A microfluidic cartridge and method for specific detection of nucleic acids from complex samples

Abstract

This invention relates to a microfluidic cartridge and method for the specific detection of nucleic acids from complex samples. The cartridge includes a top cover, a central chamber, and a lower conduit layer. The central chamber contains a tubular structure comprising a sample tube, a magnetic bead extraction tube, and a reagent tube. The sample tube is used for loading the cartridge with samples and releasing nucleic acids. The lower conduit layer includes a lower body with fluid conduits and a hollow needle structure. A reaction chamber is also located on the lower body, containing nucleic acid enrichment filter paper. The reaction chamber is used for nucleic acid enrichment and in-situ amplification. This invention combines magnetic bead-based nucleic acid purification technology with chitosan-modified nucleic acid enrichment filter paper and in-situ amplification detection technology for a fully integrated, specific, and highly sensitive nucleic acid detection from complex samples.

Description

Technical Field

[0001] This invention relates to the fields of medical and biological analysis and detection technology, and in particular to a microfluidic cartridge and method for specifically detecting nucleic acids from complex samples. Background Technology

[0002] Molecular diagnostics, based on molecular biology theory, is a technique for detecting endogenous or exogenous biomolecules in the human body. It utilizes genomic and proteomic analysis of disease-related molecular changes, taking into account alterations in their presence, structure, or expression levels, to aid in disease prevention or diagnosis. The target of detection is nucleic acid. Pathogen detection using nucleic acid generally involves four steps: sample pretreatment, nucleic acid extraction, amplification, and detection. It is the most direct, reliable, and sensitive method for early, rapid, and specific detection of pathogens, providing a scientific basis for confirming infection cases. The most traditional method for nucleic acid detection uses a magnetic bead extractor to extract nucleic acids. A portion of the extracted product is then added to a pre-prepared reaction reagent for nucleic acid amplification, amplifying the target fragment exponentially. The amplification results are presented through real-time product signal detection or end-point product detection, including real-time fluorescence detection, gel electrophoresis, colloidal gold test strips, and electrochemical detection (current-voltage detection). However, the traditional nucleic acid testing process requires multiple manual liquid transfer steps, which is cumbersome and greatly limits the efficiency of clinical testing and the further application and promotion of this technology.

[0003] Since the 1990s, microfluidic chips have seen rapid development in micrototal analytical systems, integrating basic operational units such as sample preparation, reaction, separation, and detection in biological, chemical, and medical analysis processes onto a microscale fluidic chip. These systems offer advantages such as low cost, short analysis time, and small device size, making them a valuable tool for integrating nucleic acid detection. However, traditional microfluidic chips operate within microscale channels, requiring the use of micropumps, microvalves, and complex macro- and micro-microfluidic interfaces for analytical chemistry-related detection and analysis. This process is cumbersome, requiring multiple manual steps, hindering fully integrated point-of-care diagnostics, and making it difficult to stably store the various reagents needed for the reaction. Furthermore, many clinically analyzed samples are complex and diverse, containing numerous impurities, such as mixed nasopharyngeal swabs, tissues, feces, and sputum. Traditional microfluidic chip-based nucleic acid detection technologies cannot handle these complex samples. Based on this, companies like Ceprit have developed highly representative fully integrated nucleic acid analysis systems. However, these systems still have significant shortcomings in terms of detection versatility, flexibility, reagent storage capacity, ability to handle complex samples, and detection sensitivity, further hindering the widespread adoption of fully integrated molecular diagnostic systems in clinical practice and applications. Therefore, there is an urgent need for a fully integrated microfluidic cartridge to handle various complex samples in clinical settings, meeting reagent storage requirements to achieve highly sensitive and specific detection of complex samples.

[0004] Therefore, based on years of experience and practice in related industries, the inventors have proposed a microfluidic cartridge and method for the specific detection of nucleic acids from complex samples, so as to achieve highly sensitive and specific detection of complex samples. Summary of the Invention

[0005] The purpose of this invention is to provide a microfluidic cartridge and method for specific detection of nucleic acids from complex samples. It combines magnetic bead nucleic acid purification technology with chitosan-modified nucleic acid enrichment filter paper and in situ amplification detection technology for fully integrated specific and highly sensitive nucleic acid detection from complex samples.

[0006] The objective of this invention is achieved as follows: a microfluidic cartridge for the specific detection of nucleic acids from complex samples, comprising a cartridge cover, an intermediate chamber, and a lower conduit layer; the intermediate chamber contains a tubular structure, which includes at least a sample tube, a magnetic bead extraction chamber, and a reagent tube; the sample tube is used for loading the cartridge with samples and releasing nucleic acids; the reagent tube is used to contain reaction reagents for in situ nucleic acid amplification; the lower conduit layer includes a lower body that can be fastened to the bottom of the intermediate chamber, the lower body having a fluid conduit and a hollow needle structure connected to the tubular structure and the fluid conduit; the lower body also has a reaction chamber connected to the magnetic bead extraction chamber and the reagent tube via the fluid conduit; nucleic acid enrichment filter paper can be fixedly disposed within the reaction chamber; the reagent tube communicates with the reaction chamber to provide it with reaction reagents for in situ nucleic acid amplification; the reaction chamber is used for nucleic acid enrichment and in situ nucleic acid amplification; the cartridge cover is detachably fastened to the top of the intermediate chamber.

