Multiple nucleic acid detection device and use method

Abstract

The invention discloses a multiple nucleic acid detection device and a use method, the multiple nucleic acid detection device comprises a card box and multiple chips which are communicated with each other, multiple detection of the whole process is realized in a form of integrating the extraction card box and the multiple detection chips, the sample transfer step is reduced, the pollution risk is reduced, the detection sensitivity is improved, and the detectable target range is widened. A plurality of independent functional chambers are arranged in the card box, under the cooperation of a near-end flow channel opening and a far-end flow channel opening, a reaction chamber is communicated with other functional chambers and sample injection holes required by nucleic acid reaction, and tedious magnetic bead method nucleic acid extraction, nucleic acid release and removal of impurities in a sample are realized in the card box to obtain pure nucleic acid. Meanwhile, the nucleic acid concentration is improved, the subsequent detection sensitivity can be improved, the internal part of the extraction card box, the internal part of the detection chip and the connection between the card box and the chip are fully sealed, and cross contamination among multiple reaction chambers and aerosol pollution to the environment during amplification are prevented.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology and relates to a multiplex nucleic acid detection device and its usage method. Background Technology

[0002] In recent years, nucleic acid-based detection methods have been accepted in clinical diagnostics due to their high specificity, ease of configuration to detect virtually any target, minimal sample size requirements (high sensitivity), and automation capabilities. Polymerase chain reaction (PCR) has become the method of choice for rapid diagnosis of infectious agents, as it can be used to detect pathogens with high sensitivity and specificity. However, nucleic acid amplification detection requires a series of complex processing steps, including collecting clinical samples and transporting them to a central laboratory, lysis / digestion, nucleic acid binding, buffer washing and nucleic acid elution to extract pathogenic DNA / RNA, reverse transcription of RNA, and then PCR. This method typically requires specialized equipment and highly trained technicians in a laboratory setting. Furthermore, the manual handling process, especially the multiple steps of reopening test tubes and pipetting, can lead to accidental laboratory-acquired infections or aerosol contamination between samples.

[0003] To meet the demand for rapid nucleic acid testing in off-site laboratory environments, point-of-care (POCT) systems highly integrate the entire nucleic acid testing process into a single small device. They are easy to operate, significantly reducing reliance on professional personnel and dedicated laboratory environments, thus effectively expanding testing accessibility. Miniaturization, portability, and ease of operation are the main characteristics of POCT, which can also shorten testing time, providing timely diagnosis and treatment for patients in acute and remote areas, thereby reducing morbidity and mortality. Currently, POCT still faces some challenges in clinical and field applications. The testing cost of POCT is relatively high, and there are gaps in the stability and reliability of POCT testing.

[0004] Furthermore, with the expansion of the disease spectrum and the accelerated mutation rate of pathogens, single-target detection can no longer meet clinical and research needs. For example, infectious disease control requires simultaneous screening for multiple pathogens (such as influenza virus, SARS-CoV-2 and its variants). Traditional nucleic acid detection technologies (such as single PCR) suffer from low throughput, high cost, and cumbersome operation. Therefore, there is a need to develop a rapid, portable, and more automated integrated multiplex target point-of-care testing (POCT) device that can provide nucleic acid test results.

[0005] Currently, many microfluidic devices can perform integrated and automated molecular diagnostics. Utilizing internal microchannel networks and microvalve structures, combined with external control units such as temperature, magnetic, and optical systems, various operations that previously required manual intervention are integrated onto a single microchip. This automates the entire detection and analysis process and reduces the risk of cross-contamination caused by manual operation. The increasing maturity of integrated and automated microfluidic chip detection systems facilitates the development of more portable, sensitive, reliable, and low-cost point-of-care testing (POCT). Furthermore, microfluidic chips, through integrated design, enable high-throughput parallel detection, simplifying multiplex detection processes and reducing costs. While mature microfluidic chip products integrating nucleic acid purification and multiplex amplification detection functions exist, their usage costs are high.

[0006] US Patent 8673238B2 discloses Cepheid's GeneXpert molecular diagnostic microfluidic kit and a dedicated fully automated analysis instrument for this kit. The GeneXpert molecular diagnostic system employs column-based full extraction technology and single-cavity qPCR technology, requiring no manual intervention throughout the process. The use of disposable, independent modules avoids the risk of contamination. Full extraction and real-time monitoring of qPCR amplification signals effectively ensure the product's sensitivity and specificity. GeneXpert supports multiple target detection; a single qPCR amplification cavity, combined with six fluorescence channels, allows a single GeneXpert cartridge to detect up to five indicators at a time (internal quality control uses one fluorescence channel). Therefore, GeneXpert products are only suitable for single-sample, low-indicator testing scenarios, typically in community hospitals and outpatient clinics. Furthermore, the patented technology relies entirely on Cepheid's GeneXpert platform; the GeneXpert system instruments and kits are expensive, making its widespread adoption difficult in resource-scarce areas.

[0007] US Patent 8940526B2 discloses BioFire's FilmArray microfluidic chip, a powerful, highly sensitive, and multiplex detection system that uses nested PCR and numerous individual microwells to detect multiple targets. This patent allows for a single detection of 2.0-30 pathogens. Samples (such as nasal swabs, feces, blood, etc.) are simply processed and added directly to the chip's reagent pouch. The pouch contains all the necessary lyophilized reagents (including primers, probes, enzymes, etc.). After sample addition and loading, the gasbag within the instrument works in conjunction with the chip's multi-chamber microfluidic structure. The gasbag's compression drives the liquid to flow between different zones, automatically completing the magnetic bead-based nucleic acid extraction, amplification, and detection. Specifically, the patent discloses that the chip consists of an upper reservoir section and a lower reaction layer. The reservoir section contains pre-filled lyophilized reagents, which are reconstituted with a dissolving solution before use. The reaction layer uses a flexible pouch to partition the cell lysis zone, nucleic acid purification zone, and amplification zone. Liquid flow between these zones is achieved through gasbag compression within the device. While the microfluidic chip boasts low material costs, its packaging requires sophisticated processes and maintains an extremely high level of vacuum within the chip, placing stringent manufacturing demands. Furthermore, its detection relies entirely on the FilmArray system, making it incompatible with other devices and limiting scalability. Each instrument can only process one sample at a time, necessitating multiple instruments in parallel for high-throughput scenarios, resulting in lower efficiency. Additionally, the chip and instrument are expensive, leading to higher costs per detection compared to traditional methods, potentially restricting its application in resource-constrained regions.

[0008] From the current state of technological development, microfluidic chip research is still mainly in the laboratory stage, generally adopting a highly integrated design strategy, integrating precision functional components such as microvalves inside the chip. This complex architecture not only significantly increases the complexity of the manufacturing process, leading to increased difficulty in mass production and high costs, but also requires a complex external control system for precise control, further restricting its industrialization process. To reduce the control system required for microfluidic chips, patent CN115340944A discloses a microfluidic chip for multiplex nucleic acid detection of extracted samples. It achieves directional liquid injection by setting multiple injection holes and vent holes, and seals the reaction chamber with a waterproof and breathable membrane to avoid environmental contamination. However, the waterproof and breathable membrane cannot prevent water vapor leakage, and cannot effectively prevent nucleic acid molecule leakage and aerosol contamination. In addition, the reaction chambers in this patent do not form a closed system, and do not consider the problem of cross-contamination between chambers in multiplex detection, resulting in a decrease in the accuracy of multi-target nucleic acid detection results. To address the current challenges of integrating and controlling nucleic acid extraction and multiplexing, which are difficult and costly, this invention proposes a system combining an extraction cartridge and a multiplexing chip. This system can reliably and stably achieve multiplexing, avoiding cross-contamination and aerosol contamination, while also reducing the cost of implementing multiplexing sample control. Summary of the Invention

[0009] The purpose of this invention is to solve the problems in the prior art and provide a multiplex nucleic acid detection device and its usage method.

[0010] To achieve the above objectives, the present invention employs the following technical solution:

[0011] A multiplex nucleic acid detection device includes interconnected cartridges and multiple chips;

[0012] The cartridge includes a chamber body, inside which a fluid control chamber is arranged, and around the fluid control chamber are distributed a reaction chamber, other functional chambers required for nucleic acid reaction, a gas connection column, a plunger chamber and a sample injection port;

[0013] The fluid control chamber is equipped with a rotary valve, and the bottom of the rotary valve has a proximal flow channel and a distal flow channel that are interconnected. The fluid control chamber also has a central flow channel that is connected to the reaction chamber. The central flow channel is surrounded by inner flow channels, and the fluid control chamber is connected to other functional chambers and sample injection ports required for nucleic acid reaction through the inner flow channels.

[0014] When the rotary valve is installed inside the fluid control chamber, the proximal flow port is connected to the central flow port. When the rotary valve is rotated circumferentially, the distal flow port can be connected to any one of the internal flow ports.

[0015] The plunger chamber is used to provide gas pressure, and the plunger chamber is connected to the reaction chamber through a gas connecting column;

[0016] The injection port connects to multiple chips.

[0017] A further improvement of the present invention is that:

[0018] Other functional chambers required for the nucleic acid reaction include a lysis chamber, a drying chamber, a waste liquid chamber, a first washing chamber, a second washing chamber, an elution chamber, and an oil storage chamber;

[0019] The pyrolysis chamber, drying chamber, waste liquid chamber, first washing chamber, second washing chamber, elution chamber and oil storage chamber are all provided with connecting through holes, and each connecting through hole is connected to the inner flow channels distributed circumferentially around the central flow channel.

[0020] The central flow port is located at the center of the lower end face of the fluid control chamber, the proximal flow port is opened at the center of the lower end face of the rotary valve, and the external flow port of the reaction chamber is opened inside the reaction chamber, which is connected to the central flow port.

[0021] The rotary valve has a microchannel inside, and the proximal flow port and the distal flow port are connected through the microchannel.

[0022] A plunger hole is provided at the lower end of the plunger chamber, and the connecting column has a hollow internal structure. Connecting air holes are provided at both the upper and lower ends of the connecting column.

[0023] A vent hole is provided at the upper end of the reaction chamber, through which the reaction chamber is connected to the connecting column and the plunger chamber.

[0024] The lower and upper end faces of the reaction chamber are both circular, with the diameter of the lower end face being smaller than the diameter of the upper end face.

[0025] The reaction chamber includes a first chamber and a second chamber connected together. The first chamber is cylindrical and the second chamber is truncated conical.

