inside the sorting pool 33 at the connection between the nucleic acid capture pool 32 and the sorting pool 33, and the switching valve 38 can realize the switching between the following two states: State 1: the nucleic acid capture pool 32 is connected to the extraction waste liquid pool 28 through the sorting pool 33; State 2: the nucleic acid capture pool 32 is connected to the buffer pool 34 through the sorting pool 33; Figure 6 As shown, one end of the switching valve 38 away from the nucleic acid capture pool 32 is fixed, and the other end is swingable.

[0083] Application Example 2

[0084] This application example provides a nucleic acid detection method for non-medical diagnosis purposes based on the centrifugal fully integrated nucleic acid detection microfluidic chip described in Example 2. Compared with Application Example 1, the only difference is that:

[0085] When the chip starts to rotate counterclockwise with high acceleration, the switching valve 38 adheres to the buffer tank 34 and closes the entrance of the buffer tank 34 due to inertia, and the waste liquid is transferred from the left open port to the extraction waste liquid tank 28; when the chip starts to rotate clockwise with high acceleration, the switching valve 38 adheres to the entrance of the extraction waste liquid tank 28 due to inertia, and the eluent is transferred to the buffer tank 34; thereby achieving directional transfer of the liquid.

[0086] The centrifugal fully integrated nucleic acid detection microfluidic chip described in Example 2 is used. In the nucleic acid detection method for non-medical diagnosis purposes performed in this application example, on the one hand, the volume of the total cleaning solution is 300 μL. Based on the depth of the color of the residual pigment, it can be judged that the efficiency of the graded cleaning is excellent. On the other hand, during the amplification process, the chip rotates synchronously at 2000 rpm for 60 s, and the flow resistance valve effectively intercepts the liquid in the quantitative pool and enters the amplification pool in advance. In addition, the switching valve 38 effectively ensures the directional transfer of the liquid, avoiding the loss of the liquid during the directional transfer.

[0087] Example 3

[0088] This embodiment provides a centrifugal fully integrated nucleic acid detection microfluidic chip. Compared with Embodiment 1, the only difference is that the three cleaning liquid distribution pools 29 located in the same radial direction are replaced with three cleaning liquid distribution pools 29 with different radial directions; Figure 7 As shown, the three cleaning liquid distribution pools 29 are distributed on different radii of the microfluidic chip, from near to far from the center of the circle, the cleaning liquid distribution pool 29 with the farthest radial distribution is connected to the nucleic acid capture pool 32, and the cleaning liquid distribution pool 29 with the closest radial distribution is connected to the gas buffer pool 21; the three cleaning liquid distribution pools 29 are adjacent to each other through flow channels with different resistance sizes, and the resistance size is controlled by the flow channel size, that is, the resistance of the third distribution pool flow channel 293 between the cleaning liquid distribution pool 29 with the closest radial distribution and the middle cleaning liquid distribution pool 29 is greater than the resistance of the second distribution pool flow channel 292 between the middle cleaning liquid distribution pool 29 and the cleaning liquid distribution pool 29 with the farthest radial distribution, and the resistance of the second distribution pool flow channel 292 between the middle cleaning liquid distribution pool 29 and the cleaning liquid distribution pool 29 with the farthest radial distribution is greater than the resistance of the first distribution pool flow channel 291 between the cleaning liquid distribution pool 29 with the farthest radial distribution and the nucleic acid capture pool 32; the design of the flow channel can further widen the speed difference between graded cleaning and adapt to cleaning liquids with more liquid properties.

[0089] Application Example 3

[0090] This application example provides a nucleic acid detection method for non-medical diagnosis purposes based on the centrifugal fully integrated nucleic acid detection microfluidic chip described in Example 3. Compared with Application Example 1, the only difference is that:

[0091] When the liquid in the total cleaning liquid pool 24 is distributed to the three cleaning liquid distribution pools 29, the chip is first centrifugally rotated at a low speed, and the cleaning liquid in the cleaning liquid distribution pool 29 with the farthest radial distribution is transferred to the nucleic acid capture pool 32 through the first distribution pool flow channel 291 to complete the first cleaning; then the chip is rotated at a medium speed, and the cleaning liquid in the cleaning liquid distribution pool 29 in the middle is transferred to the nucleic acid capture pool 32 through the second distribution pool flow channel 292 and the first distribution pool flow channel 291 in turn to complete the second cleaning; finally, the chip is rotated at a high speed, and the cleaning liquid in the cleaning liquid distribution pool 29 with the closest radial distribution is transferred to the nucleic acid capture pool 32 through the third distribution pool flow channel 293, the second distribution pool flow channel 292, and the first distribution pool flow channel 291 in turn to complete the third cleaning, thereby achieving multi-step effective cleaning.