[0007] In a preferred embodiment of the present invention, the magnetic bead extraction chamber is provided with a reuse chamber, and magnetic beads are pre-placed in the reuse chamber. The magnetic beads are used for magnetic capture and extraction of nucleic acids, and the reuse chamber is used for nucleic acid extraction, washing and elution.

[0008] In a preferred embodiment of the present invention, the tubular structure further includes a washing liquid tube, an elution liquid tube, and an enrichment liquid tube. The washing liquid tube contains washing liquid and is connected to the reuse chamber to provide washing liquid to the reuse chamber. The elution liquid tube contains elution liquid and is connected to the reuse chamber to provide elution liquid to the reuse chamber. The enrichment liquid tube contains nucleic acid resuspension solution and is connected to the reuse chamber to provide nucleic acid resuspension solution to the reuse chamber.

[0009] In a preferred embodiment of the present invention, a waste liquid pool pipe is further provided in the intermediate chamber, the waste liquid pool pipe being able to communicate with the reaction chamber, and the waste liquid pool pipe being used to contain the waste liquid after flowing through the nucleic acid enrichment filter paper.

[0010] In a preferred embodiment of the present invention, a switching valve is further provided in the intermediate chamber, the switching valve being used to connect or disconnect the fluid pipes on the reuse chamber and the lower body.

[0011] In a preferred embodiment of the present invention, the fluid conduit includes a first flow channel, a second flow channel, and a third flow channel. A first hollow ejector pin, a second hollow ejector pin, a third hollow ejector pin, a fourth hollow ejector pin, a fifth hollow ejector pin, and a sixth hollow ejector pin are provided on the lower part body and communicate with the first flow channel. A seventh hollow ejector pin and an eighth hollow ejector pin are provided on the lower part body and communicate with the second flow channel. A ninth hollow ejector pin is provided on the lower part body and communicates with the third flow channel.

[0012] The sample tube is connected to the magnetic bead extraction chamber via the first hollow pin and the first flow channel; the washing solution tube is connected to the magnetic bead extraction chamber via the second hollow pin and the first flow channel; the elution solution tube is connected to the magnetic bead extraction chamber via the third hollow pin and the first flow channel; the enrichment solution tube is connected to the magnetic bead extraction chamber via the fourth hollow pin and the first flow channel; the magnetic bead extraction chamber is connected to the first flow channel via the fifth hollow pin; the switching valve is connected to the first flow channel via the sixth hollow pin; the switching valve is connected to the reaction chamber via the seventh hollow pin and the second flow channel; the reagent tube is connected to the reaction chamber via the eighth hollow pin and the second flow channel; and the waste liquid tank tube is connected to the reaction chamber via the ninth hollow pin and the third flow channel.

[0013] In a preferred embodiment of the present invention, the lower body is provided with a plurality of insertion holes for inserting a first hollow ejector pin, a second hollow ejector pin, a third hollow ejector pin, a fourth hollow ejector pin, a fifth hollow ejector pin, a sixth hollow ejector pin, a seventh hollow ejector pin, an eighth hollow ejector pin, and a ninth hollow ejector pin. Each insertion hole has a needle tip solution opening at its bottom end and an adhesive dispensing hole with an increasing diameter at its top end. The adhesive dispensing hole is used to dispense adhesive to fix the first hollow ejector pin, the second hollow ejector pin, the third hollow ejector pin, the fourth hollow ejector pin, the fifth hollow ejector pin, the seventh hollow ejector pin, the eighth hollow ejector pin, and the ninth hollow ejector pin, respectively.

[0014] In a preferred embodiment of the present invention, a first lower rubber stopper and a first upper rubber stopper are provided at an interval from bottom to top at the bottom end of the sample tube. A sample cavity is provided above the first upper rubber stopper. A first communicating cavity communicating with the sample cavity is provided between the first lower rubber stopper and the first upper rubber stopper. The tip of the first hollow ejector pin can pass through the first lower rubber stopper to communicate with the first communicating cavity, and the tip of the first hollow ejector pin can be inserted into the first upper rubber stopper to seal it. A pneumatic rubber stopper is provided at the top end of the sample tube, and an air source needle channel is provided on the pneumatic rubber stopper.

[0015] In a preferred embodiment of the present invention, a reusable cavity plug is provided at the bottom end of the magnetic bead extraction cavity tube, and the reusable cavity is provided above the reusable cavity plug. The top end of the fifth hollow pin can pass through the reusable cavity plug and communicate with the reusable cavity. A pneumatic plug is provided at the top end of the magnetic bead extraction cavity tube, and an air source needle channel is provided on the pneumatic plug.

[0016] In a preferred embodiment of the present invention, a second lower rubber plug and a second upper rubber plug are provided at intervals from bottom to top at the bottom end of the waste liquid tank pipe. A waste liquid chamber is provided inside the waste liquid tank pipe. A second communicating cavity is provided between the second lower rubber plug and the second upper rubber plug, which communicates with the waste liquid chamber. The top end of the ninth hollow pin can pass through the second lower rubber plug to communicate with the second communicating cavity, and the top end of the ninth hollow pin can be inserted into the second upper rubber plug to seal it. A waste liquid tank pipe cover is provided at the top end of the waste liquid tank pipe, and an air vent is provided on the waste liquid tank pipe cover to allow the waste liquid chamber to communicate with the outside atmosphere.