[0026] The multi-chip includes a cover plate, an airflow layer, and a sample introduction layer connected sequentially from top to bottom;

[0027] The sample injection layer has a first liquid main channel, one end of which is connected to the sample injection port and the other end is connected to the connection port. The first liquid main channel is also connected to several first reaction chambers, and the first reaction chambers are connected to the outlet end of the short exhaust channel.

[0028] The airflow layer has a first exhaust hole that is connected to the outlet end of the short exhaust channel. The first exhaust hole is connected to the first main gas channel. One end of the first main gas channel is connected to the outlet of the first channel, and the other end is connected to the valve control hole.

[0029] The cover plate is provided with an air chamber and a valve control chamber. The air chamber is connected to the outlet of the first flow channel, and a sliding plunger is provided in the air chamber.

[0030] The valve control chamber is connected to the valve control hole and the connection hole in sequence. A valve control device is installed inside the valve control chamber. When the valve control device moves downward to the bottom, the lower end of the valve control device is embedded into the valve control hole and the connection hole in sequence, and the connection hole is closed at this time. When the valve control device moves upward, the valve control device is separated from the connection hole and the valve control hole, and the connection hole is opened at this time. The first liquid main channel, the connection hole, the valve control hole, the first gas main channel, the first flow channel outlet and the gas chamber are connected.

[0031] The upper end of the outlet of the short exhaust channel is covered with a first waterproof and breathable membrane.

[0032] A method for using a multiplex nucleic acid detection device includes the following steps:

[0033] Step 1: With the rotary valve closed, inject the sample into the corresponding functional chamber. The sample is then subjected to lysis, adsorption, washing, drying, and elution processes in sequence. During processing, the rotary valve is rotated according to the reaction requirements of each stage. The reaction chamber is connected to any corresponding functional chamber through the coordination of the proximal flow port, the distal flow port, and the inner flow port. Sample transfer is accomplished using the gas pressure provided by the plunger chamber.

[0034] Step 2: After elution, the reaction solution is located in the reaction chamber. Turn the rotary valve to connect the reaction chamber and the injection port, and inject the reaction solution into the multi-chip for detection through the injection port.

[0035] Step 1 includes the following steps:

[0036] Lysis: The sample is injected into the lysis chamber, and the sample begins to lyse with the lysis buffer pre-stored in the lysis chamber;

[0037] Adsorption: Rotate the rotary valve to connect the reaction chamber and the waste liquid chamber through the distal flow port, and transfer the pre-stored magnetic bead buffer solution in the reaction chamber to the waste liquid chamber;

[0038] Rotate the rotary valve to connect the reaction chamber and the lysis chamber through the distal flow port, and transfer the lysed sample into the reaction chamber. Close the rotary valve and start nucleic acid adsorption through the magnetic beads pre-stored in the reaction chamber. After adsorption is complete, rotate the rotary valve to connect the reaction chamber and the waste liquid chamber through the distal flow port, and transfer the liquid in the reaction chamber to the waste liquid chamber.

[0039] Washing: Turn the rotary valve to connect the reaction chamber and the washing chamber through the distal flow port, and transfer the washing liquid in the washing chamber to the reaction chamber for washing. After washing is completed, turn the rotary valve to connect the reaction chamber and the waste liquid chamber through the distal flow port, and transfer the liquid to the waste liquid chamber.

[0040] Drying: Turn the rotary valve to connect the reaction chamber and the drying chamber through the distal flow port, and blow the residual liquid in the reaction chamber into the drying chamber;

[0041] Elution: Rotate the rotary valve to connect the reaction chamber and the elution chamber through the distal flow port, and transfer the elution solution to the reaction chamber for nucleic acid elution.

[0042] Step 2 includes the following steps:

[0043] Sample injection: After elution, rotate the rotary valve to connect the reaction chamber and the injection port through the distal flow channel. The liquid in the reaction chamber enters each of the first reaction chambers in sequence through the injection port and the first liquid main channel. A first waterproof and breathable membrane 13 is set at the first exhaust port. The first waterproof and breathable membrane 13 is used to block the liquid in the first reaction chamber.

[0044] Exhaust: During sample injection, the gas inside the injection layer is propelled by the liquid through the first reaction chamber, the outlet end of the short exhaust channel, the first exhaust hole, the first gas main channel, and the outlet of the first channel into the gas chamber. At this time, the lower end of the valve control will block the connection hole, and the connection hole will be closed.

[0045] Reaction chamber separation: After the sample injection is completed, rotate the rotary valve to connect the reaction chamber and the oil storage chamber through the distal flow channel port, and draw the hydrophobic reagent in the oil storage chamber into the reaction chamber. Rotate the rotary valve to connect the reaction chamber and the injection port through the distal flow channel port, and transfer the hydrophobic reagent from the reaction chamber to the first liquid main flow channel. Move the valve control upward to separate the valve control from the connection hole, open the connection hole, and the hydrophobic reagent enters the gas chamber through the connection hole, valve control hole, first gas main flow channel and first flow channel outlet.

[0046] After the reaction chambers are separated, the nucleic acid reaction begins.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] This invention discloses a multiplex nucleic acid detection device, comprising interconnected cartridges and multiplex chips. The device integrates the extraction cartridge and the multiplex detection chip to achieve full-process multiplex detection, reducing sample transfer steps, lowering the risk of contamination, and improving detection sensitivity and the detectable target range. The cartridge contains multiple independent functional chambers. With the cooperation of proximal and distal flow channels, the reaction chamber is connected to other functional chambers and injection wells required for nucleic acid reaction. This allows for the cumbersome magnetic bead-based nucleic acid extraction within the cartridge, releasing nucleic acid and removing impurities from the sample to obtain pure nucleic acid. Simultaneously, it increases the nucleic acid concentration, contributing to improved sensitivity in subsequent detection. The internal structure of the extraction cartridge, the internal structure of the detection chip, and the connections between the cartridge and the chip are all kept airtight to prevent internal cross-contamination during amplification and aerosol pollution to the environment.

[0049] Furthermore, in this invention, multiple chips support independent amplification and detection of multiple target nucleic acids through independent reaction chambers, thus avoiding cross-contamination.

[0050] Furthermore, in this invention, the detection chip is equipped with a waterproof and breathable membrane at the exhaust end of the reaction chamber, which avoids cross-contamination while enabling precise fluid control: Firstly, the waterproof and breathable membrane allows gas to escape while effectively blocking liquid leakage, eliminating the risk of cross-contamination between adjacent chambers; secondly, by maintaining a stable total amount of reaction liquid, it eliminates the loss of reaction liquid caused by liquid overflow, ensuring that the reaction liquid is completely and directionally introduced into the chamber and achieves a uniform and equal distribution, thereby solving the problem of multiplex detection quantitative error caused by incomplete filling or local concentration deviation, and improving the reliability and stability of multiplex nucleic acid quantitative detection.

[0051] Furthermore, in this invention, the detection chip integrates multiple independent reaction chambers, supporting parallel amplification and detection of multiple nucleic acids. The oil storage chamber is used for hydrophobic agents. The two ends of each reaction chamber are physically separated by hydrophobic reagents. This reduces the number of control valves required for the chip and the complexity of the chip, while achieving full enclosure of the reaction chambers. This avoids the evaporation and reduction of the reaction liquid at high temperatures and prevents cross-contamination between the reaction chambers.

[0052] Furthermore, in this invention, an airflow drying step is added to the nucleic acid extraction step after washing to accelerate the volatilization of inhibitors such as ethanol in the washing solution and reduce the inhibitory effect of residual alcohols in nucleic acid extraction on downstream nucleic acid amplification. Attached Figure Description

[0053] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0054] Figure 1 This is a schematic diagram of the overall assembly isometric view of the present invention.

[0055] Figure 2 This is a front view of the overall assembly of the present invention.

[0056] Figure 3 This is a top view of the overall assembly of the present invention.

[0057] Figure 4 This is an exploded view of the overall assembly of the present invention (where a is the exploded view). Figure 1 ; b is an explosion symbol Figure 2 ).

[0058] Figure 5 This is a bottom view of the integrated sample introduction layer structure of the extraction cartridge body and the detection chip of the present invention.

[0059] Figure 6 This is a front view of the integrated sample introduction layer structure of the extraction cartridge body and the detection chip of the present invention.

[0060] Figure 7 This is a top view of the integrated sample introduction layer structure of the extraction cartridge body and the detection chip of the present invention.

[0061] Figure 8 This is a schematic diagram of the rotary valve of the present invention.

[0062] Figure 9 This is a schematic diagram of the rotary valve of the present invention (where a is a top view of the rotary valve; b is a cross-sectional view along line AA of figure a).

[0063] Figure 10 This is a top view of the assembly of the present invention. Figure 1 (Where, a is a top view of the assembly after the waterproof and breathable membrane has been removed; b is a cross-sectional view showing the connection between the reaction chamber and the pyrolysis chamber).

[0064] Figure 11Top view of the assembly of the present invention Figure 2 (Where, a is a top view of the assembly after removing the plunger; b is a cross-sectional view of the gas passage connecting the plunger chamber and the reaction chamber).

[0065] Figure 12 This is the overall assembly of the present invention (where a is a top view of the assembly of the invention; b is a cross-sectional view of the amplification injection of the nucleic acid extraction and multiplex detection device).

[0066] Figure 13 A schematic diagram of the internal flow channels of the detection chip assembly.

[0067] Figure 14 This is a top view of the airflow layer chip.

[0068] Figure 15 This is a bottom view of the airflow layer chip.

[0069] Figure 16 This is a chip assembly diagram for Example 2.

[0070] Figure 17 This is a top view of the chip sample introduction layer in Example 2.

[0071] Figure 18 This is a top view of the chip sample introduction layer in Example 2.

[0072] Figure 19 This is a top view of the chip airflow layer in Example 2.

