An all-integrated microfluidic nucleic acid detection device and detection method

Abstract

The present application relates to the technical field of nucleic acid instant detection, and in particular to a full-integrated microfluidic nucleic acid detection device and a detection method, comprising: a base; a cartridge, which is inserted and installed along the horizontal direction of the base, and at the same time, a rotary valve at the bottom of the cartridge is inserted and installed with a valve driving part installed on the base to realize coaxial rotary connection of the two; a gas path assembly, comprising a syringe pump and a gas pump, the syringe pump drives the fluid in the process of sample lysis, nucleic acid extraction and purification, and nucleic acid amplification by adjusting the air pressure in the central cavity, and the gas pump and a heating part are used to realize drying separation of ethanol remaining after nucleic acid extraction; an ultrasonic assembly; a temperature control assembly; and the technical problems of poor control accuracy and low ultrasonic efficiency caused by the layout problem of the rotary valve and the ultrasonic module, poor sample compatibility and influence on detection effect caused by ethanol remaining in the reagent, and unreasonable temperature control and heat dissipation measures are solved.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid point detection technology, and in particular to a fully integrated microfluidic nucleic acid detection device and detection method. Background Technology

[0002] Molecular diagnostic technologies, especially polymerase chain reaction (PCR)-based nucleic acid detection, have become the "gold standard" for diagnosing infectious diseases due to their high sensitivity and specificity. However, traditional PCR nucleic acid detection procedures typically involve several fragmented steps: 1. Sample pretreatment: lysing samples using centrifugation, heating, or chemical methods in a biosafety cabinet; 2. Nucleic acid extraction and purification: extracting nucleic acids using magnetic beads, silica gel membrane columns, or organic solvents; 3. Reaction system preparation: manually or semi-automatically preparing the amplification reaction system in the PCR preparation area; 4. Amplification and detection: performing amplification and acquiring fluorescence signals in real time using a real-time quantitative PCR instrument. These procedures suffer from the following significant drawbacks: cumbersome and time-consuming operation; the entire process typically takes 3-6 hours and relies on skilled technicians; high risk of contamination: repeated opening, pipetting, and sample transfer can easily generate aerosol contamination, leading to false positives; high laboratory requirements: requiring separate laboratories (reagent preparation area, sample processing area, amplification area), making deployment difficult in primary healthcare institutions or field environments; long result waiting times: failing to meet the immediate testing needs of emergency and outpatient departments.

[0003] The GeneXpert system (Cepheid) is a benchmark product in the field of fully automated nucleic acid detection with a "sample in, result out" process. Its core solution involves: using a disposable microfluidic cartridge to pre-package all reagents required for nucleic acid extraction, purification, amplification, and detection; achieving precise microfluidic control through a rotary valve and piston pump; releasing nucleic acids by lysing sample cells / pathogens using ultrasound; capturing nucleic acids using a silica gel filter membrane, followed by washing and elution before entering the PCR reaction chamber; and completing real-time fluorescence PCR amplification and detection in a closed environment. This system offers advantages such as high integration, ease of operation, and low risk of contamination. However, the aforementioned technologies still have many problems, such as: ① The ultrasonic lysis module and the rotary valve drive module are highly integrated coaxially, which means that the rotary valve drive motor must be connected to the rear via a reduction gear transmission mechanism. The additional reduction gear transmission may increase the transmission error, which is not conducive to the precise control of the rotary valve; ② The ultrasonic module can only act on a small number of samples in the cavity area at the bottom of the rotary valve. Only a portion of the samples can be processed, and after transfer, another portion of the samples are released to the ultrasonic area for lysis, resulting in reduced ultrasonic efficiency; ③ When performing nucleic acid extraction, it is impossible to remove the ethanol residue on the nucleic acid adsorption carrier in the reagent kit. Only alcohol-free or low-alcohol nucleic acid extraction reagents can be used, resulting in poor sample compatibility; ④ The heating and cooling module uses air cooling, which places high demands on the internal air duct layout of the instrument. Furthermore, if multiple amplification channels are added inside the instrument, it is difficult to effectively arrange the space of the air cooling duct. Summary of the Invention

[0004] The purpose of this invention is to provide a fully integrated microfluidic nucleic acid detection device and method to solve the following technical problems commonly found in existing nucleic acid detection devices: poor control accuracy and low ultrasonic efficiency due to unreasonable layout of rotary valve and ultrasonic module; poor sample compatibility and affected detection effect due to ethanol residue in reagents; and unreasonable temperature control and heat dissipation measures.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A fully integrated microfluidic nucleic acid detection device, comprising: Base; The card box is horizontally inserted into the base and installed in place. At the same time, the rotary valve at the bottom of the card box is inserted into the valve drive unit installed on the base to achieve coaxial rotational connection between the two. The card box has an independent central cavity and several functional cavities inside. The rotation of the rotary valve realizes the communication between the central cavity and different functional cavities. The gas path assembly includes an injection pump and an air pump, both mounted on a base, and a hollow plunger that reciprocates and is sealed within the central cavity of the cartridge. The injection pump and the air pump are connected in parallel and then connected in series with a heating element via a solenoid valve, and finally connected to the central cavity via the hollow plunger. The injection pump adjusts the gas pressure within the central cavity to achieve fluid drive during sample lysis, nucleic acid extraction and purification, and nucleic acid amplification. The air pump and the heating element are used to heat the gas entering the central cavity to dry and separate the residual ethanol after nucleic acid extraction. An ultrasound assembly, mounted on a base, includes a reciprocating probe that, when extended, contacts and couples with the sidewall of the outer side of the cartridge to perform lysis of the sample solution within the cartridge. The temperature control component is mounted on the base and includes a fixed temperature control component that is fixedly mounted relative to the base and a movable temperature control component that is slidably mounted relative to the base. When the cartridge is inserted into place, one side of the amplification strip of the cartridge contacts the surface of the fixed temperature control component. The displacement of the movable temperature control component cooperates with the fixed temperature control component to squeeze and contact both sides of the amplification strip of the cartridge, so as to achieve consistent temperature control of the nucleic acid template solution entering the amplification strip. The direction in which the probe of the ultrasound component acts on the cartridge is perpendicular to the insertion direction of the cartridge. The direction in which the hollow plunger moves along the cartridge is perpendicular to the insertion direction of the cartridge and also perpendicular to the direction in which the probe acts on the cartridge. The direction in which the moving temperature control component moves is perpendicular to the insertion direction of the cartridge, while being parallel to the direction in which the probe acts on the cartridge and perpendicular to the plane where the amplification film is located.

[0006] Furthermore, the rotary valve is sealed and rotated on the bottom surface of the cartridge body, and the amplification chip is inserted into the cartridge body. Both the amplification chip and the rotary valve are limited to the cartridge body by vertical insertion and pressing of the cap. The two outer sides of the cap are trapezoidal and are horizontally inserted into the base plate mounted on the base. The bottom surface of the cap and the bottom surface of the rotary valve are both provided with trapezoidal grooves. The rotary valve protrudes from the center of the cap, and the trapezoidal grooves of both constitute a large trapezoidal groove. The base plate is provided with clearance holes for the locking block of the connecting seat to pass through. The locking block is set with a trapezoidal structure. When the cap of the cartridge is inserted and installed on the base plate, the trapezoidal groove of the rotary valve and the locking block of the connecting seat are inserted and engaged. The connecting seat is fixedly installed on the power end of the valve drive unit.

[0007] Furthermore, the card holder's central cavity and the bottom of its functional cavities are both provided with connecting channels penetrating the box body. The top of the rotary valve is provided with a central connecting hole located at the center and a side connecting hole located at the edge. The rotary valve has a connecting channel inside, and the central connecting hole and the side connecting hole are respectively connected to the two ends of the connecting channel. The central connecting hole is connected to the connecting channel at the bottom of the central cavity. The side connecting hole is connected to the connecting channels of different functional cavities by rotating the rotary valve to different angles.

[0008] Furthermore, both the ultrasound component and the moving temperature control component are slidably mounted on the slide plate. The slide plate is fixedly mounted relative to the slide base, and the slide base is slidably mounted on the base. Elastic elements are installed between the ultrasound component, the moving temperature control component, and the upright plate of the slide plate. The elastic elements are used to apply a pre-tightening force to the probe or the moving temperature control component so that the probe is in full contact with the side wall of the housing or the moving temperature control component is in full contact with the amplification wafer. The probe is directly facing the lysis buffer chamber in the functional cavity inside the housing. Other functional cavities are provided on both sides of the lysis buffer chamber.