[0017] The object of the present invention can also be achieved as follows: a method for specifically detecting nucleic acids from complex samples, comprising the following steps:

[0018] Step a, Reagent pre-storage: Pre-store reagents in the sample tube, washing solution tube, elution solution tube, enrichment solution tube, magnetic bead extraction chamber tube and reagent tube of the aforementioned microfluidic cartridge for specific detection of nucleic acids from complex samples;

[0019] Step b, Sample collection: Place the collected sample into a sample tube;

[0020] Step c, Nucleic acid release: The sample undergoes pathogen lysis within the sample tube, releasing nucleic acid;

[0021] Step d, Nucleic acid capture: Prepare to use a fully integrated instrument, press down to connect the sample tube and the magnetic bead extraction chamber, blow air into the sample tube, and the solution in the sample tube flows into the reuse chamber of the magnetic bead extraction chamber and mixes with the magnetic beads. The process of magnetic beads capturing nucleic acid is completed with the help of the fully integrated instrument.

[0022] Step e, Nucleic acid washing: Press down the washing solution tube to connect it with the magnetic bead extraction chamber tube, blow air into the washing solution tube, and the washing solution in the washing solution tube flows into the reuse chamber of the magnetic bead extraction chamber tube to wash the magnetic beads. With the help of the fully integrated instrument, the washing solution is withdrawn to complete the washing process of the magnetic beads capturing nucleic acid.

[0023] Step f, Nucleic acid elution: Press down the elution tube to connect it with the magnetic bead extraction chamber tube, blow air into the elution tube, and the elution solution in the elution tube flows into the reuse chamber of the magnetic bead extraction chamber tube to elute the magnetic beads. The elution process of magnetic beads capturing nucleic acid is completed with the help of the fully integrated instrument.

[0024] Step g, Nucleic Acid Enrichment: Press down the enrichment liquid tube to connect it with the magnetic bead extraction chamber tube, blow air into the enrichment liquid tube to allow the solution inside to enter the reuse chamber of the magnetic bead extraction chamber tube; press down the switch valve to blow air into the magnetic bead extraction chamber tube, and the purified nucleic acid sample solution in the magnetic bead extraction chamber tube will pass through the switch valve and flow through the fluid pipe to the reaction chamber. The nucleic acid sample solution flows through the nucleic acid enrichment filter paper and is enriched by the filter paper. The enriched solution enters the waste liquid tank tube.

[0025] Step h, Nucleic acid amplification: Press down the reagent tube to connect it to the reaction chamber through the fluid tube, blow air into the reagent tube so that the solution inside enters and fills the reaction chamber and the nucleic acid enrichment filter paper enriched with nucleic acid samples;

[0026] Step i, Output detection results: Real-time fluorescence detection is performed on the reaction chamber and nucleic acid enrichment filter paper in the lower pipeline layer, and fluorescence values ​​are continuously plotted into fluorescence curves. Finally, quantitative judgment is made based on the Ct value.

[0027] As described above, the microfluidic cartridge and method for specifically detecting nucleic acids from complex samples of the present invention have the following beneficial effects:

[0028] In this invention, a sample tube, a magnetic bead extraction chamber, a reagent tube providing nucleic acid in situ amplification reaction reagents, a nucleic acid enrichment filter paper, and a reaction chamber for nucleic acid in situ amplification are integrated. This combines magnetic bead nucleic acid purification technology with chitosan-modified nucleic acid enrichment filter paper and in situ amplification detection technology for a fully integrated, specific, and highly sensitive nucleic acid detection from complex samples.

[0029] The advantages of this invention's integrated magnetic bead method for nucleic acid purification are that it can purify low-load nucleic acids from complex samples, avoiding the problems of complex procedures, long processing times, high dependence on operators, and low nucleic acid extraction efficiency associated with conventional manual nucleic acid extraction. It also avoids the shortcomings of conventional silica membranes and filter papers in handling complex samples. Furthermore, this invention integrates chitosan-modified nucleic acid enrichment filter paper for secondary nucleic acid enrichment, which avoids the problem of incomplete sample loading and low sensitivity caused by incomplete elution during conventional magnetic bead extraction.

[0030] This invention can be applied to nucleic acid detection of multiple mixed swabs, such as 50-to-1 or 100-to-1, avoiding the low sensitivity problem of conventional methods when detecting 50-to-1 or 100-to-1 samples, and also reducing costs. In addition, this invention can be further extended to the field of liquid biopsy, such as for the specific and highly sensitive detection of cancer. Attached Figure Description

[0031] The accompanying drawings are intended only to illustrate and explain the present invention and do not limit the scope of the invention.

[0032] in:

[0033] Figure 1 : This is an exploded view of the microfluidic cartridge for specific detection of nucleic acids from complex samples according to the present invention.

[0034] Figure 2 This is a schematic diagram of the assembly of the card box cover, the lower pipe layer, and the intermediate chamber of the present invention.

[0035] Figure 3: An exploded view of the sample tube of the present invention and the first hollow ejector pin and ejector pin fixing hole below it.

[0036] Figure 4 : This is an exploded view of the magnetic bead extraction cavity tube of the present invention and the fifth hollow ejector pin and ejector pin fixing hole below it.

[0037] Figure 5 : This is an exploded view of the waste liquid tank pipe of the present invention and the ninth hollow ejector pin and ejector pin fixing hole below it.

[0038] Figure 6 : This is an exploded view of the lower part body and nucleic acid enrichment filter paper of the present invention.