[0073] Wherein: 1-Clamping component, 2-Rotary valve, 3-Sealing gasket, 4-Second waterproof and breathable membrane, 5-Top cover, 6-Plunger, 7-Cavity body, 8-Sample injection layer, 9-Airflow layer, 10-Cover plate, 11-Sliding plunger, 12-Valve control, 13-First waterproof and breathable membrane, 14-Sample injection layer two, 15-Airflow layer two, 201-Proximal flow channel opening, 202-Microchannel, 203-Distal flow channel opening, 204-Wedge-shaped groove, 501-Reaction chamber vent, 502-Gas channel, 503-Through hole, 504-Cycle chamber vent, 505-Drying chamber vent Holes, 506-Vent hole for waste liquid chamber, 507-Vent hole for first washing chamber, 508-Vent hole for second washing chamber, 509-Vent hole for elution chamber, 510-Vent hole for oil storage chamber, 701-Reaction chamber, 702-Pyrolysis chamber, 703-Drying chamber, 704-Waste liquid chamber, 705-Fluid control chamber, 706-First washing chamber, 707-Second washing chamber, 708-Elution chamber, 709-Oil storage chamber, 710-Gas connection column, 711-Plunger chamber, 7100-Liquid flow channel, 721-Outlet of outer flow channel of reaction chamber, 722-Outlet of outer flow channel of pyrolysis chamber, 72 3-Drying chamber outer flow channel inlet, 724-Waste liquid outer flow channel inlet, 725-First washing chamber outer flow channel inlet, 726-Second washing chamber outer flow channel inlet, 727-Eluting chamber outer flow channel inlet, 728-Oil storage chamber outer flow channel inlet, 729-Plunger chamber bottom through hole, 731-Central flow channel inlet, 780-Sample injection hole, 71-Reaction chamber bottom groove, 72-Cyclic chamber bottom groove, 801-First liquid main flow channel, 802-Liquid branch flow channel, 803-Short exhaust flow channel, 804-First reaction chamber, 805-Short exhaust flow channel outlet end, 806-Connecting hole, 90 1-First exhaust port, 902-First gas branch channel, 903-First channel outlet, 904-First gas main channel, 905-Valve control port, 906-Counter hole, 1001-Gas chamber, 1002-Valve control chamber, 1401-Sample injection port two, 1402-Second liquid main channel, 1403-Liquid branch channel, 1404-Intermediate distribution chamber, 1405-Second reaction chamber, 1501-Second channel outlet, 1502-Second gas main channel, 1503-Second gas branch channel, 1504-Intermediate chamber, 1505-Second exhaust port. Detailed Implementation

[0074] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0075] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0076] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0077] In the description of the embodiments of the present invention, it should be noted that if terms such as "upper," "lower," "horizontal," or "inner" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product of the invention is in use, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. Furthermore, terms such as "first" and "second" are only used to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0078] Furthermore, the use of the term "horizontal" does not imply that the component must be absolutely horizontal, but rather that it can be slightly tilted. For example, "horizontal" simply means that its direction is more horizontal than "vertical," and does not mean that the structure must be completely horizontal, but can be slightly tilted.

[0079] In the description of the embodiments of the present invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in the present invention according to the specific circumstances.

[0080] The present invention will now be described in further detail with reference to the accompanying drawings:

[0081] See Figure 1 This invention discloses a multiplex nucleic acid detection device that can reliably and stably achieve multiplex detection, avoiding cross-contamination and aerosol contamination problems, while reducing the implementation cost of multiplex detection sample control. The purpose of this invention is to provide a multiplex nucleic acid detection method for rapid screening of various pathogens, improve detection efficiency and stability through nucleic acid extraction, shorten detection time through automated operation, and reduce the risk of cross-contamination through a closed system design.

[0082] The technical solution adopted in this invention is to provide a nucleic acid detection kit, including an extraction cartridge and an amplification detection chip packaged together. The extraction cartridge consists of multiple chambers and a rotatable rotary valve 2. The chambers include a reagent chamber (lysis chamber 702, washing chamber, elution chamber 708), a waste liquid chamber 704, and a reaction chamber 701. The flow path of each chamber is controlled by rotating the rotary valve 2, and positive and negative driving pressure is provided to the reaction chamber 701 under the action of the plunger 6 or an air pump, realizing the directional transfer of liquid between the reaction chamber 701 and other chambers. The reaction solution pre-stored in the reagent chamber is transferred to the reaction chamber 701 through the rotary valve 2-plunger 6 linkage mechanism to complete the reaction, and then removed from the reaction chamber 701, thus completing the various steps of nucleic acid extraction in sequence, thereby realizing nucleic acid extraction based on the magnetic bead method.

[0083] Furthermore, the reaction chamber 701 and lysis chamber 702 integrate ultrasonic generators, allowing seamless docking with external ultrasonic probes. Through ultrasonic cavitation, they enhance cell lysis, accelerate cell membrane disruption, and release nucleic acids; promote homogenization of the magnetic beads and reaction solution, significantly improving extraction efficiency. After sample extraction, the channels of the detection chip are opened, and the extracted sample is injected into the chip. A sealed flow channel connects the detection chip and the extraction cartridge, controlled by a rotary valve 2 and driven by a plunger 6 to achieve directional fluid transport. The detection chip consists of liquid channels, multiple amplification chambers, and gas channels. After extraction, the sample uniformly fills each amplification chamber. After injection, a hydrophobic reagent is injected to seal the amplification chambers, ensuring complete sealing of each chamber, preventing cross-contamination, preventing evaporation of the reaction solution, reducing liquid levels within the amplification chambers to avoid affecting fluorescence detection, and ensuring environmental contamination.

[0084] The extraction cartridge simplifies the operation process through a linkage mechanism between rotary valve 2 and plunger 6: rotating rotary valve 2 to the target position, pulling plunger 6 extracts the reagent, and then rotating it a second time before pushing it down to complete the transfer, thus simplifying the cumbersome nucleic acid extraction steps and enabling automated completion under closed conditions. After nucleic acid extraction, rotating rotary valve 2 connects the intermediate plunger 6 chamber to the detection chip. The plunger 6 chamber provides positive pressure to the reaction chamber 701, injecting the extracted sample into the detection chip, where multiple target nucleic acid detections are performed.

[0085] This invention, based on a cartridge and chip combined structure, is used for nucleic acid extraction and simultaneous amplification and quantitative detection of multiplex nucleic acids. Its core functions include:

[0086] First, nucleic acid extraction and purification: Based on the cartridge structure design, the cumbersome magnetic bead method for nucleic acid extraction is realized within the cartridge, releasing nucleic acid and removing impurities from the sample to obtain pure nucleic acid, while increasing the nucleic acid concentration, which helps to improve the sensitivity of subsequent detection.

[0087] Second, ultrasonic lysis and mixing: biological samples are lysed and nucleic acids are released through non-contact ultrasonic disruption; at the same time, ultrasonic waves are used to mix magnetic beads and reaction reagents during the nucleic acid extraction process to improve the nucleic acid extraction efficiency.

[0088] Third, spatial multiplex nucleic acid detection: independent reaction chambers support independent amplification and detection of multiple target nucleic acids, avoiding cross-contamination.

[0089] Fourth, quantitative control of fluid in the amplification chamber: The fluid control system, which combines branched flow channels and a waterproof and breathable membrane, achieves uniform sample distribution and fluid control through a gas-liquid isolation barrier, ensuring a constant volume of amplification reaction liquid to achieve accurate quantitative detection.

[0090] Fifth, pollution protection: The nucleic acid extraction cartridge uses a waterproof and breathable membrane to isolate the reaction system from the environment, avoiding leakage and contamination of biological reagents. The interior of the extraction cartridge, the interior of the detection chip, and the connection between the cartridge and the chip are all kept fully sealed to prevent internal cross-contamination during amplification and aerosol pollution to the environment.

[0091] Sixth, suppressing heating evaporation: The detection chip employs hydrophobic reagent separation technology to suppress the evaporation of the reaction solution at high temperatures and achieve physical isolation between chambers, ensuring detection stability. Using hydrophobic reagent separation technology to achieve the sealing of several chambers in one step reduces the complexity of microvalve structures and chip fabrication and control.

[0092] Example 1

[0093] The nucleic acid extraction cartridge adopts a waterproof and breathable membrane design to prevent contamination. The cartridge is equipped with ventilation holes and uses a waterproof and breathable membrane to prevent liquid leakage in the reaction system, thus balancing the air pressure balance inside the cartridge and biosafety.

[0094] The nucleic acid extraction cartridge uses a rotary valve 2 as the core of fluid control, and the connection between the functional chambers is realized through the microchannel inside the rotary valve 2.

[0095] The main chamber 7 comprises a plunger chamber 6 711, a lysis chamber 702, a washing chamber, an elution chamber 708, a waste liquid chamber 704, and a reaction chamber 701 with pre-embedded functionalized magnetic beads. The plunger 6 device integrated in the plunger chamber 6 711, in conjunction with the rotary valve 2, enables precise transfer of liquid across chambers, suitable for controlling large volumes of liquid in high-throughput processing. The magnetic beads are surface-modified with specific ligands for efficient adsorption of nucleic acids; by placing a magnet outside the reaction chamber, the magnetic beads can be fixed to achieve liquid-solid separation.

[0096] The nucleic acid extraction cartridge has a reaction chamber 701 connected to a plunger chamber 711. A rotary valve 2 directs the flow of a microchannel between the reaction chamber 701 and a specific chamber. The positive and negative pressure applied to the reaction chamber 701 by the positive and negative movement of the plunger 6 allows for the discharge or injection of reagents within the reaction chamber. An external magnet then adheres and fixes magnetic beads to achieve nucleic acid separation. Each chamber has a vent at the top and is covered by a second waterproof and breathable membrane 4, ensuring pressure balance and smooth liquid transfer while preventing liquid / biomolecule leakage, thus achieving leak-proof and contamination-proof functions.

[0097] The nucleic acid extraction cartridge achieves broad-spectrum lysis through chemical-ultrasonic lysis. At the start of the reaction, the sample is added to the lysis chamber 702 while the rotary valve 2 is closed. The ultrasonic generator is in close contact with the outer wall of the lysis chamber 702. After the generator is started, the lysis chamber 702 is assisted by chemical reagents for ultrasonic lysis, and the heat generated by the ultrasound accelerates the rapid lysis of most sample types.

[0098] The nucleic acid extraction cartridge promotes nucleic acid adsorption and purification through ultrasonic mixing. During the nucleic acid extraction process, when separating the magnetic beads from the liquid, an external magnetic field is required to adsorb and fix the magnetic beads. After the magnetic beads agglomerate under the action of the magnetic field, they are difficult to redisperse in the solution even if the external magnetic field disappears. Ultrasonic mixing can achieve redispersing of the magnetic beads within 10 seconds. Ultrasonic mixing is used in the nucleic acid adsorption, washing and elution steps during the nucleic acid extraction reaction to promote nucleic acid adsorption and purification.