[0009] Furthermore, the housing is made of PP material and the probe is in planar contact with the outer wall of the housing. The ultrasonic module operates at a frequency of 35–40 kHz, with an ultrasonic power of 20%–80% of the operating frequency, a duty cycle of 50%–80%, and an ultrasonic processing time of 30–180 s. The housing wall thickness is 1–1.5 mm, and the width L of the lysis fluid chamber is 3.8–4.3 mm. The cross-sectional area of ​​the lysis fluid chamber is 25–30 mm², and the liquid depth in the lysis fluid chamber during lysis is 16 mm. The width of the probe's emitting surface is greater than the width L of the lysis fluid chamber.

[0010] Furthermore, the fixed temperature control component is fixedly installed on the vertical surface of the side support plate, the bottom of the side support plate is vertically installed on the base, one side of the base plate is vertically installed on the vertical surface of the side support plate, the other side is installed on a side vertical plate, the side vertical plate is vertically installed on the base, and a guide rail for the reciprocating movement of the fluorescence detection component is also installed on the side support plate.

[0011] Furthermore, the mobile temperature control assembly includes a mobile water-cooled plate fixedly installed relative to the mobile support and a mobile temperature control plate installed on the mobile water-cooled plate. One side of the mobile temperature control plate is in contact with the surface of the mobile water-cooled plate, and the other side is in contact with the surface of the amplification plate during amplification operations. The fixed temperature control assembly includes a fixed water-cooled plate fixedly installed relative to the side support plate and a fixed temperature control plate installed on the fixed water-cooled plate. One side of the fixed temperature control plate is in contact with the surface of the fixed water-cooled plate, and the other side is in contact with the surface of the amplification plate during amplification operations. Both the mobile water-cooled plate and the fixed water-cooled plate are connected to the water storage tank through pipelines.

[0012] Furthermore, the inlet of the hollow plunger is connected to the heating unit via a pipeline, and the inlet of the heating unit is connected to a solenoid valve. The solenoid valve includes a first solenoid valve and a second solenoid valve. The normally open end of the first solenoid valve is connected to the syringe pump, and the normally closed end of the first solenoid valve is connected to the atmosphere. The common end of the first solenoid valve is connected to the normally open end of the second solenoid valve, and the normally closed end of the second solenoid valve is connected to the air pump. The heating unit is connected in series between the common end of the second solenoid valve and the hollow plunger. The first and second solenoid valves work together to realize the syringe pump's actions of drawing negative pressure, inputting positive pressure, and performing precision infusion in the central cavity.

[0013] Furthermore, the amplification chip contains multiple independently arranged amplification chambers connected in parallel. Each amplification chamber extends through both sides of the amplification chip. Each amplification chamber is arranged in parallel and connected to the liquid inlet of the amplification chip. The liquid inlet of the amplification chip is connected to the liquid outlet channel of the box body. The liquid outlet channel of the box body extends through the bottom of the box body and is connected to the side connection hole of the rotary valve when the rotary valve is rotated to a specific angle. Both sides of the amplification chip are sealed by a sealing film.

[0014] A detection method for a fully integrated microfluidic nucleic acid detection device, comprising the following steps: S1. Cartridge assembly: Assemble the cartridge body, rotary valve, amplification strip and cap. After assembly, the functional chambers inside the cartridge are pre-filled with reagents and sealed with a membrane on top. Then, close the lid. At this time, the protrusions on the lid will puncture the membrane on the top of each functional chamber. S2. Card box installation: Insert the assembled card box along the base plate and install it. At the same time, insert the card block of the connecting seat of the valve drive unit at the bottom of the rotary valve and install it. The amplification chip side contacts the fixed temperature control plate. S3. Rotate the rotary valve until its side connection hole connects to the sample chamber. The sample liquid in the sample chamber is input into the lysis buffer chamber through the central chamber by the action of the solenoid valve and the injection pump. S4. The slide block moves the slide plate, and the probe of the ultrasonic component on the slide plate contacts the outer wall of the box. At the same time, the moving temperature control plate contacts the side of the amplification plate. Together with the fixed temperature control plate, they squeeze and contact the two sides of the amplification cavity. The ultrasonic component is driven to perform ultrasonic lysis on the solution in the lysis buffer cavity, complete the nucleic acid lysis, and release the nucleic acid template. S5. After lysis, the binding liquid in the binding liquid chamber is first drawn into the central chamber and then into the lysis liquid chamber by rotating the rotary valve in conjunction with the injection pump. Then the mixture is introduced into the high-level inlet of the extraction chamber. After the mixture is filtered by the filter membrane, it is discharged into the waste liquid chamber. The first and second washing liquid chambers are switched sequentially by rotating the rotary valve. The positive pressure drives the washing liquid to wash the lysis products twice to remove impurities and interferences, thus completing the nucleic acid purification. S6. The rotary valve and solenoid valve work together to connect the air pump and the central chamber, start the heating unit, and the gas blown out by the air pump is heated to 60°C by the heating unit. The hot gas enters the central chamber through the hollow plunger and then enters the filter membrane of the extraction chamber, so that the residual ethanol in the nucleic acid adsorbed by the filter membrane is dried and evaporated. S7. The rotary valve and solenoid valve work together to connect the injection pump to the central chamber, switch the elution chamber path, and drive the elution solution through the central chamber, through the low inlet and filter membrane of the extraction chamber, to elute the purified nucleic acid and obtain a high-purity nucleic acid template solution. S8, the rotary valve and the solenoid valve work together to draw negative pressure into the amplification chamber of the amplification plate, and then the nucleic acid template solution in the extraction chamber is transported to the amplification chamber through the positive pressure of the central chamber, and fully reconstituted with the PCR cake-shaped lyophilized reaction reagent pre-placed in the amplification chamber to form a PCR reaction system; S9. When the temperature control component is working, the fixed temperature control plate and the moving temperature control plate heat up evenly to make contact with the amplification cavity. When cooling is required, the fixed temperature control plate and the moving temperature control plate are powered in opposite directions to cool down, which, together with the fixed water cooling plate and the moving water cooling plate, cools down the amplification cavity evenly. The heat is transferred to the water storage tank through heat transfer. During the amplification process, the fluorescence detection component continuously detects and collects signals to achieve qualitative and quantitative detection of nucleic acids.

[0015] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1). The present invention sets the ultrasonic component as an independently reciprocating module, the probe moving direction is perpendicular to the card insertion direction, and is completely separated from the rotary valve drive part. The rotary valve is directly inserted into the connecting seat block through the trapezoidal groove at the bottom of the card, and the valve drive part directly drives the rotation without going through the deceleration transmission mechanism. The above layout eliminates the coaxial integration constraint between the ultrasonic module and the rotary valve drive module. The valve drive part directly drives the connecting seat, avoiding the backlash error and cumulative transmission error introduced by the additional deceleration transmission mechanism, significantly improving the angle positioning accuracy and repeatability of the rotary valve, and ensuring the accuracy and sealing reliability of the flow channel switching between the central cavity and each functional cavity. (2). In this invention, the ultrasonic probe is positioned on the outer wall of the cartridge, facing the lysis fluid chamber in the functional cavity inside the cartridge. When the probe extends, it directly contacts and couples with the plane of the outer wall of the cartridge. The lysis fluid chamber is designed with a specific width and cross-sectional area and limits the liquid depth. At the same time, the width of the probe's emitting surface is greater than the width of the lysis fluid chamber. The above structure enables the ultrasonic energy to cover the entire liquid area of ​​the lysis fluid chamber at one time, realizing the batch synchronous lysis of the full sample without the need to transfer the sample in batches. Compared with the ultrasonic method that only acts on a small number of samples in the bottom cavity of the rotary valve, this invention greatly increases the sample processing capacity of a single lysis, shortens the total lysis time, and improves the overall efficiency of nucleic acid extraction. (3). The present invention adds a heating part to the gas circuit assembly. The heating part is connected in parallel with the injection pump and then connected in series with the hollow plunger through a solenoid valve. After the cleaning step is completed, the solenoid valve is switched to connect the gas pump and the hollow plunger. The heating part is started to heat the gas blown out by the gas pump. The hot gas enters the central cavity through the hollow plunger and then passes into the filter membrane of the extraction chamber. The residual ethanol in the nucleic acid adsorbed on the filter membrane is actively dried and evaporated by hot air. The above design enables the device to be compatible with conventional nucleic acid extraction reagents containing ethanol. It is not limited by the low extraction efficiency of alcohol-free or low-alcohol reagents. It effectively eliminates the inhibitory effect of ethanol on the subsequent PCR amplification reaction, and broadens the range of reagent selection and sample compatibility. (4). The present invention adopts liquid cooling for the temperature control components. The fixed temperature control components and the movable temperature control components are respectively equipped with fixed water cooling plates and movable water cooling plates. Both are connected to the water storage tank through pipelines. When the expansion requires cooling, the fixed temperature control plate and the movable temperature control plate are energized in opposite directions to cool down. The heat is transferred to the water storage tank through the coolant in the water cooling plate and then concentrated for heat dissipation by the water storage tank. This liquid cooling method completely eliminates the internal air duct structure required for air cooling, avoids the occupation of the internal space of the instrument by the air duct and the complex layout requirements, and significantly improves the internal space utilization of the instrument. Attached Figure Description