[0039] In the picture:

[0040] 1. Card box lid;

[0041] 2. Intermediate chamber body;

[0042] 200. Pneumatic rubber stopper; 201. Air source needle channel; 2021. First upper rubber stopper; 2022. Second upper rubber stopper; 2031. First lower rubber stopper; 2032. Second lower rubber stopper; 204. Waste liquid tank pipe cover; 205. Air outlet; 206. Reusable cavity rubber stopper;

[0043] 21. Outer casing; 22. Side panel; 23. Tube support;

[0044] 241. Sample tube; 242. Washing solution tube; 243. Elution solution tube; 244. Enrichment solution tube; 245. Magnetic bead extraction chamber tube; 246. Switch valve; 247. Reagent tube; 248. Waste liquid tank tube;

[0045] 3. Lower piping layer;

[0046] 31. Short hollow thimble;

[0047] 32. Long hollow thimble;

[0048] 33. Pin fixing hole; 331. Dispensing hole; 332. Pin insertion hole; 333. Solution opening at the tip of the needle;

[0049] 34. Lower body;

[0050] 35. Nucleic acid enrichment filter paper;

[0051] 36. Fluid conduit; 361. First flow channel; 362. Second flow channel; 363. Third flow channel;

[0052] 37. Filter paper fixing holes;

[0053] 38. Reaction chamber. Detailed Implementation

[0054] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention will now be described with reference to the accompanying drawings.

[0055] The specific embodiments of the present invention described herein are for illustrative purposes only and should not be construed as limiting the invention in any way. Under the teachings of this invention, those skilled in the art can conceive of any possible modifications based on the invention, all of which should be considered within the scope of the invention. It should be noted that when an element is referred to as being "set on" another element, it can be directly on the other element or there may be an intervening element. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or there may be an intervening element. The terms "mounted," "connected," and "linked" should be interpreted broadly; for example, they can refer to mechanical or electrical connections, or internal communication between two elements, and can be direct or indirect connections through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms according to the specific circumstances. The terms "vertical," "horizontal," "upper," "lower," "left," "right," and similar expressions used herein are for illustrative purposes only and do not represent the only possible embodiments.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0057] like Figures 1 to 6 As shown, the present invention provides a microfluidic cartridge for specific detection of nucleic acids from complex samples, including a cartridge cover 1, an intermediate chamber 2 and a lower conduit layer 3;

[0058] The intermediate chamber 2 is equipped with a tubular structure that can move along the axial direction. The tubular structure includes multiple solution tubes, each of which can store a certain amount of reagents to supply the reagents required for sample processing and detection. The tubular structure includes at least a sample tube 241, a magnetic bead extraction chamber tube 245, and a reagent tube 247.

[0059] Sample tube 241 is used for sample loading and nucleic acid release from the cartridge; the sample source can be samples containing DNA or RNA, such as pharyngeal swab washing fluid, blood, animal tissue, food, pathogenic microorganisms, etc.; before loading, lyophilized proteinase K can also be pre-filled inside for digestion of impurity proteins;

[0060] The magnetic bead extraction chamber 245 uses magnetic bead nucleic acid purification technology. The magnetic bead extraction chamber 245 is equipped with a reuse chamber, in which magnetic beads are pre-placed. The magnetic beads are used to magnetically capture and extract nucleic acids, and the reuse chamber is used for nucleic acid extraction, washing and elution.

[0061] Reagent tube 247 is used to contain reaction reagents for in situ amplification of nucleic acids; these reagents can be PCR reagents or isothermal amplification reagents, such as RPA, Lamp, etc.

[0062] The lower part of the pipeline layer 3 includes a lower part body 34 that can be fastened to the bottom of the intermediate chamber. The lower part body 34 is provided with a fluid pipeline 36 and a hollow needle structure. The hollow needle structure can connect the pipeline structure and the fluid pipeline 36, and play the role of opening the flow path. The fluid pipeline 36 can perform micro-operations on the reagent.

[0063] The lower body 34 is also provided with a reaction chamber 38. The reaction chamber 38 can be connected to the magnetic bead extraction chamber 245 and the reagent tube 247 through the fluid pipe 36. The nucleic acid enrichment filter paper 35 can be fixedly placed in the reaction chamber 38. The nucleic acid enrichment filter paper 35 is modified with chitosan and can enrich nucleic acids and amplify nucleic acids in situ on the nucleic acid enrichment filter paper 35. The reagent tube 247 can be connected to the reaction chamber 38 to provide it with reaction reagents for nucleic acid in situ amplification. The reaction chamber 38 is used for nucleic acid enrichment and nucleic acid in situ amplification. The reaction chamber 38 can fix the nucleic acid enrichment filter paper 35 for nucleic acid capture and enrichment, and the captured nucleic acid can be used for in situ amplification.

[0064] The card holder cover 1 can be detachably fastened to the top of the intermediate chamber body 2. The card holder cover 1 is used to secure the tubular structure inside the intermediate chamber body, ensuring that no parts are lost when the chamber is inverted.

[0065] This invention constitutes an integrated structure for processing highly complex samples: all steps of conventional magnetic bead-based nucleic acid purification are concentrated in the cartridge, which avoids the time-sharing, step-by-step and manual intervention required by conventional magnetic bead extractors. Only one sample loading step is needed to complete the magnetic bead extraction in a fully automatic and fully enclosed manner with the cartridge, with a low possibility of external interference.