[0099] Ultrasonic waves are used to promote nucleic acid lysis, adsorption, and purification, with the waste liquid being centrally stored in a 704 waste liquid chamber via a dedicated channel. This approach achieves a fully closed-loop nucleic acid extraction process while maintaining operational flexibility, improving the sensitivity and stability of subsequent detection, and is suitable for high-sensitivity detection scenarios.

[0100] The bottom of the reaction chamber 701 and the pyrolysis chamber 702 of the cartridge are respectively provided with bottom grooves 71 and 72, which reduce the container wall thickness at the point of close contact with the ultrasonic generator and improve ultrasonic efficiency.

[0101] During nucleic acid extraction, the maximum volume of liquid that needs to be ultrasonically mixed in reaction chamber 701 is 1700 μL for washing solution and the minimum is 300 μL for elution solution. To avoid excessive ultrasonication due to a low liquid level in reaction chamber 701 during elution, the internal space of reaction chamber 701 is conical, which ensures a high liquid level even when the amount of reaction solution is small. This also prevents the reaction solution from oscillating and splashing under ultrasonic waves, thus reducing the amount of reaction solution transferred downstream.

[0102] Meanwhile, the washing solution used in magnetic bead nucleic acid extraction typically contains alcohols such as ethanol. During nucleic acid extraction, these alcohols remain in the reaction chamber and on the magnetic beads, eventually entering the elution solution and participating in the final amplification reaction. These alcohols can inhibit nucleic acid amplification. In this invention, to reduce the amount of alcohol entering the downstream reaction, a drying process is added after the initial washing. After washing and removing the waste liquid, the rotary valve 2 is connected to the drying chamber 703. A plunger 6 or an air pump generates airflow to accelerate the evaporation of residual alcohols in the reaction chamber 701 and blow any remaining trace amounts of liquid from the flow channels or dead corners of the chamber into the drying chamber 703.

[0103] The detection chip is based on a multi-layer microfluidic structure design, with the main body of the chip consisting of three layers: upper, middle, and lower.

[0104] Sample injection layer 8 is equipped with multiple independent reaction chambers, which are connected by the injection port through the main liquid channel and the branch liquid channel. The sample uniformly fills the reaction chambers and reacts.

[0105] The middle airflow layer 9 integrates an exhaust port array. Each reaction chamber forms a "one chamber, one port" directional exhaust system through a microchannel and a corresponding exhaust port, ensuring that the gas in the reaction chamber is discharged in one direction.

[0106] The upper cover plate 10 seals the middle airflow layer 9 and provides an air chamber to ensure air pressure balance and avoid resistance during sample injection.

[0107] In addition to the three main layers, the chip also has a sealing layer on its lower surface. The thin film sealing reduces the thickness and improves the heat transfer efficiency when the reaction chamber is heated.

[0108] A first waterproof and breathable membrane 13 is provided at the exhaust port connecting the sample injection layer 8 and the airflow layer 9 on the detection chip. This membrane allows gas to pass through while blocking liquid, forming a gas-liquid isolation barrier. This prevents the reaction liquid from entering the airflow layer 9, avoiding contamination and sample waste. At the same time, it restricts the flow of liquid to ensure that it completely fills all reaction chambers, maintaining a constant volume of reaction liquid within the reaction chambers and improving the accuracy of quantitative detection.

[0109] The main gas channel connects to the gas chamber to balance the internal gas pressure of the chip. The gas chamber adopts a deformable material (such as silicone film) or a plunger 6-chamber 711 structure. By changing the volume, it adapts to the dynamic pressure changes of the fluid while maintaining the chip's sealing, ensuring that the chip is absolutely sealed and avoiding aerosol contamination.

[0110] After sample injection, a hydrophobic reagent is injected through injection port 780, and the flow path between sample injection layer 8 and gas flow layer 9 is controlled by the valve body.

[0111] During the sample introduction phase, the connection holes of the gas and liquid main channels are closed, and the hydrophilic sample solution fills the reaction chamber through the liquid branch channel. During the separation phase, the connection holes of the gas and liquid main channels are opened, and the hydrophobic reagent enters the gas and liquid main channels, forming an immiscible interface with the hydrophilic sample solution, physically separating the reaction chambers. The injection of the hydrophobic reagent creates an immiscible phase interface between the reaction chambers, and simultaneously forms a gas-liquid two-phase interface at the outlet of the gas branch channel. Sealing the sample inlet and outlet ends of the reaction chambers achieves a double-end seal, preventing crosstalk between the reaction chambers, suppressing evaporation of the reaction liquid during heating, and ensuring the stability of the reaction system.

[0112] The entire process of nucleic acid extraction and detection is as follows: After sampling, the sample is injected into the lysis chamber 702. The rotary valve 2 is rotated to open the fluid channel. Under the action of the plunger 6, the liquid transfer is controlled. The sample is transferred to the reaction chamber 701 and then the sample is extracted using the magnetic bead method (lysis, washing, and elution of nucleic acid). After extraction, the extracted sample is transferred to the plunger 6 chamber 711. The rotary valve 2 is rotated to connect the plunger 6 chamber 711 to the injection port. The plunger 6 is pressed down to inject the extracted sample into the detection chip. The extracted sample enters several reaction chambers of the chip. At the same time, the gas is discharged from the exhaust port through the first waterproof and breathable membrane 13, while the reaction liquid cannot pass through the exhaust port and is blocked and restricted in the reaction chamber by the first waterproof and breathable membrane 13. The gas enters the gas flow layer 9 from the exhaust port and then enters the gas chamber from the main gas flow channel of the gas flow layer 9.

[0113] After sample injection, rotating valve 2 transfers the pre-stored hydrophobic reagent in the cartridge to plunger chamber 6 (711) and injects it into the detection chip through the injection port. At this point, the connection port opens, allowing the hydrophobic reagent to enter the gas flow channel 9 from the liquid main channel of the sample injection layer 8 via the connection port. It forms a stable phase interface with the hydrophilic reaction liquid of the sample injection layer 8 and the gas of the gas flow layer 9, creating a double-ended physical barrier at the sample injection and exhaust ends of the reaction chamber. This ensures independent sealing between each reaction chamber and between the reaction chamber and the outside environment. Each reaction chamber contains pre-embedded lyophilized PCR reagents for different targets, enabling the detection of different targets in different chambers. The temperature control module heats the chip to initiate the amplification reaction and performs real-time fluorescence detection, achieving simultaneous quantitative detection of multiple targets in space. The entire extraction and detection process is operated by a matching automated device, ensuring a rapid, uniform, and highly reliable process.

[0114] This invention, combined with peripheral devices, enables automated nucleic acid extraction and detection, achieving multi-target nucleic acid detection from sample input to result output.

[0115] Example 2

[0116] This invention relates to a fully enclosed nucleic acid extraction cartridge, which adopts a ring chamber structure and a rotary valve 2 linkage control design to achieve multi-step liquid directional transfer and sealing to prevent contamination. Its structural design and working process are as follows: The cartridge adopts an integrated injection-molded medical-grade polypropylene or polycarbonate shell, which integrates a lysis chamber 702 (pre-packaged with lyophilized lysis reagent containing 4M guanidine hydrochloride and 1% Triton X-100), a first washing chamber 706 (pre-packaged with 0.1mol / L to 2mol / L EDTA, 0.5mol / L to 2mol / L sodium chloride, and 10% to 40% volume ratio of isopropanol), a second washing chamber 707 (pre-packaged with 80% ethanol), an elution chamber 708 (filled with TE buffer), a reaction chamber 701 with built-in silica-coated magnetic beads, a waste liquid chamber 704, and a plunger chamber 711 with a precision plunger. Each chamber has a through hole at the bottom and a 0.8mm diameter vent hole at the top, which is covered with a hydrophobic ePTFE second waterproof and breathable membrane 4 (pore size 0.1μm). This design effectively prevents liquid leakage while maintaining air pressure balance, ensuring smooth liquid transfer during operation and avoiding aerosol pollution.

[0117] The core of the fluid control system is the rotary valve 2, which has a microchannel 202 (0.5×0.5mm cross-section) inside. 2 The microchannel is fixedly connected to the reaction chamber 701 at its proximal end, and the other end is selectively aligned with the bottom through-holes of different chambers by rotation. The plunger 6 in the plunger chamber 711 generates bidirectional hydraulic pressure in the reaction chamber 701 under drive: when negative pressure is applied, the reagent is drawn from the reagent chamber into the reaction chamber 701, and when positive pressure is applied, the liquid in the reaction chamber 701 is removed, realizing the cross-chamber transfer of 100-2000μL of liquid. In addition, the plunger chamber 711 can be connected to an air pump to replace the plunger movement.

[0118] The bottom of the lysis chamber 702 and the reaction chamber 701 are equipped with a bottom groove 71 for the reaction chamber and a bottom groove 72 for the lysis chamber (with a wall thickness reduced to 0.5 mm) to improve ultrasonic efficiency. When lysis is initiated, the rotary valve 2 closes the passage of the lysis chamber 702, and the external ultrasonic head works in close contact with the bottom groove 72 of the lysis chamber, working in conjunction with chemical reagents to achieve >95% pathogen disruption within 2 minutes. Low-power ultrasound is simultaneously used during nucleic acid adsorption, washing, and elution in the reaction phase. Vibration for 10 seconds during magnetic bead mixing increases the adsorption rate by 40%, the washing phase breaks up magnetic bead aggregation, and the elution phase accelerates nucleic acid dissociation (efficiency up to 95%). During nucleic acid extraction, the ultrasonic generator comes into contact with the reaction chamber 701. When transferring the reaction solution, the rotary valve 2 is rotated to connect the reaction chamber. After the reaction solution is transferred to the reaction chamber, the rotary valve 2 is closed, and the ultrasonic generator is started. After the magnetic beads and reaction solution are fully mixed and reacted, the magnetic beads are fixed using an external magnet. The rotary valve 2 is then rotated to connect the reaction chamber 701 to the waste liquid chamber 704. Under the positive pressure provided to the reaction chamber 701 by the plunger chamber 711, the reaction solution in the reaction chamber 701 is transferred to the waste liquid chamber 704. This process is repeated sequentially to achieve the adsorption, washing, and elution of the magnetic beads, thus completing nucleic acid extraction using the magnetic bead method.