[0092] Using the centrifugal fully integrated nucleic acid detection microfluidic chip described in Example 3, in the nucleic acid detection method for non-medical diagnosis purposes in this application example, the total volume of the cleaning solution is 300 μL, Figure 8 (a) shows the dye dry spots on the silicon membrane in the nucleic acid capture pool to characterize the cleaning effect, that is, the area indicated by the arrow is lighter in color, indicating that the efficiency of graded cleaning is higher.

[0093] Comparative Example 1

[0094] This comparative example provides a centrifugal fully integrated nucleic acid detection microfluidic chip. Compared with Example 1, the only difference is that the three cleaning liquid distribution pools 29 are completely omitted, that is, the total cleaning liquid pool 24 is directly connected to the nucleic acid capture pool 32.

[0095] Comparative application example 1

[0096] This comparative application example provides a nucleic acid detection method for non-medical diagnostic purposes based on the centrifugal fully integrated nucleic acid detection microfluidic chip described in Comparative Example 1. Compared with Application Example 1, the only difference is that: during the cleaning process, the cleaning liquid in the total cleaning liquid pool 24 is directly transferred to the nucleic acid capture pool 32 at one time to complete a single cleaning, and the waste liquid after cleaning enters the extraction waste liquid pool 28 from the nucleic acid capture pool 32 through the left side of the sorting pool 33.

[0097] Using the centrifugal fully integrated nucleic acid detection microfluidic chip described in Comparative Example 1, in the nucleic acid detection method for non-medical diagnosis purposes performed in this comparative application example, the volume of the total cleaning solution is 300 μL, Figure 8 (b) shows the dye dry spots on the silicon membrane in the nucleic acid capture pool to characterize the cleaning effect, that is, the area indicated by the arrow is darker in color, indicating that the efficiency of a single wash is lower.

[0098] Depend on Figure 8 Comparing (a) and (b), it can be seen that the depth of the residual pigment color can clearly show that the efficiency of graded cleaning is higher than that of single cleaning under the same volume of cleaning liquid.

[0099] Comparative Example 2

[0100] This comparative example provides a centrifugal fully integrated nucleic acid detection microfluidic chip. Compared with Example 1, the only difference is that the flow resistance valve 36 is completely omitted, and the quantitative pool 35 and the amplification pool 37 are connected through a microchannel with a smaller cross-section.

[0101] Comparative Application Example 2

[0102] This comparative application example provides a nucleic acid detection method for non-medical diagnostic purposes based on the centrifugal fully integrated nucleic acid detection microfluidic chip described in Comparative Example 2. Compared with Application Example 1, the only difference is that during the amplification process, the eluted sample in each quantitative pool 35 directly enters the corresponding amplification pool 37 through a microchannel with a smaller cross-section.

[0103] Fig. 9 (a) and (b) respectively show the interception effect of the quantitative pool liquid in Application Example 1 and Comparative Application Example 2. The quantitative pool and the amplification pool of both are distributed at the same radial distance from the center of the circle. Fig. 9(a) corresponds to the flow resistance valve. Fig. 9 (b) corresponds to an ordinary microchannel with a smaller cross-section. It rotates synchronously at 2000 rpm for 60 s. It can be seen that the flow resistance valve can effectively intercept the liquid in the quantitative pool and prevent it from entering the amplification pool prematurely. However, at the same speed, in comparison with Application Example 2, the liquid in the quantitative pool is basically transferred to the amplification pool in advance.