[0066] This invention achieves ultra-high sensitivity nucleic acid detection through a two-stage nucleic acid extraction and enrichment process. Conventional nucleic acid extraction based on magnetic beads cannot fully load the purified sample after elution during detection, thus limiting the potential for further sensitivity improvement due to methodological limitations. This invention integrates chitosan-modified filter paper after magnetic bead extraction, allowing for further enrichment and full loading of the sample after extraction and elution, followed by in-situ amplification, significantly improving the sensitivity of nucleic acid detection. In a specific embodiment of this invention, the nucleic acid detection sensitivity reaches 20 copies / ml, a substantial improvement over the existing 100 copies / ml.

[0067] The microfluidic cartridge for specific nucleic acid detection from complex samples of the present invention integrates a sample tube, a magnetic bead extraction chamber, a reagent tube for providing nucleic acid in situ amplification reaction reagents, a nucleic acid enrichment filter paper, and a reaction chamber for in situ nucleic acid amplification. It combines magnetic bead nucleic acid purification technology with chitosan-modified nucleic acid enrichment filter paper and in situ amplification detection technology for fully integrated, specific, and highly sensitive nucleic acid detection from complex samples.

[0068] The advantages of this invention's integrated magnetic bead method for nucleic acid purification are that it can purify low-load nucleic acids from complex samples, avoiding the problems of complex procedures, long processing times, high dependence on operators, and low nucleic acid extraction efficiency associated with conventional manual nucleic acid extraction. It also avoids the shortcomings of conventional silica membranes and filter papers in handling complex samples. Furthermore, this invention integrates chitosan-modified nucleic acid enrichment filter paper for secondary nucleic acid enrichment, which avoids the problem of incomplete sample loading and low sensitivity caused by incomplete elution during conventional magnetic bead extraction.

[0069] This invention can be applied to nucleic acid detection of multiple mixed swabs, such as 50-to-1 or 100-to-1, avoiding the low sensitivity problem of conventional methods when detecting 50-to-1 or 100-to-1 samples, and also reducing costs. In addition, this invention can be further extended to the field of liquid biopsy, such as for the specific and highly sensitive detection of cancer.

[0070] Furthermore, such as Figure 1 As shown, the tubing structure also includes a washing liquid tube 242, an eluent tube 243, and an enrichment liquid tube 244. The washing liquid tube 242 contains washing liquid and can communicate with the reuse chamber to provide washing liquid to the reuse chamber. The washing liquid is an alcohol-containing washing liquid, which can wash away impurities such as proteins that are not specifically adsorbed on the magnetic beads in the reuse chamber of the magnetic bead extraction chamber tube 245.

[0071] The eluent tube 243 contains eluent and can be connected to the reuse chamber to provide eluent to the reuse chamber. It can elute nucleic acid from the nucleic acid-magnetic bead complex after removing impurities for enrichment, amplification and detection.

[0072] The enrichment tube 244 contains a nucleic acid resuspension solution and can be connected to the reuse chamber to provide the nucleic acid resuspension solution to the reuse chamber. The nucleic acid resuspension solution is a slightly acidic DEPC solution, which can resuspend the eluted nucleic acid and then flow through the nucleic acid enrichment filter paper 35 for nucleic acid enrichment.

[0073] Furthermore, such as Figure 1 As shown, a waste liquid pool pipe 248 is also provided inside the intermediate chamber body 2. The waste liquid pool pipe 248 can be connected to the reaction chamber 38. The waste liquid pool pipe 248 is used to contain the waste liquid after flowing through the nucleic acid enrichment filter paper, so as to avoid opening the intermediate chamber body 2 during the detection process and reduce contamination.

[0074] Furthermore, such as Figure 1 As shown, a switching valve 246 is also provided in the intermediate chamber 2. The switching valve 246 is used to connect or disconnect the fluid pipe 36 on the reuse chamber and the lower body 34. The switching valve has a connecting structure, which can put the flow channels before and after the switching valve into a connected or closed state.

[0075] Furthermore, such as Figure 6 As shown, the fluid conduit 36 ​​includes a first flow channel 361, a second flow channel 362, and a third flow channel 363. The first flow channel 361 is not connected to the second flow channel 362 and the third flow channel 363. The second flow channel 362 and the third flow channel 363 are both connected to the reaction chamber 38. The fluid conduit 36 ​​can perform microliter-nanoliter fluid manipulation.

[0076] like Figure 1 As shown, the lower body 34 is provided with a first hollow ejector pin, a second hollow ejector pin, a third hollow ejector pin, a fourth hollow ejector pin, a fifth hollow ejector pin and a sixth hollow ejector pin that communicate with the first flow channel 361; the lower body 34 is provided with a seventh hollow ejector pin and an eighth hollow ejector pin that communicate with the second flow channel 362; and the lower body 34 is provided with a ninth hollow ejector pin that communicates with the third flow channel 363.

[0077] The first, second, third, fourth, sixth, seventh, eighth, and ninth hollow thimbles are short hollow thimbles 31, and the fifth hollow thimble is a long hollow thimble 32. Each hollow thimble is hollow, allowing the reagent solution to flow through. Each short hollow thimble has a beveled end, which can pierce the bottom of each solution tube and valve (sample tube 241, washing solution tube 242, elution solution tube 243, enrichment solution tube 244, switch valve 246, reagent tube 247, and waste liquid tank tube 248) to open and close the solution flow path in conjunction with the inner cavity of each solution tube, thereby controlling the fluid flow.