[0016] Figure 1 This is a three-dimensional structural diagram of the present invention; Figure 2 This is a three-dimensional structural schematic diagram from another perspective of the present invention; Figure 3 This is a top view of the structure of the present invention; Figure 4 This is a three-dimensional structural diagram of the card box of the present invention; Figure 5 This is a top view of the card holder of the present invention. Figure 6 for Figure 5 Schematic diagram of the cross-sectional structure at point AA; Figure 7 This is a bottom view of the card holder structure of the present invention; Figure 8 This is a schematic cross-sectional view of the rotary valve of the present invention; Figure 9 This is a three-dimensional structural diagram of the base plate of the present invention; Figure 10 This is a front view schematic diagram of the base plate of the present invention; Figure 11 for Figure 10 Schematic diagram of the cross-sectional structure at point BB; Figure 12 This is a three-dimensional structural diagram of the connector of the present invention; Figure 13 This is a top view of the connecting seat of the present invention; Figure 14 This is a three-dimensional structural diagram of the ultrasonic component and the movable temperature control component of the present invention; Figure 15 This is a three-dimensional structural schematic diagram of the ultrasonic component and the mobile temperature control component of the present invention from another perspective. Figure 16 This is a three-dimensional structural diagram of the side support plate of the present invention; Figure 17 This is a three-dimensional structural diagram of the gas path assembly of the present invention.

[0017] In the diagram: 100, base; 101, base plate; 1011, body; 1012, side limiting part; 10121, side limiting groove; 1013, edge limiting part; 1014, clearance hole; 102, connecting seat; 1021, seat body; 1022, locking block; 1023, valve drive part; 103, slide; 104, sliding plate; 1041, elastic element; 105, side support plate; 1051, first mounting surface; 1052, second mounting surface; 1053. Side panel; 200, Card holder; 201, Box body; 2011, Central cavity; 2012, Sample cavity; 2013, Lysis buffer cavity; 2014, First washing buffer cavity; 2015, Extraction cavity; 2016, Second washing buffer cavity; 2017, Binding buffer cavity; 2018, Waste liquid cavity; 2019, Elution buffer cavity; 2020, Discharge channel; 202, Box lid; 2021, Protrusion; 203, Cap; 204, Amplification plate; 2041, Amplification cavity; 2 042. Liquid inlet; 205. Rotary valve; 2051. Valve body; 2052. Seal; 2053. Connection channel; 2054. Center connection hole; 2055. Side connection hole; 2056. Flange; 300. Ultrasonic assembly; 301. Ultrasonic generator; 302. Probe; 400. Gas path assembly; 401. Gas path support platform; 402. Injection pump; 403. Heating unit; 404. Air pump; 405. Solenoid valve; 4051. First electric... 4052, Solenoid Valve; 406, Plunger Power Unit; 407, Hollow Plunger; 500, Temperature Control Component; 501, Fixed Temperature Control Component; 5011, Fixed Temperature Control Board; 5012, Fixed Water-Cooling Plate; 502, Movable Temperature Control Component; 5021, Movable Temperature Control Board; 5022, Movable Water-Cooling Plate; 5023, Movable Support; 600, Fluorescence Detection Component; 601, Detection Component Drive Unit; 700, Water Storage Tank; 800, Power Module. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] The accompanying drawings are for illustrative purposes only and should not be construed as limiting the scope of this patent. The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the embodiments of this application. The singular forms “a,” “described,” and “…” used in the embodiments of this application and the appended claims are also considered. The word "the" is also intended to include the majority form unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0020] In the following description, when referring to the accompanying drawings, the same numbers in different drawings denote the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application as detailed in the appended claims.

[0021] In the description of this application, it should be understood that the terms "first," "second," "third," etc., are used only to distinguish similar objects and are not necessarily used to describe a specific order or sequence, nor should they be construed as indicating or implying relative importance. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0022] Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more. "And / or" The description of the relationships between related objects indicates that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. The character " / " generally indicates that the preceding and following related objects have an "OR" relationship. The invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] To address the limitations of existing technologies, this embodiment provides a technical solution. The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0024] This invention primarily addresses the technical problems commonly found in existing nucleic acid point-of-care testing devices. These devices suffer from poor control precision of the rotary valve 205, low ultrasonic efficiency, poor sample compatibility, and inadequate temperature control and heat dissipation measures. This invention provides a fully integrated microfluidic nucleic acid detection device and method, avoiding errors caused by the deceleration mechanism, improving transmission precision, employing an injection pump 402 for higher control accuracy, being less affected by batch-to-batch variations in the cartridge 200, being compatible with conventional extraction reagents, improving sample compatibility, avoiding the problem of air cooling failing to meet arrangement requirements, and achieving better ultrasonic side-mounted cavitation effects. Example 1

[0025] See appendix Figure 1-3A fully integrated microfluidic nucleic acid detection device includes: a base 100, which serves as a support platform for a power module 800, a water tank 700, a fluorescence detection component 600, a temperature control component 500, a gas path component 400, an ultrasonic component 300, and a cartridge 200. The power module 800 provides electrical power to the device, the water tank 700 provides heat exchange for cooling the temperature control component 500, the fluorescence detection component 600 performs fluorescence detection on the amplification plate 204, the temperature control component 500 controls the temperature of the amplification plate 204, the gas path component 400 adjusts the gas pressure in the central cavity 2011 to achieve fluid drive during sample lysis, nucleic acid extraction and purification, and nucleic acid amplification, and the ultrasonic component 300 performs ultrasonic lysis of the liquid in the lysis chamber 2013 of the cartridge 200. The cartridge 200 contains various functional liquids and achieves sample lysis, nucleic acid extraction and purification, and elution through the rotation of the rotary valve 205, ultimately obtaining a nucleic acid template solution.