[0104] The present invention illustrates the detailed structural features of the present invention through the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must rely on the above detailed structural features to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the components selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0105] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0106] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0107] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A centrifugal fully integrated nucleic acid detection microfluidic chip, characterized in that: It comprises a reagent layer (1) located at an upper layer and a structural layer (2) located at a lower layer, wherein the upper and lower layers are arranged around the same centrifugal axis and are tightly combined; The reagent layer (1) comprises at least one group of liquid capsule reagent components, wherein the liquid capsule reagent components comprise a lysing liquid capsule (15), a cleaning liquid capsule (17) and an eluent liquid capsule (16); the reagent layer (1) is also provided with a sample addition tube (13), and an air hole (14) is provided next to the sample addition tube (13) to ensure smooth entry of fluid during the sample addition process; The structural layer (2) comprises at least one group of sample lysis functional modules, nucleic acid extraction and purification functional modules, and amplification functional modules, which are sequentially distributed along the radial direction; the sample lysis functional module comprises a sample pool (22), a lysis liquid pool (23), and a mixing pool (27); the sample pool (22) and the lysis liquid pool (23) are independently connected to the mixing pool (27); the nucleic acid extraction and purification functional module comprises a nucleic acid capture pool (32), an eluent pool (26), a total cleaning liquid pool (24), a sorting pool (33), a buffer The nucleic acid capture pool (32) is connected to the mixing pool (27), and the elution pool (26) is connected to the nucleic acid capture pool (32); the total cleaning liquid pool (24) is connected to at least two cleaning liquid distribution pools (29), respectively, and at least two of the cleaning liquid distribution pools (29) are sequentially distributed and connected in radial direction, and the cleaning liquid distribution pool (29) with the farthest radial distribution is connected to the nucleic acid capture pool (32); the nucleic acid capture pool (32) is connected to the sorting pool (3 3), the left and right sides of the sorting pool (33) are respectively connected to the extraction waste liquid pool (28) and the buffer pool (34); the amplification function module is connected to the buffer pool (34) for performing quantitative and amplification reactions; the amplification function module includes a quantitative pool (35), an amplification pool (37), and an amplification waste liquid pool (39); at least one quantitative pool (35) is arranged along the circumference, and the quantitative pools (35) are interconnected; the quantitative pool (35) farthest from the buffer pool (34) is connected to the amplification pool (39); The quantitative pool (35) and the amplification pool (37) are connected in a one-to-one correspondence and are sequentially distributed along the radial direction; the structural layer (2) further comprises at least one gas buffer pool (21), the gas buffer pool (21) is connected to the extraction waste liquid pool (28), at least two of the cleaning liquid distribution pools (29), and the amplification waste liquid pool (39) through gas channels, and the mixing pool (27) is connected to the extraction waste liquid pool (28) through a gas channel, so that gas-liquid circulation is achieved inside the entire chip; The sample addition tube (13), lysing solution capsule (15), cleaning solution capsule (17), and eluent capsule (16) located in the upper reagent layer (1) correspond to the sample pool (22), lysing solution pool (23), total cleaning solution pool (24), and eluent pool (26) located in the lower structural layer (2) respectively.

2. The centrifugal fully integrated nucleic acid detection microfluidic chip according to claim 1, characterized in that: The mixing pool (27) is connected to the nucleic acid capture pool (32) via a first siphon valve (30); the quantitative pool (35) closest to the buffer pool (34) is connected to the buffer pool (34) via a second siphon valve (31); the quantitative pool (35) is connected to the amplification pool (37) via a flow resistance valve (36); the flow resistance valve (36) comprises an inlet channel (361), a breakthrough valve port (362) and an overflow channel (363); the inlet channel (361) is located at the bottom of the quantitative pool (35) and enables the bottom of the quantitative pool (35) to be connected to the breakthrough valve port (362); the overflow channel (363) is located at the top of the amplification pool (37) and enables the top of the amplification pool (37) to be connected to the breakthrough valve port (362); and there is gas in the breakthrough valve port (362).

3. The centrifugal fully integrated nucleic acid detection microfluidic chip according to claim 1, characterized in that: Located inside the sorting pool (33), a switching valve (38) is provided at the connection point between the nucleic acid capture pool (32) and the sorting pool (33), and the switching valve (38) can realize switching between the following two states: State 1: the nucleic acid capture pool (32) is connected to the extraction waste liquid pool (28) through the sorting pool (33); State 2: The nucleic acid capture pool (32) is connected to the buffer pool (34) through the sorting pool (33).

4. The centrifugal fully integrated nucleic acid detection microfluidic chip according to claim 1, characterized in that: The lysate pool (23), the total cleaning liquid pool (24), and the eluent pool (26) are each provided with a sac puncture needle (25), so that the sealed aluminum foil located in the upper sac can be punctured and the internal reagent can be released under the action of pressing.

5. The centrifugal fully integrated nucleic acid detection microfluidic chip according to claim 1, characterized in that: A water-absorbing material is arranged in the extraction waste liquid pool (28) to increase the liquid holding volume and prevent the waste liquid from flowing back.