[0078] The long hollow ejector pin (the fifth hollow ejector pin) has a beveled end and is located at the lower end of the reuse chamber of the magnetic bead extraction tube 245, allowing for fluid manipulation within the reuse chamber. Slightly longer than the short hollow ejector pin, it ensures that when the lower end of the reuse chamber is punctured, the long hollow ejector pin can penetrate a certain distance and create a space with the lower end of the reuse chamber of the magnetic bead extraction tube 245. This prevents the magnetic bead from settling and blocking the long hollow ejector pin during magnetic bead manipulation.

[0079] Sample tube 241 is connected to magnetic bead extraction chamber 245 via a first hollow pin and a first flow channel 361; washing solution tube 242 is connected to magnetic bead extraction chamber 245 via a second hollow pin and a first flow channel 361; elution solution tube 243 is connected to magnetic bead extraction chamber 245 via a third hollow pin and a first flow channel 361; enrichment solution tube 244 is connected to magnetic bead extraction chamber 245 via a fourth hollow pin and a first flow channel 361; magnetic bead extraction chamber 245 is connected to first flow channel 361 via a fifth hollow pin; switching valve 246 is connected to first flow channel 361 via a sixth hollow pin; switching valve 246 is connected to reaction chamber 38 via a seventh hollow pin and a second flow channel 362; reagent tube 247 is connected to reaction chamber 38 via an eighth hollow pin and a second flow channel 362; waste liquid tank tube 248 is connected to reaction chamber 38 via a ninth hollow pin and a third flow channel 363.

[0080] Furthermore, such as Figure 1 As shown, the lower body 34 is provided with multiple ejector pin fixing holes 33 for inserting and fixing the first hollow ejector pin, the second hollow ejector pin, the third hollow ejector pin, the fourth hollow ejector pin, the fifth hollow ejector pin, the sixth hollow ejector pin, the seventh hollow ejector pin, the eighth hollow ejector pin and the ninth hollow ejector pin;

[0081] like Figure 3 , Figure 4 , Figure 5As shown, each of the pin fixing holes 33 includes a pin insertion hole 332. The bottom end of each pin insertion hole 332 is provided with a needle end solution opening 333, and the top end of each pin insertion hole 332 is provided with a dispensing hole 331 with an increasing diameter. The dispensing hole 331 is used for dispensing glue to fix the first hollow pin, the second hollow pin, the third hollow pin, the fourth hollow pin, the fifth hollow pin, the seventh hollow pin, the eighth hollow pin, and the ninth hollow pin, respectively.

[0082] The pin insertion hole 332 is used for inserting the pin. Its diameter is slightly larger than that of the hollow ejector pin to ensure the pin does not wobble during dispensing. When the hollow ejector pin is inserted into the pin insertion hole 332, dispensing adhesive into the dispensing hole 331 will fix the hollow ejector pin in place and seal it completely. UV-curing adhesive is preferred for dispensing.

[0083] Furthermore, such as Figure 3 As shown, a first lower rubber stopper 2031 and a first upper rubber stopper 2021 are spaced apart from bottom to top at the bottom of the sample tube 241. A sample cavity is provided above the first upper rubber stopper 2021. A first connecting cavity communicating with the sample cavity is provided between the first lower rubber stopper 2031 and the first upper rubber stopper 2021. In the initial state, the tip of the first hollow ejector pin is inserted into the first lower rubber stopper 2031 without piercing it, and the tip of the first hollow ejector pin is closed. In the working state, the tip of the first hollow ejector pin can pass through the first lower rubber stopper 2031 to communicate with the first connecting cavity, and the tip of the first hollow ejector pin can be inserted into the first upper rubber stopper 2021 to close it, so that the sample cavity is not connected to the flow channel and fluid manipulation is not possible. A pneumatic rubber stopper 200 is provided at the top of the sample tube 241, and an air source needle channel 201 is provided on the pneumatic rubber stopper 200. The lower end of the air source needle puncture channel 201 has a thin wall, so that when a hollow needle connected to an external air source punctures the rubber plug, there is not much puncture resistance.

[0084] The pneumatic stopper 200 has two annular grooves on its outer periphery for sealing the top of the sample tube 241; the pneumatic stopper 200 has an air source needle channel 201 inside, which allows an external air source to be connected to another piercing hollow needle to pierce the pneumatic stopper 200 and connect to the internal cavity (sample cavity); when the first hollow needle at the lower end of the sample tube pierces the first lower stopper 2031, fluid manipulation can be performed.

[0085] The structures of the washing solution tube 242, the elution solution tube 243, the enrichment solution tube 244, and the reagent tube 247 are the same as or similar to the structure of the sample tube 241.

[0086] Furthermore, such as Figure 4 As shown, a reuse chamber plug 206 is provided at the bottom of the magnetic bead extraction chamber tube 245, and a reuse chamber is provided above the reuse chamber plug 206. The top of the fifth hollow ejector pin can pass through the reuse chamber plug 206 to connect to the reuse chamber. A pneumatic plug 200 is provided at the top of the magnetic bead extraction chamber tube 245, and an air source needle channel 201 is provided on the pneumatic plug 200.