[0026] See appendix Figure 4-8 The card holder 200 is horizontally inserted into the base 100 and installed in place. At the same time, the rotary valve 205 at the bottom of the card holder 200 is inserted into the valve drive part 1023 installed on the base 100 to achieve coaxial rotational connection between the two. It can be understood that the installation of the rotary valve 205 and the valve drive part 1023 at the bottom of the card holder 200 is completed at the same time as the card holder 200 is inserted into place. Compared with the complex structure of the prior art, the structure of the card holder 200 of the present invention is simpler and easier to assemble and disassemble. Specifically, the cartridge 200 has an independent central cavity 2011 and several functional cavities inside. The functional cavities include a sample cavity 2012, a lysis buffer cavity 2013, a first cleaning buffer cavity 2014, an extraction cavity 2015, a second cleaning buffer cavity 2016, a binding buffer cavity 2017, a waste liquid cavity 2018, and an elution buffer cavity 2019. The functional cavities are arranged around the central cavity 2011. The lysis buffer cavity 2013 is located between the sample cavity 2012 and the first cleaning buffer cavity 2014. The rotation of the rotary valve 205 enables communication between the central cavity 2011 and the different functional cavities. Specifically, the rotary valve 205 is sealed and rotated to the bottom surface of the cartridge body 201 of the cartridge 200. The amplification chip 204 is inserted and installed into the cartridge body 201. Both the amplification chip 204 and the rotary valve 205 are limited to the cartridge body 201 by the vertical insertion and pressing of the cap 203 into the cartridge body 201. It can be understood here that the bottom of the cartridge 200 has a groove, the top of the rotary valve 205 is tightly fitted to the bottom of the cartridge body 201, the pressure cap 203 is inserted into the bottom and side of the cartridge body 201, the pressure cap 203 is used to apply pressure to the rotary valve 205 to make it seal against the bottom of the cartridge body 201, the amplification chip 204 is inserted into the side wall of the cartridge body 201, and then the pressure cap 203 is inserted into the side wall of the cartridge body 201 to limit the position of the amplification chip 204. See appendix. Figure 7 and attached Figure 9-11The two outer sides of the pressure cap 203 are trapezoidal and are horizontally inserted into the base plate 101 mounted on the base 100. Two side limiting parts 1012 are provided on the upper surface of the base plate 101. Each side limiting part 1012 forms a side limiting groove 10121 with the body 1011 of the base plate 101. The side limiting grooves 10121 of the two side limiting parts 1012 are inserted into the two outer sides of the pressure cap 203. The cross-sectional shape of the two side limiting grooves 10121 is shown in the attached figure. Figure 11 A side limiting part 1013 is provided on one side of the upper surface of the base plate 101, located on both sides of the limiting part 1012. The side limiting part 1013 is located on the side away from the card holder 200 entering the base plate 101, and its purpose is to provide auxiliary limiting for the end of the card holder 200 after it is inserted. For details, please refer to the appendix. Figure 7-13 Both the bottom surface of the pressure cap 203 and the bottom surface of the rotary valve 205 are provided with trapezoidal grooves. The trapezoidal grooves on the bottom surface of the pressure cap 203 are discontinuous. The center of the pressure cap 203 is provided with a through hole for the rotary valve 205 to be inserted and installed. The rotary valve 205 protrudes from the center of the pressure cap 203, and the trapezoidal grooves of both together form a large trapezoidal groove. The bottom of the rotary valve 205 is provided with a flange 2056 to facilitate the pressing of the pressure cap 203. The base plate 101 is provided with a locking block 1 for the connecting seat 102. The clearance hole 1014 through which 022 passes is designed with a trapezoidal structure for the locking block 1022. When the pressure cover 203 of the card box 200 is inserted and installed on the base plate 101, the trapezoidal groove of the rotary valve 205 engages with the locking block 1022 of the connecting seat 102. The seat body 1021 of the connecting seat 102 is fixedly installed with the power end of the valve drive unit 1023. The valve drive unit 1023 uses a servo motor to directly drive the connecting seat 102 to rotate, thereby driving the rotary valve 205. See Appendix Figure 4-8The card holder 200 has a connecting channel through the box body 201 in the central cavity 2011 and the bottom of the functional cavity. The rotary valve 205 has a central connecting hole 2054 and a side connecting hole 2055 at the top. The rotary valve 205 has a connecting channel 2053 inside. The central connecting hole 2054 and the side connecting hole 2055 are respectively connected to the two ends of the connecting channel 2053. The central connecting hole 2054 is connected to the connecting channel at the bottom of the central cavity 2011. The side connecting hole 2055 is connected to the connecting channel of different functional cavities by rotating the rotary valve 205 to different angles. It can be understood that the valve body 2051 of the rotary valve 205 has a sealing layer at the top. The sealing layer is a two-color injection molded part. The connecting channel 2053 is formed by the bottom of the two-color sealing part 2052 being recessed inward. The amplification wafer 204 contains multiple independently arranged amplification chambers 2041 connected in parallel. Preferably, the number of amplification chambers 2041 is eight. The amplification chambers 2041 extend through both sides of the amplification wafer 204. Each amplification chamber 2041 is arranged in parallel and communicates with the liquid inlet 2042 of the amplification wafer 204. The liquid inlet 2042 of the amplification wafer 204 is connected to the liquid outlet channel 2020 of the housing 201. The liquid outlet channel 2020 of the housing 201 extends through the bottom of the housing 201 and communicates with the side connection hole 2055 of the rotary valve 205 when the rotary valve 205 is rotated to a specific angle. Both sides of the amplification chambers 2041 are sealed by a sealing film. The channels between the amplification chambers 2041 and the liquid inlet 2042 are formed by an inward indentation on one side of the amplification wafer 204 and are sealed by a sealing film in the later stage.

[0027] It is understood that the present invention adopts a trapezoidal inclined guide structure, which utilizes the self-centering effect of the inclined plane to transform the linear motion of horizontal insertion into precise alignment of the axis of the rotary valve 205 and the axis of the valve drive part 1023. The vertical pressing limit and the horizontal insertion guide are decoupled, reducing the cumulative tolerance of assembly and realizing the blind insertion function of one insertion. No additional alignment operation is required. The wedge fit of the trapezoidal structure can compensate for the assembly deviation of ±0.5mm, ensuring that the coaxiality of the rotary valve 205 and the valve drive part 1023 is ≤0.1mm, avoiding uneven wear and leakage during rotation.

[0028] Participate in the attached Figure 1-3 and attached Figure 17The gas path assembly 400 includes an injection pump 402 and an air pump 404, both mounted on the base 100 via a gas path support platform 401, and a hollow plunger 407 reciprocatingly sliding and sealed within the central cavity 2011 of the cartridge 200. The injection pump 402 and air pump 404 are connected in parallel and then connected in series with a heating unit 403 via a solenoid valve 405, which in turn connects to the central cavity 2011. The heating unit 403 uses a ceramic heating tube. The hollow plunger 407 is driven by a lead screw pair driven by a plunger power unit 406 to move the hollow plunger 407 up and down. The injection pump 402 adjusts the gas pressure within the central cavity 2011 to achieve fluid drive during sample lysis, nucleic acid extraction and purification, and nucleic acid amplification. The air pump 404 and heating unit 403 are used to heat the gas entering the central cavity 2011 to remove residual gases after nucleic acid extraction. Ethanol is dried and separated. Specifically, the inlet of the hollow plunger 407 is connected to the heating unit 403 through a pipeline. The inlet of the heating unit 403 is connected to the solenoid valve 405. The solenoid valve 405 includes a first solenoid valve 4051 and a second solenoid valve 4052. The normally open end of the first solenoid valve 4051 is connected to the syringe pump 402, and the normally closed end of the first solenoid valve 4051 is connected to the atmosphere. The common end of the first solenoid valve 4051 is connected to the normally open end of the second solenoid valve 4052. The normally closed end of the second solenoid valve 4052 is connected to the air pump 404. The heating unit 403 is connected in series between the common end of the second solenoid valve 4052 and the hollow plunger 407. The first solenoid valve 4051 and the second solenoid valve 4052 work together to realize the syringe pump 402 to draw negative pressure, input positive pressure, and perform precision infusion in the central cavity 2011.

[0029] Understandably, when both the first solenoid valve 4051 and the second solenoid valve 4052 are de-energized, the syringe pump 402 precisely pushes and pulls, and through the micro-step of the hollow plunger 407, it achieves precise adjustment of the gas pressure in the central cavity 2011, which is used for quantitative transfer of samples and quantitative distribution of elution solution. When the first solenoid valve 4051 is de-energized and the second solenoid valve 4052 is energized, the gas pump 404 is connected to the hollow plunger 407, and at the same time the heating unit 403 is energized to heat the gas flow to the set temperature (the set temperature of the heating unit 403 in this application is 60°C). The hot gas flow enters the central cavity 2011 and the extraction cavity 2015 through the hollow plunger 407 for ethanol drying. When the first solenoid valve 4051 is energized and the second solenoid valve 4052 is de-energized, the syringe pump 402 retracts, creating a negative pressure in the central cavity 2011, which extracts the gas in the amplification cavity 2041 to facilitate subsequent liquid aspiration. By combining the on / off states of two solenoid valves 405, a high-precision micro-injection pump 402 and a high-flow air pump 404 are integrated into the same pipeline and connected in series with a heating unit 403. The injection pump 402 is responsible for accurate liquid volume measurement, while the air pump 404 is responsible for high-flow gas purging. The two complement each other and do not interfere with each other, solving the problem in the prior art that a single pump source cannot simultaneously achieve both accuracy and flow rate. Hot air drying effectively avoids the inhibition of PCR polymerase by ethanol.