6. A method for nucleic acid detection for non-medical diagnosis purposes based on the centrifugal fully integrated nucleic acid detection microfluidic chip according to any one of claims 1 to 5, characterized in that: The lysing liquid capsule (15) contains a lysing liquid, the cleaning liquid capsule (17) contains a cleaning liquid, the eluent liquid capsule (16) contains an eluent, and the buffer pool (34) contains pre-embedded freeze-dried enzyme spheres; The nucleic acid detection method comprises the following steps: Step S1: injecting the sample liquid into the sample pool (22) of the microfluidic chip through the sample tube (13), and sealing the air hole (14) next to the sample tube (13), so that the microfluidic chip and the sample tube (13) form a fully enclosed internal environment; Step S2: cleavage: The microfluidic chip is centrifuged to completely transfer the residual sample liquid in the sample tube (13) to the sample pool (22); the lysing liquid capsule (15) is pressed to release the lysing liquid into the lysing liquid pool (23); the microfluidic chip is centrifuged to transfer the sample liquid and the lysing liquid to the mixing pool (27) for full mixing and lysing; the lysed liquid enters the nucleic acid capture pool (32) to achieve nucleic acid capture; the microfluidic chip is centrifuged to allow the nucleic acid captured liquid to enter the extraction waste liquid pool (28) from the nucleic acid capture pool (32) through one side of the sorting pool (33); Step S3: graded cleaning: The cleaning liquid capsule (17) is pressed to release the cleaning liquid into the total cleaning liquid pool (24), and under the action of the squeezing pressure of the liquid capsule, the cleaning liquid enters at least two cleaning liquid distribution pools (29) respectively, and the microfluidic chip is centrifuged to achieve the cleaning liquid in the at least two cleaning liquid distribution pools (29) being graded and entering the nucleic acid capture pool (32) in the order of distance from the center of the circle from far to near, so as to complete graded cleaning, and the waste liquid after cleaning enters the extraction waste liquid pool (28) from the nucleic acid capture pool (32) through one side of the sorting pool (33); Step S4 Elution: The eluent capsule (16) is pressed to release the eluent into the eluent pool (26); the microfluidic chip is centrifuged in the opposite direction to the graded cleaning, and the eluent passes through the nucleic acid capture pool (32) to elute the captured nucleic acid, and then passes from the nucleic acid capture pool (32) through the other side of the sorting pool (33) into the buffer pool (34), and the eluted nucleic acid is reconstituted and mixed with the freeze-dried enzyme pellet in the buffer pool (34); Step S5 Amplification: The microfluidic chip is centrifuged in a direction opposite to the graded cleaning, and the eluted sample is distributed into at least one quantitative pool (35), and the excess eluted sample enters the amplification waste liquid pool (39), so that the reconstituted and mixed liquid enters the amplification function module for quantitative and amplification reactions.

7. The nucleic acid detection method according to claim 6, characterized in that: In step S2, after the lysis is completed, the centrifugal rotation of the microfluidic chip is stopped, and the lysed liquid breaks through the first siphon valve (30) under the capillary action and enters the nucleic acid capture pool (32) from the mixing pool (27); In step S5, after the re-dissolving and mixing is completed, the centrifugal rotation of the microfluidic chip is stopped, and under the capillary action, the re-dissolving and mixed liquid breaks through the second siphon valve (31) and enters the quantitative pool (35) of the amplification function module from the buffer pool (34); The microfluidic chip is centrifuged in a direction opposite to the graded cleaning, and the eluted sample is distributed to at least one quantitative pool (35), and the excess eluted sample enters the amplification waste liquid pool (39); the high-speed centrifugation breaks through the gas resistance of the breakthrough valve port (362) of the flow resistance valve (36), so that the eluted sample in each quantitative pool (35) passes through the inlet channel (361), the breakthrough valve port (362) and the overflow channel (363) of the flow resistance valve (36) in sequence, and enters the corresponding amplification pool (37), thereby completing the quantitative and amplification reactions.

8. The nucleic acid detection method according to claim 6, characterized in that: In step S2, the sample solution and the lysate are transferred to a mixing pool for full mixing and lysis through an instrument ultrasonic module and / or forward and reverse reciprocating centrifugation.

9. The nucleic acid detection method according to claim 6, characterized in that: In step S4, the eluted nucleic acid is reconstituted and mixed with the freeze-dried enzyme pellet in the buffer pool by reciprocating forward and reverse centrifugation.

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