[0087] The magnetic bead extraction chamber 245 differs from the preceding solution tubes (sample tube 241, washing solution tube 242, elution solution tube 243, enrichment solution tube 244, and reagent tube 247) in two ways: First, the reusable chamber stopper 206 is a single-layer stopper, lacking an upper stopper. This is because the reusable chamber is pre-filled with magnetic beads. Before use, the tip of the fifth hollow pin (long hollow pin) is inserted into the reusable chamber stopper 206, sealing the interior of the reusable chamber. Before performing the magnetic bead operation, the magnetic beads must be... The bead extraction chamber 245 is pressed down a certain distance, causing the fifth hollow ejector pin (long hollow ejector pin) to pierce the reusable chamber stopper 206 and pass through a certain distance, so that the reusable chamber is connected to the first flow channel 361; secondly, the pneumatic stopper at the upper end of the magnetic bead extraction chamber is initially in a loose state and is not tightly sealed. It is sealed with aluminum foil to ensure that it will not leak. When using it, the aluminum foil needs to be torn open to ensure that its interior is connected to the external atmosphere, thus ensuring smooth fluid flow during magnetic bead operation.

[0088] Furthermore, such as Figure 5 As shown, the bottom end of the waste liquid tank pipe 248 is provided with a second lower rubber plug 2032 and a second upper rubber plug 2022 spaced apart from bottom to top. A waste liquid chamber is provided inside the waste liquid tank pipe 248. A second connecting cavity is provided between the second lower rubber plug 2032 and the second upper rubber plug 2022, which communicates with the waste liquid chamber. The top end of the ninth hollow pin can pass through the second lower rubber plug 2032 to communicate with the second connecting cavity, and the top end of the ninth hollow pin can be inserted into the second upper rubber plug 2022 to seal it. A waste liquid tank pipe cover 204 is provided at the top end of the waste liquid tank pipe 248. A vent hole 205 is provided on the waste liquid tank pipe cover 204. The vent hole 205 is a small circular hole that allows the waste liquid chamber to communicate with the outside atmosphere, maintaining the internal pressure during liquid inflow.

[0089] In the initial state, the tip of the ninth hollow ejector pin is pierced through the second lower rubber stopper 2032, and this state continues until all fluid control in the cartridge is completed and no more waste liquid needs to be added. Then, the waste liquid pool tube 248 is pressed down so that the tip of the ninth hollow ejector pin is inserted into the second upper rubber stopper 2022, sealing the waste liquid pool tube 248.

[0090] Furthermore, such as Figure 2 As shown, the intermediate chamber body 2 includes an outer shell 21, a side patch 22 is provided on the outer wall of the outer shell 21, a tube rack 23 is provided inside the outer shell 21, and multiple receiving channels are provided inside the tube rack 23. The multiple receiving channels are respectively slidably arranged with a washing liquid tube 242, an elution liquid tube 243, a enrichment liquid tube 244, a magnetic bead extraction chamber tube 245, a switch valve 246, a reagent tube 247, and a waste liquid pool tube 248. The card box cover 1 is detachably fastened to the top of the tube rack; the sample tube 241 is provided on the outside of the tube rack; and the lower tube layer 3 is fastened to the bottom of the outer shell.

[0091] Furthermore, such as Figure 6As shown, the reaction chamber 38 is equipped with filter paper fixing holes 37 for fixing nucleic acid enrichment filter paper 35. The chitosan-modified nucleic acid enrichment filter paper 35 is used to enrich nucleic acids purified from magnetic beads and to perform in-situ amplification of the nucleic acids on the filter paper. The reaction chamber 38 contains reaction reagents, providing the raw materials and primer / probe system required for nucleic acid amplification.

[0092] Taking the detection of SARS-CoV-2 in a "50-to-1" oral swab rinse solution by PCR as an example, the method for specifically detecting nucleic acid from complex samples using the microfluidic cartridge of the present invention includes:

[0093] Step a, Reagent pre-storage: Pre-store reagents in the sample tube, washing solution tube, elution solution tube, enrichment solution tube, magnetic bead extraction chamber tube and reagent tube of the aforementioned microfluidic cartridge for specific detection of nucleic acids from complex samples;

[0094] Specifically, each individual solution tube (sample tube 241, washing solution tube 242, elution solution tube 243, enrichment solution tube 244, magnetic bead extraction chamber tube 245, and reagent tube 247) has reagents pre-stored for subsequent experiments.

[0095] During the pre-storage process, the pneumatic rubber stoppers at the top of the sample tube 241, washing solution tube 242, elution solution tube 243, enrichment solution tube 244, and reagent tube 247 are matched with the first upper rubber stopper and the first lower rubber stopper at the bottom. The pneumatic rubber stopper at the top of the magnetic bead extraction chamber tube 245 is matched with the reusable chamber rubber stopper 206 at the bottom.

[0096] The sample tube 241 contains 20 μL of proteinase K, the washing solution tube 242 contains 700 μL of optimized alcohol-containing washing solution, the elution solution tube 243 contains 200 μL of elution solution, the enrichment solution tube 244 contains 1 mL of DEPC water (DNase, RNase free) with pH 5.5, the magnetic bead extraction chamber tube 245 contains 10 μL of magnetic beads, and the reagent tube 247 stores reagents that can be used for PCR amplification MIX reagents.