[0030] See appendix Figure 1-2 and attached Figure 14-15 The ultrasonic generator 301 of the ultrasonic component 300 is mounted on the base 100 and includes a reciprocating probe 302. When the probe 302 extends, it contacts and couples with the outer sidewall of the cartridge 200 to perform lysis of the sample solution inside the cartridge 200. The probe 302 is coated with silicone grease or fitted with a silicone sleeve. The cartridge 201 is made of PP material, and the probe 302 makes planar contact with the outer sidewall of the cartridge 201. Utilizing the stress deformation characteristics of PP material combined with the planar contact between the probe 302 and the outer sidewall of the cartridge 201, the fault tolerance of the cartridge 200 is high, and the yield rate of the cartridge 200 is increased. The operating frequency of the ultrasonic module is 35-40. The ultrasonic power setting is 20%–80% of the working frequency, the duty cycle is 50%–80%, the ultrasonic processing time is 30–180 s, the wall thickness of the housing 201 is 1–1.5 mm, the width L of the lysis chamber 2013 is 3.8–4.3 mm, the cross-sectional area of ​​the lysis chamber 2013 is 25–30 mm², the liquid depth in the lysis chamber 2013 during lysis is 16 mm, the width of the emitting surface of the probe 302 is greater than the width L of the lysis chamber 2013, and the width of the probe 302 is greater than the width of the chamber, so that the ultrasonic energy is precisely concentrated in the liquid in the chamber, reducing the attenuation and scattering of energy on the wall of the housing 201. It is important to understand that for different types of clinical samples commonly used in POCT molecular diagnostics, due to significant differences in their cell structure, matrix viscosity, target nucleic acid type, and thermal sensitivity, differentiated combinations of ultrasonic lysis parameters are required. Table 1-1 below shows the ultrasonic lysis parameters for each sample type: Table 1-1 shows the ultrasonic lysis parameters for each sample type: throat swab washing fluid 40 20~30 4~6 3 3 50 30~45 Whole blood / plasma 40 30~40 6~8 5 3 62.5 60~90 urine 40 40~50 8~10 5 2 71.4 60~120 Bacterial culture medium 40 60~80 12~16 8 2 80 90~180 Sputum (after expectorant pretreatment) 35 60~70 12~14 8 2 80 90~150 fecal suspension 40 50~60 10~12 5 3 62.5 60~120 Fungal culture medium / oral swab 35 70~80 14~16 8 2 80 120~180 The parameter design for each sample type in the table above is explained below.

[0031] The targets of throat swab eluent (viral RNA / DNA extraction) are viral particles and exfoliated epithelial cells. Mammalian cell membranes consist only of a phospholipid bilayer (approximately 5–10 nm thick) and lack a cell wall structure. The lipid envelope of most enveloped viruses is also fragile. A 40 kHz high frequency is used to generate small cavitation bubbles (resonance radius approximately 82 μm), which provides gentle mechanical shearing force and avoids nucleic acid fragmentation. The power is set at 20%–30% (4–6 W), corresponding to a power density of 9.3–13.9 W / mL, which is sufficient to disrupt the lipid membrane structure. A duty cycle of 50% (3 s on / 3 s off) ensures intermittent heat dissipation and prevents RNA degradation at high temperatures. Sufficient lysis can be achieved in a total time of 30–45 s.

[0032] Whole blood / plasma samples (genomic DNA extraction): Red blood cells in whole blood are anucleate cells, and no nucleic acid is released after lysis; white blood cells (5-10×10⁻⁶) 9The target cell for lysis is leukocytes ( / L), whose cell membranes also lack cell walls, but the nuclear membrane needs to be breached; plasma may contain free pathogens (bacteria, viruses); power is set at 30%–40% (6–8W), power density is 13.9–18.5 W / mL, which is about 50% higher than the throat swab protocol, in order to breach the leukocyte nuclear membrane; duty cycle is 62.5% (5s on / 3s off) to prolong the effective action time; total time is 60–90s; heme in the blood is a PCR inhibitor, and moderate sonication helps release nucleic acids but requires subsequent purification steps.

[0033] Urine sample (bacterial DNA extraction): Urine pathogens are mainly Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, etc.), whose cell walls contain a thin layer of peptidoglycan (2-7 nm) and an outer membrane lipopolysaccharide layer; although more robust than mammalian cells, they are far less thick-walled than Gram-positive bacteria; power setting 40%-50% (8-10 W), power density 18.5-23.1 W / mL; 40 kHz frequency combined with medium power can fully disrupt the three-layer structure of Gram-negative bacteria: outer membrane-peptidoglycan-inner membrane; duty cycle 71.4% (5s on / 2s off), urine matrix has low viscosity (close to water), high cavitation efficiency, no need for excessively high duty cycle; total time 60-120s, taking into account the sufficient lysis of samples with low bacterial concentration.

[0034] Bacterial culture medium (for both Gram-positive and Gram-negative bacteria): The culture medium contains a high concentration of live bacteria (10... 8 ~10 9 (CFU / mL) is required to disrupt the thick peptidoglycan layer (20–80 nm) of Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus pneumoniae; power setting is 60%–80% (12–16 W), with a power density of 27.8–37.0 W / mL, which is the highest gradient among all sample types; high frequency of 40 kHz combined with high power is used to generate a high-intensity steady-state cavitation effect. When the cavitation bubble implodes, it generates local high temperature (~5000 K), high pressure (~1000 atm), and strong shear microjets (velocity up to 100 m / s), which is sufficient to break the peptidoglycan glycosidic bonds; duty cycle of 80% (8 s on / 2 s off) ensures continuous high energy input, with a total time of 90–180 s; it is recommended to use lysozyme pretreatment to improve efficiency.

[0035] Sputum samples (pretreated with expectorant): After expectorant pretreatment with N-acetyl-L-cysteine ​​(NALC) or dithiothreitol (DTT), the viscosity of sputum decreased but remained higher than that of aqueous solutions; high-viscosity media inhibited cavitation bubble formation and movement, requiring stronger mechanical force; using a low frequency of 35kHz (resonant cavitation bubble radius of approximately 94μm, about 14% larger than 40kHz) generated larger cavitation bubbles and stronger shock waves, resulting in better penetration of viscous media; power setting of 60%–70% (12–14W), power density of 27.8–32.4 W / mL; duty cycle of 80% (8s on / 2s off), total time of 90–150s; targets included difficult-to-lyse pathogens such as Mycobacterium tuberculosis with special cell wall structures (rich in mycolic acid).

[0036] Fecal suspension: Fecal matrix is ​​complex, containing PCR inhibitors such as bile acids, polysaccharides, and humic acids. The supernatant needs to be collected after dilution. Targets include enteropathogenic bacteria (Salmonella, Shigella, Campylobacter, etc.) and viruses. Power settings: 50%–60% (10–12 W), power density: 23.1–27.8 W / mL; duty cycle: 62.5% (5s on / 3s off), medium duty cycle for equilibrium lysis; excessive lysis will release large amounts of polysaccharides and other inhibitors, affecting downstream PCR. Total time: 60–120 s.

[0037] Fungal culture medium / oral swabs (Candida, etc.): The fungal cell wall consists of a three-layer network structure composed of chitin (β-1,4-polyN-acetylglucosamine), β-glucan, and mannoprotein. Its mechanical strength far exceeds that of bacterial peptidoglycan, making it the most difficult sample type to lyse. A low-frequency (35kHz) + 70%–80% high power (14–16W) approach is used, with a power density of 32.4–37.0 W / mL. Large cavitation bubbles exert a stronger mechanical impact on the thick-walled structure. A duty cycle of 80% (8s on / 2s off), with a total time of 120–180s, is the longest lysis protocol in this system. Pretreatment with a cell-wall-disrupting enzyme (such as a cell-wall-lysing enzyme) is recommended.