[0119] The magnetic beads are manipulated via an electromagnet array on the outer wall of the cartridge. After nucleic acid binding, the electromagnets are activated, adsorbing and fixing the magnetic beads onto the side wall of reaction chamber 701. Waste liquid is discharged into waste liquid chamber 704 under positive pressure within reaction chamber 701. When washing liquid is drawn into reaction chamber 701 by negative pressure, the magnetic field is turned off and ultrasonic dispersion of the magnetic beads is initiated. During the elution stage, the eluent is drawn into reaction chamber 701 by negative pressure. Finally, the extracted nucleic acid solution is transferred to the downstream detection module under positive pressure within reaction chamber 701. The entire process, through the coordinated switching of the rotary valve 2 pathway, the pressure drive of the plunger 6 or air pump, ultrasonic assistance, and magnetic field control, completes the lysis → binding → washing → elution process in a closed environment, eliminating contamination from manual operation, and achieving a nucleic acid recovery rate of >95%.

[0120] In addition, the nucleic acid extraction cartridge incorporates an alcohol residue removal mechanism in the magnetic bead purification process. After the final washing step and waste liquid removal, rotary valve 2 rotates to a preset angle, connecting reaction chamber 701 and drying chamber 703 via an internal microchannel. A plunger drive device or external micro-pump is activated, generating a directional airflow that enters reaction chamber 701 from plunger chamber 711 and purges for 30 seconds. The airflow acts on the magnetic bead aggregates and chamber walls within reaction chamber 701, forcibly accelerating the volatilization of residual ethanol and other alcohols. Simultaneously, the fluid dynamics design creates vortices at channel bends, propelling trace amounts of liquid (≤5μL) trapped in dead corners of the chamber to the absorbent material at the bottom of the drying chamber. The top of the drying chamber is covered with a waterproof and breathable membrane, allowing volatile alcohols to exit the system through the membrane pores, while liquid residues are retained by the adsorption layer. After the purge is complete, rotary valve 2 is switched to the elution position, and elution buffer is injected to perform nucleic acid dissociation. This step significantly reduces the amount of residual ethanol in the elution buffer and eliminates the inhibitory effect on downstream nucleic acid amplification (qPCR amplification efficiency is increased by ≥92%).

[0121] After nucleic acid extraction, the extracted sample is located in reaction chamber 701. Rotating rotary valve 22 connects to the injection port 780, driving plunger 6 to press down (or using a gas pump to provide positive pressure) to inject the extracted sample into the main liquid channel 801 of the chip, and then distributing it to multiple amplification reaction chambers via the first liquid branch channel 802. Each amplification reaction chamber contains a pre-set differential target amplification detection lyophilized reagent system, and real-time fluorescence monitoring is used to achieve simultaneous quantitative detection of multiple targets.

[0122] The detection chip is a microfluidic chip used for simultaneous amplification and detection of multiple target nucleic acids. The chip body adopts a multi-layer microfluidic structure. The sample introduction layer 8 is provided with several independent first reaction chambers 804 as reaction chambers for amplification and detection; a sample injection port 780 is provided and connected to the reaction chamber 701 of the extraction cartridge through a rotary valve 2. In addition, the sample injection port is connected to the main liquid channel 801 and is connected to all the first reaction chambers 804 through the first liquid branch channel 802. The chip airflow layer is provided with several first exhaust ports 901. The first exhaust ports 901 are connected to the short exhaust channel 803 of the sample introduction layer 8 and the first gas branch channel 902 of the airflow layer 9. Each first reaction chamber 804 forms a "one chamber, one port" directional communication system with the corresponding first exhaust port 901 through a microchannel, ensuring unidirectional fluid flow and directional gas discharge; all the first exhaust ports 901 are connected to the first gas main channel 904 through the first gas branch channel 902, and finally connected to the gas chamber 1001 by the first gas main channel 904. The chip's upper cover plate 10 is equipped with a gas chamber 1001 and a valve control chamber 1002. The gas chamber 1001 has a variable volume and is not connected to the atmosphere to ensure the chip's airtightness. The gas chamber 1001 can be in the form of a plunger chamber 711 or a chamber sealed with a deformable material such as a thin film or silicone. The valve control chamber 1002 and the movable valve control 12 together form a flow control valve. The flow control valve controls the position of the valve control 12 to open and close the connection hole 806. When the valve control 12 is in the upper position, the connection hole 806 is open. When the sample extract is injected, the valve control 12 is pressed down to close the connection hole 806.

[0123] See Figure 5 and Figure 15 The first exhaust port 901, located at the outlet end 805 of the short exhaust channel connected to the first reaction chamber 804, is covered with a waterproof and breathable membrane. This membrane allows gas to pass through while simultaneously blocking liquid flow. It forms a complete gas-liquid barrier, confining the reaction liquid within the first reaction chamber 804, preventing it from entering the chip's airflow layer and avoiding cross-contamination and waste of sample extract. Simultaneously, the membrane blocks the liquid pathway, ensuring a sealed environment in the sample injection layer 8. Since there is no leakage path, after the sample extract is injected, regardless of the filling order, all reaction chambers will eventually be filled with the sample extract, ensuring that the volume of reaction liquid in each chamber is the same and constant, thus guaranteeing the accuracy of quantitative detection.

[0124] After all the sample extract has entered the amplification chamber, the rotary valve 2 is rotated to draw the hydrophobic reagent from the oil storage chamber 703 into the reaction chamber 701. Then, the hydrophobic reagent is injected from the reaction chamber 701 into the first liquid mainstream channel 801 of the sample injection layer through the injection port 780. At this time, the flow control valve of the control connection port is opened, so that the first liquid mainstream channel 801 is connected to the first gas mainstream channel 904. Under the positive pressure provided by the plunger chamber 711, the hydrophobic reagent enters the first gas mainstream channel 904 from the first liquid mainstream channel 801 and finally enters the gas chamber 1001. The hydrophobic reagent fills the gas and liquid mainstream channels. The hydrophobic reagent in the first liquid main channel 801 and the hydrophilic reaction liquid in the first liquid branch channel 902 form an immiscible two-phase interface, achieving physical separation between each reaction chamber 804; the hydrophobic reagent enters the first gas main channel 904 of the gas flow layer and forms a gas-liquid two-phase interface between the first gas main channel 904 of the gas flow layer and the first gas branch channel 902 of the gas flow layer, forming a barrier at the outlet of the first gas branch channel 902 connected to the first exhaust port 901, thus sealing the outlet of the first exhaust port 901, thereby blocking both the sample inlet and exhaust outlet of the reaction chamber, ensuring that the reaction chambers are fully independent, and preventing the evaporation of the reaction liquid under heating conditions.

[0125] Example 3

[0126] This embodiment provides a multi-layer integrated microfluidic chip combined with an extraction cartridge, such as... Figure 1-4 As shown, the extraction cartridge and the microfluidic chip respectively realize the functions of nucleic acid extraction and purification, and multiplex nucleic acid amplification and detection.

[0127] The extraction cartridge includes a chamber body 7, such as... Figure 5-7 As shown, the chamber body 7 and the sample injection layer 8 are integrated. The chamber body 7 of the extraction cartridge part consists of multiple annularly arranged functional chambers, namely, a reagent chamber for storing reaction liquid (including a lysis chamber 702, a first washing chamber 706, a second washing chamber 707, an elution chamber 708, and an oil storage chamber 709), a waste liquid chamber 704 for storing reaction waste liquid, and a blowing chamber 703 for discharging gas flow when drying the reaction chamber. In addition to the functional chambers, there is also a reaction chamber 701 that provides the reaction site, a plunger chamber 711 that provides driving gas pressure, and a gas connecting column 710 (a narrow through hole with a diameter of 2 mm × 27 mm) connecting the reaction chamber 701 and the plunger chamber 711. The lysis chamber 702, the first washing chamber 706, the second washing chamber 707, and the elution chamber are pre-encapsulated with lysis chamber 702, the first washing chamber 706, the second washing chamber 707, and the eluent, respectively. The bottom of the pyrolysis chamber 702 and the bottom of the reaction chamber are provided with a bottom groove 71 for the reaction chamber and a bottom groove 72 for the pyrolysis chamber (the wall thickness is reduced to 0.5 mm) to improve the ultrasonic efficiency.

[0128] like Figure 5 and Figure 7Each functional chamber and reaction chamber 701 has an external flow channel at its bottom, including an external flow channel 721 for the reaction chamber, an external flow channel 722 for the pyrolysis chamber, an external flow channel 723 for the drying chamber, an external flow channel 724 for the waste liquid chamber, an external flow channel 725 for the first washing chamber, an external flow channel 726 for the second washing chamber, an external flow channel 727 for the elution chamber, and an external flow channel 728 for the oil storage chamber. The diameter of the through-holes for the external flow channels 721, 722, 723, 724, 725, 726, 727, and 728 is Φ1mm.

[0129] In addition to the annularly arranged chambers, there is also a central circular fluid control chamber 705. Inside the fluid control chamber 705, a central flow channel 731 is formed. Around the central flow channel 731, circumferentially distributed inner flow channels, a pyrolysis outer flow channel 722, a drying outer flow channel 723, a waste liquid outer flow channel 724, a first washing outer flow channel 725, a second washing outer flow channel 726, an elution outer flow channel 727, and an oil storage outer flow channel 728 are connected via microchannels, each corresponding to a Φ1mm through-hole in the fluid control chamber.

[0130] A central flow port 731 is located at the center of the fluid control chamber, and the central flow port 731 is connected to the external flow port 721 at the bottom of the reaction chamber through a microchannel at the bottom of the chamber body 7. The nucleic acid extraction cartridge achieves liquid transfer function through the connection between the fluid control chamber and each chamber.

[0131] The 701 reaction chamber's structural design employs a conical space optimization scheme to address the liquid splashing problem during ultrasonic mixing. Figure 10 As can be seen from b, the internal cavity of the reaction chamber 701 is an inverted frustum-shaped cone with an upper port diameter of 13mm and a bottom port diameter of 6mm. The bottom area of ​​the chamber is larger than the contact area between the ultrasonic generator and the container wall. This geometry ensures dynamic adjustment of the liquid level when accommodating different volumes of liquid. When the maximum volume of washing solution (1700μL) is injected, the liquid level reaches 16mm, while when the minimum volume of elution solution (300μL) is injected, the liquid level remains above 5mm. This liquid level design effectively suppresses liquid splashing during the ultrasonic mixing stage, avoiding the risk of sample reduction after extraction and subsequent amplification failure due to unexpected liquid reduction. Simultaneously, the conical reaction chamber reduces liquid residue within the chamber compared to a traditional cylindrical cavity.