[0038] See appendix Figure 1-3 and attached Figure 14-16The temperature control component 500 is mounted on the base 100 and includes a fixed temperature control component 501 fixedly mounted relative to the base 100 and a movable temperature control component 502 slidably mounted relative to the base 100. When the cartridge 200 is inserted into place, one side of the amplification strip 204 of the cartridge 200 contacts the surface of the fixed temperature control component. The movable temperature control component 502, in coordination with the fixed temperature control component, squeezes and contacts both sides of the amplification strip 204 of the cartridge 200 to achieve consistent temperature control of the nucleic acid template solution entering the amplification strip 204. The probe 302 of the ultrasound component 300 acts on the cartridge 200 in a direction perpendicular to the insertion direction of the cartridge 200. The hollow plunger 407 moves along the direction of movement of the cartridge 200, perpendicular to the insertion direction of the cartridge 200 and aligned with the probe. The direction in which probe 302 acts on cartridge 200 is perpendicular to the direction in which the moving temperature control component 502 is displaced is perpendicular to the insertion direction of cartridge 200, and is parallel to the direction in which probe 302 acts on cartridge 200 and perpendicular to the plane where amplification plate 204 is located. It can be understood that the moving direction of probe 302 of ultrasound component 300, the reciprocating motion direction of hollow plunger 407 and the displacement direction of moving temperature control component 502 are perpendicular to each other in space, and form a three-dimensional layout with the insertion direction of cartridge 200. This effectively avoids mechanical interference between the moving parts in a narrow space, and at the same time ensures that probe 302 and the side wall of cartridge 200, temperature control plate and amplification plate 204 can independently apply the best pre-tightening force in their respective dimensions, ensuring the reliability and repeatability of physical coupling. Both the ultrasound component 300 and the moving temperature control component 502 are slidably mounted on the slide plate 104 via a slider and slide rail assembly. The slide plate 104 is fixedly mounted relative to the slide base 103. The slide base 103 is slidably mounted on the base 100 via a combination of slider and slide rail driven by a motor. Elastic elements 1041 are installed between the ultrasound component 300 and the moving temperature control component 502 and the upright plate of the slide plate 104. The elastic elements 1041 are preferably springs. The elastic elements 1041 are used to apply a pre-tightening force to the probe 302 or the moving temperature control component 502 so that the probe 302 is in full contact with the side wall of the housing 201 or the moving temperature control component 502 is in full contact with the amplification plate 204. The probe 302 is directly facing the lysis fluid chamber 2013 in the functional cavity inside the housing 201. Other functional cavities are provided on both sides of the lysis fluid chamber 2013 to ensure that the cavitation effect is maximized, achieving efficient and uniform cell / virus lysis, and avoiding unintended effects on reagents in adjacent cavities.

[0039] See appendix Figure 16The fixed temperature control component 501 is fixedly mounted on the vertical surface of the side support plate 105, i.e., the first mounting surface 1051. The bottom of the side support plate 105 is vertically mounted on the base 100. One side of the base plate 101 is vertically mounted on the vertical surface of the side support plate 105, i.e., the second mounting surface 1052, and the other side is mounted on a side vertical plate 1053. The side vertical plate 1053 is vertically mounted on the base 100. A guide rail for the reciprocating movement of the fluorescence detection component 600 is also mounted on the side support plate 105. It can be understood that the side support plate 105, as a core reference component, provides a unified mounting and positioning reference for the base plate 101, the fixed temperature control component 501, and the fluorescence detection component 600, constructing a compact architecture with multiple uses on one plate. This significantly shortens the dimensional chain between the temperature control bonding surface and the optical detection path, eliminates the cumulative tolerance caused by the dispersed installation of multiple components, and ensures that the heating uniformity of the amplification chip 204 and the consistency of fluorescence signal acquisition remain stable during thermal cycling. Meanwhile, the base plate 101, relying on the side support plate 105, is arranged in a bridge-like suspended layout, reserving open space at the bottom for the valve drive unit 1023 and the gas pipeline. This structurally decouples the insertion guide of the cartridge 200 from the valve drive alignment, ensuring that insertion and removal wear does not affect the rotary seal performance. In addition, the side support plate 105 integrates multiple installation features, allowing for parallel calibration and independent module replacement during assembly, significantly reducing the difficulty of debugging and maintenance. With the side support plate 105 as the anchor point, the entire system achieves spatial coordination and precision mutual protection of the three core functions of temperature control, optical detection, and fluid drive, providing a highly rigid and interchangeable platform foundation for high-throughput expansion.

[0040] See appendix Figure 14-15 The mobile temperature control assembly 502 includes a mobile water-cooled plate 5022 fixedly mounted relative to the mobile bracket 5023 and a mobile temperature control plate 5021 mounted on the mobile water-cooled plate 5022. One side of the mobile temperature control plate 5021 contacts the surface of the mobile water-cooled plate 5022, and the other side contacts the surface of the amplification wafer 204 during amplification operations. The fixed temperature control assembly 501 includes a fixed water-cooled plate 5012 fixedly mounted relative to the side support plate 105 and a fixed temperature control plate 5021 mounted on the fixed water-cooled plate 5012. Plate 5011, the fixed temperature control plate 5011 is in contact with the fixed water cooling plate 5012 on one side, and the other side is in contact with the amplification chip 204 during amplification. The movable water cooling plate 5022 and the fixed water cooling plate 5012 are both connected to the water storage tank 700 through pipelines. Both the fixed temperature control plate 5011 and the movable temperature control plate 5021 adopt semiconductor thermoelectric module (TEC1-12706) and PID control, with a heating rate ≥12℃ / s, a cooling rate ≥8℃ / s, and a temperature control accuracy of ±0.2℃.

[0041] Understandably, the symmetrical contact on both sides eliminates the temperature gradient along the thickness of the amplification wafer 204 (temperature difference between center and surface <0.2℃) that occurs with single-sided heating. Liquid cooling is used instead of air cooling because liquid has a much higher specific heat capacity than air, resulting in a higher heat transfer coefficient. Heat is concentrated in the external water tank 700 through pipes and discharged, without relying on the complex air duct layout inside the instrument. The temperature uniformity within the amplification chamber 2041 is ≤±0.1℃, significantly better than the ±0.5℃ of single-sided heating. The heating and cooling rate (≥1.5℃ / s) meets the requirements of rapid PCR (total time for 40 cycles <30min). The liquid cooling solution eliminates the need for internal air inlets and outlets, achieves an IP52 dust and water resistance rating, and only requires parallel cooling pipes when expanding to multiple channels, increasing space utilization by 50%. Example 2