[0132] like Figure 10 As shown in b, the upper surface of the fluid control chamber 705 is bonded and fixed to the rubber sealing gasket 3. A rotary valve 2 is installed on the sealing gasket 3 and inside the fluid control chamber 705. A schematic diagram of the rotary valve 2 is shown in [reference needed]. Figure 8 and Figure 9The rotary valve 2 contains a microchannel 202 with 1mm openings at both ends, designated as a proximal flow port 201 and a distal flow port 203. The distal flow port 203 can be rotated to connect with different internal flow ports in the fluid control chamber 705. The proximal flow port 201, located at the center of the rotary valve 2, is always connected to the central flow port 731 of the fluid control chamber and is unaffected by the rotation of the rotary valve. When rotating the rotary valve changes the position of the distal flow port 203 and its connection to an internal flow port in the fluid control chamber, the flow path between the reaction chamber 701 and the corresponding chamber is opened, thereby enabling the connection between the reaction chamber 701 and other chambers. Figure 10 b is a cross-sectional view showing the connection between reaction chamber 701 and pyrolysis chamber 702. The wedge-shaped groove 204 at the upper end of rotary valve 2 is used to cooperate with the drive device to facilitate accurate control of rotary valve 2.

[0133] A plunger 6 or an air pump is installed in the plunger chamber 711 to achieve positive and negative air pressure control.

[0134] The chamber body 7 and the upper cover 5 are connected and fixed by gluing or hot pressing. The upper cover 5 is provided with several vent holes, which correspond to the reaction chamber 701 and other functional chambers (such as...). Figure 11 (Top view after removing the second waterproof and breathable membrane 4 from the top cover 5) To achieve air pressure balance in the chamber, the second waterproof and breathable membrane 4 covers the vent holes, as shown in the image. Figure 11 As shown.

[0135] Furthermore, a through hole 503 is provided above the gas connecting column 710 on the upper cover 5, and it is connected to the vent hole 501 of the reaction chamber through the gas channel 502 on the upper cover 5, such as... Figure 11 As shown, the liquid flow channel 7100 at the bottom of the gas connecting column 710 chamber body is connected to the through hole 729 at the bottom of the plunger chamber 711, as... Figure 11 The diagram shows a cross-sectional view of the gas path connecting the plunger chamber 711 and the reaction chamber 701, which realizes a closed connection between the plunger chamber 711 and the reaction chamber 701, enables pressure control of the reaction chamber 701, and reduces contamination of the plunger chamber 711 or the air pump.

[0136] Such as assembly Figure 1 The cylindrical clamping element 1 is installed and snapped into the edge groove of the protruding upper end of the cover 5, and applies a clamping force to the rotary valve in the axial direction, so that the rotary valve 2 and the sealing gasket 3 are in close contact to ensure the sealing of the rotary valve fluid control.

[0137] Nucleic acid extraction is achieved through the aforementioned cartridge section. After nucleic acid extraction is completed, rotating the rotary valve to the designated position establishes connection between the reaction chamber and the detection chip, and then injects the sample extract into the detection chip. Figure 12DD is a cross-sectional view of the amplification sample introduction of the nucleic acid extraction and multiplex detection device. The detection chip, which performs nucleic acid amplification and fluorescence detection, is a multilayer integrated microfluidic chip with a rectangular outline design. It is assembled through a lamination process, consisting of a lower sample introduction layer 8, a middle gas flow layer 9, an upper cover plate 10, and a sealing film on the lower surface of the chip. The lower, middle, and upper layers respectively complete the functions of sample introduction, gas exhaust, and gas-liquid control. Figure 12 DD is a schematic diagram of the flow channel cut along the main flow channel of the chip. Figure 13 This is a schematic diagram of the internal flow channels of the assembly containing the card cartridge body, chip injection layer 8, chip airflow layer 9, and cover plate 10.

[0138] like Figure 5-7 The sample introduction layer 8 at the bottom of the chip is integrated with the extraction cartridge body 7. The sample introduction layer 8 adopts a double-sided microstructure design. Figure 7 The right side shows a bottom view of the chip sample introduction layer 8. The sample introduction layer 8 is integrally formed with the extraction cartridge. The main structure consists of a first liquid main channel 801, liquid branch channels 802, and reaction chambers 804. The first liquid main channel 801 (0.8 mm wide, 0.5 mm deep) is the core fluid distribution path. The inlet (sample introduction end) of the first liquid main channel 801 is connected to the injection hole 780 (a through hole with a diameter of Φ1 mm) at the bottom of the fluid control chamber 705 of the extraction cartridge. The other end of the first liquid main channel 801 is connected to the connection hole 806. Twelve tree-like liquid branch channels 802 (7 mm long, 0.5 mm wide, 0.5 mm deep) extend symmetrically from both sides of the first liquid main channel 801, and their ends are connected to the bottom of twelve circular reaction chambers 804 (Φ4 mm in diameter), forming a "one main channel with multiple branches" symmetrical microfluidic network. Figure 5 The right side is a top view of the chip sample introduction layer 8. The top of each reaction chamber 804 is connected to a short exhaust channel 803 (2mm in length and 0.3mm in width). The end of the short exhaust channel 803 corresponds to the exhaust hole 901 on the gas flow layer, realizing the directional exhaust of gas.

[0139] The airflow layer 9 in the middle of the chip also adopts a double-sided microstructure design. Figure 14 The image shows a top view of the gas flow layer chip. Parallel and equidistant gas branch channels 902 (0.3mm wide) extend from both sides of the first gas main channel 904 (0.5mm wide). The end of the first gas branch channel 902 is connected to the first exhaust port 901, forming a gas emission network corresponding to the sample injection layer. One end of the first gas main channel 904 is connected to the first channel outlet 903, and the other end is connected to the valve control port 905. The valve control port is located above and communicates with the connection port 806 of the sample injection layer 8. Figure 15The image shows a bottom view of the airflow layer chip. Twelve countersunk hole arrays 906 (0.4 mm deep) are set on the airflow layer 9. A first exhaust hole 901 (0.8 mm through hole) is opened in the center of each countersunk hole 906. A first waterproof and breathable membrane 13 (0.1 μm pore size) is bonded in the countersunk hole 906. The first waterproof and breathable membrane 13 is located above the outlet end of the short exhaust channel.

[0140] The internal structure of the cover plate 10 on the upper layer of the chip is shown below. Figure 12 b. The cover plate 10 enables fluid control within the multilayer microfluidic chip and simultaneously seals the flow channels of the airflow layer 9. Above the first fluid outlet 903 on the airflow layer, the cover plate 10 has a plunger-shaped gas chamber 1001 (0.5 mL volume). A sliding plunger 11 (maximum stroke 20 mm) is housed within the gas chamber 1001. Driven by internal positive pressure, the plunger 11 expands upwards within the gas chamber 1001 to balance the internal pressure and prevent resistance during sample injection. A valve control chamber 1002 (inner diameter Φ4 mm) is positioned on the cover plate 10 corresponding to the valve control hole 905. The axial position of the valve control 12 within the valve body controls the opening and closing of the connection hole 806. When the valve control 12 is in the depressed position, the connection hole 806 is closed; moving the valve control 12 upwards opens the connection hole 806. Figure 12 DD connects the first liquid mainstream channel 801 of the injection layer 8 to the first gas mainstream channel 904 of the gas flow layer.

[0141] Example 4

[0142] This embodiment discloses a novel multi-core structure. While the original cardholder structure remains unchanged, new chip structures can be derived from the inspiration of the aforementioned chip structure, such as... Figure 16 This is a chip assembly diagram for this embodiment. In this embodiment, the chip adopts a rectangular body and a semi-circular end integrated design. Figure 17 This is a top view of the chip sample introduction layer. The main structure consists of a second liquid main channel 1402, a circular intermediate distribution cavity 1404, a radial liquid branch channel 1403, and a second reaction chamber 1405.

[0143] The second liquid main channel 1402 is located on the center line of the detection chip, and its two ends are connected to the second injection hole 1401 (diameter Φ2mm) and the middle distribution cavity 1404 (diameter Φ6mm) respectively. The second injection hole 1401 is connected to the injection hole 780 on the extraction cartridge, thereby realizing the connection between the second liquid main channel 1402 and the extraction cartridge.

[0144] Eight branch liquid flow channels 1403 (0.5 mm wide) radiate outwards uniformly from the central distribution cavity 1404. The ends of the liquid flow channels 1403 are connected to the top of the circular second reaction chamber 1405 (4 mm in diameter), forming a "central distribution-peripheral reaction" structure.

[0145] Top view of the airflow layer structure, such as Figure 19As shown, the flow channel network distribution of the second gas flow layer 15 and the second sample introduction layer 14 is the same. One end of the second gas main flow channel 1502 is connected to the second flow channel outlet 1501, and a gas chamber (similar to the gas chamber in Example 3) is set at the position of the second flow channel outlet 1501. The other end is connected to the circular intermediate chamber 1504 (diameter Φ3.5mm). Eight second gas branch flow channels 1503 (width 500μm) are symmetrically distributed around the intermediate chamber 1504, and the ends are connected to the Φ0.8mm second exhaust port 1505. The second exhaust port 1505 is located above the second reaction chamber 1405 and corresponds to each other. The second sample introduction layer 14 of the detection chip is a liquid flow channel network, and the second gas flow layer 15 is a gas exhaust channel. The gas is discharged in a directional manner through the microchannel design, and cross-contamination between the reaction chambers is avoided.

[0146] The cover plate encapsulation layer integrates a plunger-type gas chamber, whose bottom plane is fitted with the gas flow layer 15 to achieve a physical seal. The gas chamber contains a movable plunger (during sample injection, the plunger is driven upward by the internal positive pressure to expand the volume and maintain system pressure balance).

[0147] The difference between this example and Example 3 is that no connection hole and waterproof and breathable membrane are provided. The fluid control logic is as follows: After extraction, the sample enters the intermediate distribution chamber 1404 from the second liquid main channel 1402. The circular intermediate distribution chamber 1404 achieves uniform distribution to each of the second liquid main channels 1402. During injection, the sample liquid enters the second reaction chamber 1405 from the second liquid branch channel 1403, and the gas is directionally discharged from the exhaust port at the top of the second reaction chamber 1405. When sealing with a hydrophobic reagent after injection, the hydrophobic reagent fully fills the second liquid main channel 1402 and the intermediate distribution chamber 1404, and simultaneously flows through the second liquid branch channel 1403. 03. Entering the second reaction chamber 1405, which is filled with a hydrophilic reaction liquid (the reaction chamber can also be hydrophilically treated to increase the surface tension between the hydrophilic reaction liquid and the reaction chamber, enhancing the retention capacity of the hydrophilic reaction liquid), the hydrophobic reagent, which is immiscible with the hydrophilic reaction liquid, is reversed through the second vent 1505 and the second gas branch channel 1503 of the gas flow layer to fill the intermediate chamber 1504. Finally, a hydrophobic barrier is simultaneously established at the inlet end (branch channel) and the outlet end (vent vent) of the reaction chamber, achieving a double-end physical seal. Utilizing the difference in interfacial tension between the hydrophobic reagent and the hydrophilic reaction liquid, an anti-evaporation seal is achieved without the need for a waterproof and breathable membrane.

[0148] Example 5

[0149] This embodiment also discloses a method for using a multiplex nucleic acid detection device, including the following steps:

[0150] Before use, the rotary valve of the extraction cartridge is closed. The extraction cartridge contains a pre-stored magnetic bead suspension in reaction chamber 701, a pre-stored lysis buffer in lysis chamber 702, a pre-stored washing buffer 1 in the first washing chamber 706, a pre-stored washing buffer 2 in the second washing chamber 707, a pre-stored elution buffer in elution chamber 708, and a pre-stored hydrophobic reagent in oil storage chamber 709. The drying chamber 703 and waste liquid chamber 704 are empty. The reaction chamber 804 on the detection chip contains pre-stored lyophilized qPCR reaction reagents and fluorescent probes. The plunger 6 in the plunger chamber 711 is in the highest position.

[0151] Sample addition: Draw the sample into a disposable syringe with a needle, and then insert the syringe needle into the vent 504 of the lysis chamber 702 above the lysis chamber 702. Figure 10 As shown, the sample is inserted into the lysis chamber 702, which contains lysis solution, through the second waterproof and breathable membrane 4 and the lysis chamber vent 504.

[0152] Pyrolysis: At this time, rotary valve 2 is in the closed state, and the mixture of lysis solution and sample in lysis chamber 702 is subjected to ultrasonic treatment. The external ultrasonic generator is placed in the bottom groove 72 of the lysis chamber 702. Figure 7 As shown, the ultrasonic generator is brought into close contact with the bottom of the lysis chamber. The ultrasonic generator is started and the ultrasonic treatment time is set to 180s (the pulse interval is set to 20s and the pulse width is set to 30s, that is, ultrasonic treatment is performed for 30s first, then stopped for 20s, and the cycle is repeated until the set time of 180s is reached, after which the ultrasonic treatment ends) to achieve rapid lysis of the sample.

[0153] Adsorption:

[0154] The reaction chamber 701 contains a pre-stored magnetic bead suspension. When using the magnetic beads to adsorb nucleic acids, the magnetic bead buffer solution in the magnetic bead suspension needs to be removed first, leaving only the magnetic beads in the reaction chamber 701. The magnetic beads are fixed with an external magnet, and the rotary valve 2 is rotated to connect the reaction chamber 701 and the waste liquid chamber 704. The plunger 6 in the plunger chamber 711 is pushed to push the magnetic bead buffer solution in the reaction chamber 701 into the waste liquid chamber 704.

[0155] Rotating rotary valve 2 connects reaction chamber 701 and pyrolysis chamber 702, as follows: Figure 10 b. Transfer the lysed sample to reaction chamber 701.

[0156] Close rotary valve 2; place the external ultrasonic generator at the bottom of reaction chamber 701, in the bottom recess 71 of the reaction chamber. Figure 7 As shown, the ultrasonic generator is brought into close contact with the bottom of the reaction chamber 701. The ultrasonic generator is started and operated for 10 seconds, then paused for 30 seconds. This process is repeated 3 times to complete the nucleic acid adsorption.

[0157] The magnetic beads are fixed in the reaction chamber 701 using an external magnet, while the plunger 6 in the plunger chamber 711 is pushed to transfer the liquid in the reaction chamber 701 to the waste liquid chamber 704.

[0158] Washing 1: Remove the external magnet and turn the rotary valve 2 to connect the reaction chamber 701 and the first washing chamber 706. Figure 5 As shown, the plunger 6 inside the plunger chamber 711 is pulled out to draw the washing liquid 1 into the reaction chamber 701; the ultrasonic generator is started and operates for 10 seconds, then waits for 30 seconds; the magnetic bead is fixed in the reaction chamber 701 using an external magnet, while the plunger 6 inside the plunger chamber 711 is pushed to transfer the liquid in the reaction chamber to the waste liquid chamber 704.

[0159] Washing 2: Remove the external magnet, turn rotary valve 2 to connect reaction chamber 701 and the second washing chamber 707. Figure 5 As shown, pull the plunger 6 in the plunger chamber 711 to draw the washing liquid 2 into the reaction chamber 701; start the ultrasonic generator to work for 10 seconds, and wait for 30 seconds; use an external magnet to fix the magnetic bead in the reaction chamber 701, and at the same time push the plunger 6 in the plunger chamber 711 to transfer the liquid in the reaction chamber to the waste liquid chamber 704.

[0160] Drying: Still using external magnets to fix the magnetic beads in reaction chamber 701, rotate rotary valve 2 to connect reaction chamber 701 and drying chamber 703. Figure 5 As shown, the plunger 6 in the plunger chamber 711 is slowly moved upwards, and then quickly pushed downwards, blowing the residual liquid (<5μL) in the reaction chamber 701 and the microchannel into the drying chamber 703. Repeatedly pushing and pulling the plunger 6 provides airflow within the reaction chamber 701, accelerating the evaporation of alcohols and carrying the evaporated substances into the drying chamber 703, finally expelling them through the vent. Because the residual liquid in the reaction chamber 701 and the microchannel is <5μL, it splashes onto the inner wall of the drying chamber 703 after being blown in, preventing the liquid from being re-drawn into the reaction chamber 701 when the plunger 6 is repeatedly pushed and pulled. After repeated pushing and pulling, the plunger 6 finally reaches its lowest position.

[0161] Elution: Remove the external magnet, turn the rotary valve 2 to connect the reaction chamber 701 and the elution chamber 708, pull the plunger 6 in the plunger chamber to draw the elution solution into the reaction chamber 701; start the ultrasonic generator to work for 10 seconds, pause for 30 seconds, repeat 3 times to complete the elution of nucleic acid.

[0162] Sample injection onto the detection chip: An external magnet is used to fix the magnetic bead, while rotating rotary valve 2 to open the distal flow channel 203 of the rotary valve. Figure 8 (as shown) and injection hole 780 ( Figure 7 (As shown) The connection connects the reaction chamber 701 and the detection chip. The valve control unit 12 located in the valve control chamber 1002 is in the depressed position. Figure 12b) This closes the connection hole 806; the plunger 6 inside the plunger chamber 711 is pushed, and the sample extract enters the main liquid channel 801. Figure 7 As shown), with the connection hole 806 closed, the sample extract enters the reaction chamber 804 through the liquid branch channel 802. Figure 7 (As shown). Simultaneously with sample injection, gas is discharged from the gas passage into the first reaction chamber 804.

[0163] The gas path in the detection chip is as follows: gas flows from the first reaction chamber 804 through the short exhaust channel 803 to the outlet end 805 of the short exhaust channel. Figure 5 As shown), through the exhaust port 901 connected to the upper part of the short exhaust channel outlet 805 (as shown), Figure 14 and 15 As shown, the gas enters the gas flow layer 9, and in the fluid layer 9, the gas enters the main gas flow channel 904 through the gas branch channel 902. Figure 14 As shown), it finally enters the gas chamber 1001 from the fluid outlet 903. Figure 12 (As shown in DD). During sample injection, pressing the plunger 6 inside the plunger chamber 711 provides pressure to the extracted sample to be injected. Under the action of fluid pressure, the original gas inside the chip enters the gas chamber and automatically pushes the sliding plunger 11 in the gas chamber upward, increasing the gas chamber volume to maintain the gas pressure balance inside the system. The injection is completed after the extracted sample fills all reaction chambers.

[0164] Reaction chamber separation: An external magnet is used to fix the magnetic bead. Simultaneously, rotating rotary valve 2 connects reaction chamber 701 to oil reservoir 709. Pulling the plunger 6 in plunger chamber 711 draws the hydrophobic reagent from oil reservoir 709 into the reaction chamber. Rotating rotary valve 2 opens the distal flow channel 203 of the rotary valve... Figure 8 (as shown) and injection hole 780 ( Figure 7 (As shown) Connection, i.e., connection between reaction chamber 701 and detection chip. Valve control unit 12 in valve control chamber 1002 is moved upwards. Figure 12 (As shown in b), but valve control 12 in the figure is in the depressed position, so that the connection hole 806 is opened; press the plunger 6 in the plunger chamber 711 to inject the hydrophobic reagent from the reaction chamber 701 into the liquid main channel 801 through the injection hole 780. Figure 7 As shown), and enters the first gas main channel 904 of the gas flow layer 9 through the connecting hole 806. Figure 12 (as shown in b), and finally enters air chamber 1001.

[0165] qPCR multiplex detection: After completing the above steps, each first reaction chamber 804 is filled with the extracted sample and is sealed. The extracted sample is mixed with the qPCR reaction solution pre-stored in the first reaction chamber 804, and a qPCR amplification reaction occurs at a specific external temperature. Real-time fluorescence detection is performed during the reaction process to achieve simultaneous qPCR quantitative detection in multiple reaction chambers.

[0166] In this invention, the plunger 6 inside the plunger chamber 711 can be eliminated by extracting the cartridge. An external air pump can be directly connected to the plunger chamber. The action of pushing the plunger 6 in the above steps is replaced by using the air pump to provide positive pressure (i.e., blowing air), and the action of pulling the plunger 6 is replaced by using the air pump to provide negative pressure (i.e., drawing air). The difference is that when using the air pump, the drying stage after washing 2 can use the air pump to provide a unidirectional continuous airflow for blowing air (when using the plunger, the airflow is bidirectional, including rapid blowing and slow drawing air), which is more conducive to the evaporation of liquid in the reaction chamber 701.

[0167] This invention achieves full-process multiple detection by integrating an extraction cartridge with a multiple detection chip, reducing sample transfer steps, lowering the risk of contamination, and improving detection sensitivity and the range of detectable targets.