[0042] A detection method for a fully integrated microfluidic nucleic acid detection device, comprising the following steps: S1. Assembly of cartridge 200: Assemble the cartridge body 201, rotary valve 205, amplification strip 204, and pressure cap 203. After assembly, the functional cavities inside the cartridge 200 are pre-filled with reagents and sealed with a top film. Then, close the lid 202. At this time, the protrusions 2021 on the lid 202 puncture the film on the top of each functional cavity. Specifically, first, press the rotary valve 205 and amplification strip 204 onto the cartridge body 201 through the pressure cap 203, then pre-fill each functional cavity with the corresponding reagents, such as isopropanol solution for binding solution and ethanol solution for cleaning solution. The top is heat-sealed with aluminum foil film. After closing the lid 202, the protrusions 2021 on the inside of the lid 202 puncture the film on the top of each cavity, allowing each cavity to be vented to the atmosphere. It should be noted that after puncturing, the film is still attached to the top of the cavity, and the bottom of the cavity is sealed by the rotary valve 205 to form a temporary sealed circuit to prevent liquid leakage. S2. Card holder 200 installation: Insert the assembled card holder 200 along the base plate 101 for installation. At the same time, the bottom of the rotary valve 205 is inserted into the locking block 1022 of the connecting seat 102 of the valve drive part 1023. One side of the amplification chip 204 is in contact with the surface of the fixed temperature control board 5011. The temperature control component 500 is not powered here and is at room temperature. S3. Rotary valve 205 rotates to connect its side connection hole 2055 to sample chamber 2012. Through the coordinated action of solenoid valve 405 and injection pump 402, sample liquid in sample chamber 2012 is input into lysis chamber 2013 via central chamber 2011. Specifically, the first solenoid valve 4051 and the second solenoid valve 4052 are de-energized, injection pump 402 is drawn back through hollow plunger 407, central chamber 2011 is under negative pressure (-20kPa), rotary valve 205 rotates to align side connection hole 2055 with bottom hole of sample chamber 2012, sample liquid is drawn into central chamber 2011, and then rotary valve 205 switches to bottom hole of lysis chamber 2013, injection pump 402 pushes positive pressure, pushing sample from central chamber 2011 into lysis chamber 2013; S4. The slide block 103 drives the slide plate 104 to move. The probe 302 of the ultrasonic component 300 on the slide plate 104 contacts the outer wall of the box 201. At the same time, the moving temperature control plate 5021 contacts the side of the amplification plate 204. Together with the fixed temperature control plate 5011, they squeeze and contact the two sides of the amplification chamber 2041. At this time, there is only contact and no heating. The ultrasonic component 300 is driven to perform ultrasonic lysis on the solution in the lysis chamber 2013 to complete nucleic acid lysis and release the nucleic acid template. S5. After lysis, the rotary valve 205 is rotated in conjunction with the syringe pump 402 to draw the binding fluid from the binding fluid chamber 2017 into the central chamber 2011 and then into the lysis fluid chamber 2013. The mixture is then introduced into the high-level inlet 2042 of the extraction chamber 2015. After filtration by the filter membrane, the mixture is discharged into the waste fluid chamber 2018. The rotary valve 205 sequentially switches the pathways of the first washing fluid chamber 2014 and the second washing fluid chamber 2016, driving the washing fluid under positive pressure to perform two washes on the lysis products, removing impurities and interferences, thus completing nucleic acid purification. Specifically, the rotary valve 205 switches... Switching to the binding fluid chamber 2017, the first solenoid valve 4051 and the second solenoid valve 4052 are de-energized. The syringe pump 402 draws the binding fluid into the central chamber 2011 and then pumps it into the lysis fluid chamber 2013 to mix with the lysis fluid. Subsequently, the rotary valve 205 switches to the high-position inlet 2042 of the extraction chamber 2015. The mixture is passed through the silica membrane (pore size 0.45μm, thickness 0.8mm) in the extraction chamber 2015 under positive pressure (10kPa). Nucleic acid is adsorbed onto the membrane. The mixture is then rotated to the first washing fluid chamber 2014 and the second washing fluid chamber 2016 in sequence, and the membrane is washed twice with the washing fluid under positive pressure. S6, the rotary valve 205 and the solenoid valve 405 work together to de-energize the first solenoid valve 4051 and energize the second solenoid valve 4052. The air pump 404 starts (output airflow rate 3L / min) and connects to the central cavity 2011. The heating unit 403 is activated. The gas blown out by the air pump 404 is heated to 60°C by the heating unit 403. The hot gas enters the central cavity 2011 through the hollow plunger 407 and then enters the filter membrane of the extraction cavity 2015, causing the residual ethanol in the nucleic acid adsorbed by the filter membrane to dry and evaporate. S7, rotary valve 205 and solenoid valve 405 work together to de-energize the first solenoid valve 4051 and the second solenoid valve 4052, connect the injection pump 402 and the central chamber 2011, switch the elution chamber 2019 path, and drive the elution solution through the central chamber 2011, through the low inlet and filter membrane of the extraction chamber 2015, to elute the purified nucleic acid and obtain a high-purity nucleic acid template solution; S8. Rotary valve 205 and solenoid valve 405 work together. The first solenoid valve 4051 is energized, and the second solenoid valve 4052 is de-energized. The injection pump 402 retracts, creating a negative pressure (-40kPa) in the amplification chamber 2041 of the amplification wafer 204 through the central cavity 2011, placing the amplification chamber 2041 in a vacuum negative pressure state. Then, rotary valve 205 disconnects from the amplification chamber 2041. At this time, the negative pressure state of the amplification chamber 2041 can be maintained. Then, rotary valve 205 and solenoid valve 405 work together, and rotary valve 205 connects to the extraction chamber... The low inlet of 2015 is connected, and the nucleic acid template solution in the extraction chamber 2015 is first drawn into the central chamber 2011. Then, the rotary valve 205 is connected to the amplification chamber 2041, and the negative pressure draws the nucleic acid template solution into each amplification chamber 2041. At the same time, the first solenoid valve 4051 and the second solenoid valve 4052 are de-energized, and the syringe pump 402 is driven by positive pressure to pressurize the amplification chamber 2041, causing the sealing membrane on both sides of the amplification chamber 2041 to expand and fully reconstitute with the PCR cake-shaped lyophilized reaction reagent pre-placed in the amplification chamber 2041 to form a PCR reaction system. S9. Temperature control component 500 operates, with fixed temperature control plate 5011 and moving temperature control plate 5021 uniformly heating the amplification chamber 2041. The process involves 45 cycles of denaturation at 95℃ for 10 seconds, annealing at 55℃ for 20 seconds, and extension at 72℃ for 20 seconds, in contact with the chamber. When cooling is required, fixed temperature control plate 5011 and moving temperature control plate 5021 are energized in opposite directions for cooling, working in conjunction with fixed water cooling plate 5012 and moving water cooling plate 5022 to uniformly cool the amplification chamber 2041. Heat is transferred to the water storage tank 700 via heat transfer. During amplification, fluorescence detection component 600 continuously reciprocates along the guide rail under the drive of detection component drive unit 601, which is fixedly installed relative to the side support plate 105, sequentially reading the fluorescence signals of each amplification chamber 2041 to achieve qualitative and quantitative nucleic acid detection.

[0043] It is understood that the device also includes a control system, which includes a main control circuit board (such as a PLC or embedded microprocessor) and a human-machine interface. The main control circuit board is electrically connected to the valve drive unit 1023, the injection pump 402, the air pump 404, the first solenoid valve 4051405, the second solenoid valve 4052405, the heating unit 403, the ultrasonic component 300, the temperature control component 500, and the fluorescence detection component 600. The main control circuit board has a timing control program pre-stored in it, corresponding to the detection method steps S1 to S9.

[0044] When the cartridge 200 is inserted into place via the base plate 101, the host computer is operated, the preset execution program is selected, and the program is started. According to the preset timing sequence, the following actions are automatically executed. The host computer and program settings are existing technologies and will not be described in detail here: First, the rotary valve 205 is controlled to switch the flow channels in sequence, and the syringe pump 402 / air pump 404 is driven to achieve quantitative fluid transport; after the liquid enters the lysis buffer chamber 2013, the ultrasonic component 300 is automatically triggered to perform timed lysis; after lysis is completed, the flow channels are automatically switched to perform cleaning, hot air drying and elution; finally, the nucleic acid template is automatically distributed to the amplification chamber 2041, and the temperature control component 500 is started to execute the preset thermal cycling program. At the same time, the fluorescence detection component 600 is triggered to automatically collect signals. The entire sample processing and detection process does not require manual intervention after the cartridge 200 is installed.

[0045] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0046] 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, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fully integrated microfluidic nucleic acid detection device, characterized in that, include: Base; The card box is horizontally inserted into the base and installed in place. At the same time, the rotary valve at the bottom of the card box is inserted into the valve drive unit installed on the base to achieve coaxial rotational connection between the two. The card box has an independent central cavity and several functional cavities inside. The rotation of the rotary valve realizes the communication between the central cavity and different functional cavities. The gas path assembly includes an injection pump and an air pump, both mounted on a base, and a hollow plunger that reciprocates and is sealed within the central cavity of the cartridge. The injection pump and the air pump are connected in parallel and then connected in series with a heating element via a solenoid valve, and finally connected to the central cavity via the hollow plunger. The injection pump adjusts the gas pressure within the central cavity to achieve fluid drive during sample lysis, nucleic acid extraction and purification, and nucleic acid amplification. The air pump and the heating element are used to heat the gas entering the central cavity to dry and separate the residual ethanol after nucleic acid extraction. An ultrasound assembly, mounted on a base, includes a reciprocating probe that, when extended, contacts and couples with the sidewall of the outer side of the cartridge to perform lysis of the sample solution within the cartridge. The temperature control component is mounted on a base and includes a fixed temperature control component that is fixedly mounted relative to the base and a movable temperature control component that is slidably mounted relative to the base. When the cartridge is inserted into place, one side of the amplification strip of the cartridge contacts the surface of the fixed temperature control component. The displacement of the movable temperature control component cooperates with the fixed temperature control component to squeeze and contact both sides of the amplification strip of the cartridge, so as to achieve consistent temperature control of the nucleic acid template solution entering the amplification strip.

2. The fully integrated microfluidic nucleic acid detection device according to claim 1, characterized in that, The direction in which the probe of the ultrasound component acts on the cartridge is perpendicular to the insertion direction of the cartridge. The direction in which the hollow plunger moves along the cartridge is perpendicular to the insertion direction of the cartridge and also perpendicular to the direction in which the probe acts on the cartridge. The direction in which the moving temperature control component moves is perpendicular to the insertion direction of the cartridge, while being parallel to the direction in which the probe acts on the cartridge and perpendicular to the plane where the amplification film is located.