[0168] In this invention, the detection chip integrates multiple independent reaction chambers, supporting parallel amplification and detection of multiple nucleic acids. The two ends of each reaction chamber are physically separated by hydrophobic reagents, which reduces the number of control valves required for the chip and the complexity of the chip, while achieving full enclosure of the reaction chambers, avoiding the evaporation and reduction of the reaction liquid at high temperatures, and preventing cross-contamination between the reaction chambers.

[0169] The detection chip features a waterproof and breathable membrane at the exhaust end of the reaction chamber, which avoids cross-contamination while enabling precise fluid control. Firstly, the waterproof and breathable membrane allows gas to escape while effectively blocking liquid leakage, eliminating the risk of cross-contamination between adjacent chambers. Secondly, by maintaining a stable total amount of reaction solution, it eliminates reaction solution loss caused by liquid overflow, ensuring that the reaction solution is completely and directionally introduced into the chamber and achieves a uniform and equal distribution. This solves the problem of multiplex detection quantification errors caused by incomplete filling or local concentration deviations, improving the reliability and stability of multiplex nucleic acid quantification.

[0170] The air chamber is constructed using deformable materials (silicone / film) to create a closed air chamber, maintaining system pressure balance while ensuring complete system sealing and preventing aerosol pollution in the environment.

[0171] In the nucleic acid extraction step of this invention, an airflow drying step is added after washing to accelerate the volatilization of inhibitors such as ethanol in the washing solution and reduce the inhibitory effect of residual alcohols in nucleic acid extraction on downstream nucleic acid amplification.

[0172] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A multiplex nucleic acid detection device, characterized in that, This includes interconnected card boxes and multiple chips; The cartridge includes a chamber body (7), inside which a fluid control chamber (705) is provided, and around the fluid control chamber (705) are distributed a reaction chamber (701), other functional chambers required for nucleic acid reaction, a gas connection column (710), a plunger chamber (711), and a sample injection port (780); The fluid control chamber (705) is equipped with a rotary valve (2). The bottom of the rotary valve (2) has a proximal flow port (201) and a distal flow port (203) that are interconnected. The fluid control chamber (705) has a central flow port (731) that is connected to the reaction chamber (701). The central flow port (731) is surrounded by inner flow ports in a circumferential manner. The fluid control chamber (705) is connected to other functional chambers and injection ports (780) required for nucleic acid reaction through the inner flow ports. When the rotary valve (2) is installed inside the fluid control chamber (705), the proximal flow port (201) is connected to the central flow port (731). When the rotary valve (2) is rotated circumferentially, the distal flow port (203) can be connected to any one of the internal flow ports. The plunger chamber (711) is used to provide gas pressure, and the plunger chamber (711) is connected to the reaction chamber (701) through a gas connecting column (710); The injection port (780) connects to multiple chips.

2. The multiplex nucleic acid detection device according to claim 1, characterized in that, Other functional chambers required for the nucleic acid reaction include a lysis chamber (702), a drying chamber (703), a waste liquid chamber (704), a first washing chamber (706), a second washing chamber (707), an elution chamber (708), and an oil storage chamber (709); The pyrolysis chamber (702), drying chamber (703), waste liquid chamber (704), first washing chamber (706), second washing chamber (707), elution chamber (708) and oil storage chamber (709) are all provided with connecting through holes, and each connecting through hole is connected to the inner flow channel openings distributed circumferentially around the central flow channel opening (731).

3. The multiplex nucleic acid detection device according to claim 2, characterized in that, The central flow port (731) is located at the center of the lower end face of the fluid control chamber (705), the proximal flow port (201) is opened at the center of the lower end face of the rotary valve (2), and the external flow port (721) is opened inside the reaction chamber (701), which is connected to the central flow port (731). The rotary valve (2) has a microchannel (202) inside, and the proximal flow port (201) and the distal flow port (203) are connected through the microchannel (202).

4. The multiplex nucleic acid detection device according to claim 1, characterized in that, The plunger chamber (711) has a plunger hole at its lower end, and the connecting column (710) has a hollow structure inside. Connecting air holes are opened at both the upper and lower ends of the connecting column (710). The upper end of the reaction chamber (701) is provided with a reaction chamber vent (501), and the reaction chamber (701) is connected to the connecting column (710) and the plunger chamber (711) through the reaction chamber vent (501).

5. The multiplex nucleic acid detection device according to claim 1, characterized in that, The lower and upper end faces of the reaction chamber (701) are both circular, with the diameter of the lower end face being smaller than the diameter of the upper end face. The reaction chamber (701) includes a first chamber and a second chamber connected together. The first chamber is cylindrical and the second chamber is truncated conical.

6. The multiplex nucleic acid detection device according to claim 1, characterized in that, The multi-chip includes a cover plate (10), an airflow layer (9), and a sample introduction layer (8) connected from top to bottom; The sample injection layer (8) has a first liquid main channel (801) connected to the sample injection hole (780) at one end and the connecting hole (806) at the other end. The first liquid main channel (801) is also connected to several first reaction chambers (804), and the first reaction chambers (804) are connected to the outlet end (805) of the short exhaust channel. The airflow layer (9) has a first exhaust hole (901) that is connected to the outlet end (805) of the short exhaust channel. The first exhaust hole (901) is connected to the first gas main channel (904). One end of the first gas main channel (904) is connected to the outlet (903) of the first channel, and the other end is connected to the valve control hole (905). The cover plate (10) is provided with an air chamber (1001) and a valve control chamber (1002). The air chamber (1001) is connected to the outlet (903) of the first flow channel. A sliding plunger (11) is provided in the air chamber (1001). The valve control chamber (1002) is connected to the valve control hole (905) and the connection hole (806) in sequence. A valve control device (12) is provided inside the valve control chamber (1002). When the valve control device (12) moves downward to the bottom, the lower end of the valve control device (12) is embedded into the valve control hole (905) and the connection hole (806) in sequence, and the connection hole (806) is closed at this time. When the valve control device (12) moves upward, the valve control device (12) is separated from the connection hole (806) and the valve control hole (905), and the connection hole (806) is opened at this time. The first liquid main channel (801), the connection hole (806), the valve control hole (905), the first gas main channel (904), the first flow channel outlet (903) and the gas chamber (1001) are connected.

7. A multiplex nucleic acid detection device according to claim 6, characterized in that, The upper end of the short exhaust channel outlet (805) is covered with a first waterproof and breathable membrane (13).

8. The method of using the device according to claim 1, characterized in that, Includes the following steps: Step 1: With the rotary valve (2) in the closed state, the sample is injected into the corresponding functional chamber. The sample is subjected to lysis, adsorption, washing, drying and elution in sequence. During the process, the rotary valve (2) is rotated according to the reaction requirements of each stage. The reaction chamber (701) is connected to any corresponding functional chamber through the cooperation of the proximal flow port (201), the distal flow port (203) and the inner flow port. The sample is transferred by the gas pressure provided by the plunger chamber (711). Step 2: After elution, the reaction solution is located in the reaction chamber (701). Rotate the rotary valve (2) to connect the reaction chamber (701) and the injection port (780) and inject the reaction solution into the multi-chip for detection through the injection port (780).

9. The method of using the multiplex nucleic acid detection device according to claim 8, characterized in that, Step 1 includes the following steps: Lysis: The sample is injected into the lysis chamber (702), and the sample and the lysis solution pre-stored in the lysis chamber (702) begin the lysis reaction; Adsorption: Rotate the rotary valve (2) to connect the reaction chamber (701) and the waste liquid chamber (704) through the distal flow port (203) to transfer the magnetic bead buffer solution pre-stored in the reaction chamber (701) to the waste liquid chamber (704); Rotate the rotary valve (2) to connect the reaction chamber (701) and the lysis chamber (702) through the distal flow port (203), transfer the lysed sample into the reaction chamber (701), close the rotary valve (2), and start nucleic acid adsorption through the magnetic beads pre-stored in the reaction chamber (701). After the adsorption is completed, rotate the rotary valve (2) to connect the reaction chamber (701) and the waste liquid chamber (704) through the distal flow port (203), and transfer the liquid in the reaction chamber (701) into the waste liquid chamber (704). Washing: Rotate the rotary valve (2) to connect the reaction chamber (701) and the washing chamber through the distal flow port (203) to transfer the washing liquid in the washing chamber to the reaction chamber (701) for washing. After washing, rotate the rotary valve (2) to connect the reaction chamber (701) and the waste liquid chamber (704) through the distal flow port (203) to transfer the liquid to the waste liquid chamber (704). Drying: Rotate the rotary valve (2) to connect the reaction chamber (701) and the drying chamber (703) through the distal flow port (203) to blow the residual liquid in the reaction chamber (701) into the drying chamber (703); Elution: Rotate the rotary valve (2) to connect the reaction chamber (701) and the elution chamber (708) through the distal flow port (203) to transfer the elution solution to the reaction chamber (701) for nucleic acid elution.

10. The method of using the multiplex nucleic acid detection device according to claim 9, characterized in that, Step 2 includes the following steps: Sample injection: After elution, rotate the rotary valve (2) to connect the reaction chamber (701) and the injection port (780) through the distal flow channel (203). The liquid in the reaction chamber (701) enters each of the first reaction chambers (804) in sequence through the injection port (780) and the first liquid main channel (801). A first waterproof and breathable membrane 13 is provided at the first exhaust port (901). The first waterproof and breathable membrane 13 is used to block the liquid in the first reaction chamber (804). Exhaust: During sample injection, the gas inside the injection layer (8) is pushed by the liquid through the first reaction chamber (804), the short exhaust channel outlet end (805), the first exhaust hole (901), the first gas main channel (904) and the first channel outlet (903) into the gas chamber (1001). At this time, the lower end of the valve control (12) blocks the connection hole (806), and the connection hole (806) is closed. Reaction chamber separation: After the sample injection is completed, rotate the rotary valve (2) to connect the reaction chamber (701) and the oil storage chamber (709) through the distal flow port (203) to draw the hydrophobic reagent in the oil storage chamber (709) into the reaction chamber (701). Rotate the rotary valve (2) to connect the reaction chamber (701) and the injection port (780) through the distal flow port (203) to transfer the hydrophobic reagent from the reaction chamber (701) to the first liquid main flow channel (801). Move the valve control (12) upward to separate the valve control (12) from the connection hole (806). The connection hole (806) is opened, and the hydrophobic reagent enters the gas chamber (1001) through the connection hole (806), the valve control hole (905), the first gas main flow channel (904), and the first flow channel outlet (903). After the reaction chambers are separated, the nucleic acid reaction begins.

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