3. The fully integrated microfluidic nucleic acid detection device according to claim 2, characterized in that, The rotary valve is sealed and rotated on the bottom surface of the cartridge body. The amplification chip is inserted into the cartridge body. Both the amplification chip and the rotary valve are limited to the cartridge body by vertical insertion and pressing of the cap. The two outer sides of the cap are trapezoidal and are horizontally inserted into the base plate. The bottom surface of the cap and the bottom surface of the rotary valve are both provided with trapezoidal grooves. The rotary valve protrudes from the center of the cap, and the trapezoidal grooves of both constitute a large trapezoidal groove. The base plate is provided with clearance holes for the locking block of the connector to pass through. The locking block is set with a trapezoidal structure. When the cap of the cartridge is inserted and installed on the base plate, the trapezoidal groove of the rotary valve and the locking block of the connector are inserted and engaged. The connector is fixedly installed on the power end of the valve drive unit.

4. The fully integrated microfluidic nucleic acid detection device according to claim 3, characterized in that, The card holder has connecting channels penetrating the box body at the bottom of its central cavity and functional cavities. The rotary valve has a central connecting hole and a side connecting hole at its top. The rotary valve has a connecting channel inside. The central connecting hole and the side connecting hole are connected to the two ends of the connecting channel, respectively. The central connecting hole is connected to the connecting channel at the bottom of the central cavity. The side connecting hole is connected to the connecting channels of different functional cavities by rotating the rotary valve to different angles.

5. The fully integrated microfluidic nucleic acid detection device according to claim 4, characterized in that, Both the ultrasound component and the moving temperature control component are slidably mounted on the slide plate. The slide plate is fixedly mounted relative to the slide base, and the slide base is slidably mounted on the base. Elastic elements are installed between the ultrasound component, the moving temperature control component, and the upright plate of the slide plate. The elastic elements are used to apply a pre-tightening force to the probe or the moving temperature control component so that the probe is in full contact with the side wall of the box or the moving temperature control component is in full contact with the amplification wafer. The probe is directly facing the lysis buffer chamber in the functional cavity inside the box. Other functional cavities are provided on both sides of the lysis buffer chamber.

6. The fully integrated microfluidic nucleic acid detection device according to claim 5, characterized in that, The fixed temperature control component is fixedly installed on the vertical surface of the side support plate. The bottom of the side support plate is vertically installed on the base. One side of the base plate is vertically installed on the vertical surface of the side support plate, and the other side is installed on a side vertical plate. The side vertical plate is vertically installed on the base. A guide rail for the reciprocating movement of the fluorescence detection component is also installed on the side support plate.

7. The fully integrated microfluidic nucleic acid detection device according to claim 6, characterized in that, The mobile temperature control assembly includes a mobile water-cooled plate fixedly installed relative to the mobile support and a mobile temperature control plate installed on the mobile water-cooled plate. One side of the mobile temperature control plate is in contact with the surface of the mobile water-cooled plate, and the other side is in contact with the surface of the amplification plate during amplification operations. The fixed temperature control assembly includes a fixed water-cooled plate fixedly installed relative to the side support plate and a fixed temperature control plate installed on the fixed water-cooled plate. One side of the fixed temperature control plate is in contact with the surface of the fixed water-cooled plate, and the other side is in contact with the surface of the amplification plate during amplification operations. Both the mobile water-cooled plate and the fixed water-cooled plate are connected to the water storage tank through pipelines.

8. The fully integrated microfluidic nucleic acid detection device according to claim 7, characterized in that, The inlet of the hollow plunger is connected to the heating unit via a pipeline. The inlet of the heating unit is connected to a solenoid valve. The solenoid valve includes a first solenoid valve and a second solenoid valve. The normally open end of the first solenoid valve is connected to the syringe pump, and the normally closed end of the first solenoid valve is connected to the atmosphere. The common end of the first solenoid valve is connected to the normally open end of the second solenoid valve, and the normally closed end of the second solenoid valve is connected to the air pump. The heating unit is connected in series between the common end of the second solenoid valve and the hollow plunger. The first and second solenoid valves work together to realize the syringe pump's actions of drawing negative pressure, inputting positive pressure, and performing precision infusion in the central cavity.

9. The fully integrated microfluidic nucleic acid detection device according to claim 8, characterized in that, The amplification chip contains multiple independently arranged amplification chambers connected in parallel. Each amplification chamber extends through both sides of the amplification chip. Each amplification chamber is arranged in parallel and connected to the liquid inlet of the amplification chip. The liquid inlet of the amplification chip is connected to the liquid outlet channel of the box body. The liquid outlet channel of the box body extends through the bottom of the box body and is connected to the side connection hole of the rotary valve when the rotary valve is rotated to a specific angle. Both sides of the amplification chamber are sealed by a sealing film.

10. A method for manufacturing a fully integrated microfluidic nucleic acid detection device, based on the fully integrated microfluidic nucleic acid detection device according to any one of claims 1-9, characterized in that, The steps of this method are as follows: S1. Cartridge assembly: Assemble the cartridge body, rotary valve, amplification strip and cap. After assembly, the functional chambers inside the cartridge are pre-filled with reagents and sealed with a membrane on top. Then, close the lid. At this time, the protrusions on the lid will puncture the membrane on the top of each functional chamber. S2. Card box installation: Insert the assembled card box along the base plate and install it. At the same time, insert the card block of the connecting seat of the valve drive unit at the bottom of the rotary valve and install it. The amplification chip side contacts the fixed temperature control plate. S3. Rotate the rotary valve until its side connection hole connects to the sample chamber. The sample liquid in the sample chamber is input into the lysis buffer chamber through the central chamber by the action of the solenoid valve and the injection pump. S4. The slide block moves the slide plate, and the probe of the ultrasonic component on the slide plate contacts the outer wall of the box. At the same time, the moving temperature control plate contacts the side of the amplification plate. Together with the fixed temperature control plate, they squeeze and contact the two sides of the amplification cavity. The ultrasonic component is driven to perform ultrasonic lysis on the solution in the lysis buffer cavity, complete the nucleic acid lysis, and release the nucleic acid template. S5. After lysis, the binding liquid in the binding liquid chamber is first drawn into the central chamber and then into the lysis liquid chamber by rotating the rotary valve in conjunction with the injection pump. Then the mixture is introduced into the high-level inlet of the extraction chamber. After the mixture is filtered by the filter membrane, it is discharged into the waste liquid chamber. The first and second washing liquid chambers are switched sequentially by rotating the rotary valve. The positive pressure drives the washing liquid to wash the lysis products twice to remove impurities and interferences, thus completing the nucleic acid purification. S6. The rotary valve and solenoid valve work together to connect the air pump and the central chamber, start the heating unit, and the gas blown out by the air pump is heated to 60°C by the heating unit. The hot gas enters the central chamber through the hollow plunger and then enters the filter membrane of the extraction chamber, so that the residual ethanol in the nucleic acid adsorbed by the filter membrane is dried and evaporated. S7. The rotary valve and solenoid valve work together to connect the injection pump to the central chamber, switch the elution chamber path, and drive the elution solution through the central chamber, through the low inlet and filter membrane of the extraction chamber, to elute the purified nucleic acid and obtain a high-purity nucleic acid template solution. S8, the rotary valve and the solenoid valve work together to draw negative pressure into the amplification chamber of the amplification disc, and then the nucleic acid template solution in the extraction chamber is transported to the amplification chamber through the positive pressure of the central chamber combined with the negative pressure, and fully reconstituted with the PCR cake-shaped lyophilized reaction reagent pre-placed in the amplification chamber to form a PCR reaction system; S9. When the temperature control component is working, the fixed temperature control plate and the moving temperature control plate heat up evenly to make contact with the amplification cavity. When cooling is required, the fixed temperature control plate and the moving temperature control plate are powered in opposite directions to cool down, which, together with the fixed water cooling plate and the moving water cooling plate, cools down the amplification cavity evenly. The heat is transferred to the water storage tank through heat transfer. During the amplification process, the fluorescence detection component continuously detects and collects signals to achieve qualitative and quantitative detection of nucleic acids.

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