[0097] Step b, Sample collection: Place the collected sample into sample tube 241;

[0098] Specifically, 50 mL centrifuge tubes (swab rinsing tubes used during sampling, existing technology) containing 30 mL of viral lysis buffer were used to collect 50 oral swabs. The swabs were rinsed directly into the centrifuge tubes after collection, and nucleic acid was released immediately within the tubes. A novel coronavirus (2019-nCoV) pseudovirus ribonucleic acid standard was used instead. During sample collection, the samples were transferred from the centrifuge tubes to sample tube 241, which had a volume of 1–2 mL.

[0099] Step c, Nucleic acid release: The sample undergoes pathogen lysis within sample tube 241 to release nucleic acid;

[0100] Specifically, the centrifuge tube contains viral lysis buffer. Once the sample is inside, the pathogen can be lysed, releasing nucleic acid.

[0101] Step d, Nucleic acid capture: Prepare to use a fully integrated instrument, press down to connect the sample tube 241 and the magnetic bead extraction chamber 245, blow air into the sample tube 241, and the solution in the sample tube 241 flows into the reuse chamber of the magnetic bead extraction chamber 245 and mixes with the magnetic beads. The process of capturing nucleic acid with magnetic beads is completed with the help of the fully integrated instrument.

[0102] Specifically, a fully integrated instrument will be used. The structure of the fully integrated instrument is not unique, but it only needs to meet the following four conditions: it has a small integrated air pump, the end of which must have a hollow needle for air inlet and outlet to ensure that it can be used with the pneumatic rubber stoppers at the top of each solution tube for fluid control; it has a controllable magnetic control module to provide a controllable magnetic field for nucleic acid extraction-washing-elution of magnetic beads in the magnetic bead extraction chamber 245; it has a temperature control module, i.e., a temperature control component, to provide the required temperature during the reaction; and it has a fluorescence detection module, i.e., a fluorescence detection component, to read the fluorescence at the reaction chamber 38 and the nucleic acid enrichment filter paper 35 in real time.

[0103] When performing nucleic acid capture, the sample tube 241 and the magnetic bead extraction chamber tube 245 need to be pressed down first. The top of the first hollow pin passes through the first lower rubber stopper 2031 at the bottom of the sample tube 241 and connects to the first connecting chamber. The top of the fifth hollow pin passes through the reusable chamber rubber stopper 206 and connects to the reusable chamber. The inner cavities of the sample tube 241 and the magnetic bead extraction chamber tube 245 are connected.

[0104] Next, the external air pump hollow needle (existing technology) is inserted into the air source needle channel 201 of the pneumatic rubber stopper 200 at the top of the sample tube 241, and air is blown into the sample tube 241. At this time, the solution in the sample tube 241 flows into the reuse chamber of the magnetic bead extraction chamber 245 and mixes with the magnetic beads. Air is blown slowly and continuously to achieve the effect of "blowing" and mixing. After a period of time, the blowing is stopped, and magnetic attraction is performed with the fully integrated instrument to make the magnetic beads adhere to the side wall of the magnetic bead extraction chamber. Then, air is drawn out of the sample tube 241 to draw the solution in the reuse chamber back into the sample tube 241. Then the sample tube 241 is pressed down, and the top of the first hollow needle is inserted into the first rubber stopper 2021 to close the first hollow needle, thus completing the entire process of magnetic bead capturing nucleic acid.

[0105] Step e, Nucleic acid washing: Press down the washing solution tube 242 to connect it with the magnetic bead extraction chamber tube 245, blow air into the washing solution tube 242, and the washing solution in the washing solution tube 242 flows into the reuse chamber of the magnetic bead extraction chamber tube 245 to wash the magnetic beads. With the help of the fully integrated instrument, the washing solution is withdrawn to complete the washing process of the magnetic beads capturing nucleic acid.

[0106] Specifically, the washing liquid tube 242 is pressed down a certain distance, and the top of the second hollow pin passes through the first lower rubber plug 2031 at the bottom of the washing liquid tube 242, connecting the washing liquid tube 242 with the magnetic bead extraction chamber tube 245.

[0107] Next, the external air pump hollow needle (existing technology) is inserted into the air source needle channel 201 of the pneumatic rubber stopper 200 at the top of the washing liquid tube 242, and air is continuously blown in. At this time, the internal solution enters the reuse chamber of the magnetic bead extraction chamber 245. Then, the magnetic adsorption module of the fully integrated instrument is removed, and air is blown in again. The magnetic beads will be fully resuspended in the washing liquid. Air is blown in for a period of time to thoroughly wash and remove non-specifically adsorbed proteins and other impurities. Air blowing is stopped, and the magnetic beads are adsorbed by the magnetic adsorption module of the fully integrated instrument to both sides of the magnetic bead extraction chamber. Then, the washing liquid tube 242 is evacuated to draw the washing waste liquid in the magnetic bead extraction chamber 245 back into the washing liquid tube 242. The washing liquid tube 242 is then pressed down to allow the second hollow needle to penetrate into the first rubber stopper 2021. The second hollow needle is then closed, thus completing the entire washing process of magnetic beads capturing nucleic acid.

[0108] Step f, Nucleic acid elution: Press down the elution tube 243 to connect it with the magnetic bead extraction chamber 245, blow air into the elution tube 243, and the elution fluid in the elution tube 243 flows into the reuse chamber of the magnetic bead extraction chamber 245 to elute the magnetic beads. The elution process of magnetic beads capturing nucleic acid is completed with the help of the fully integrated instrument.

[0109] Specifically, the eluent tube 243 is pressed down a certain distance, and the top of the third hollow pin passes through the first lower rubber stopper 2031 at the bottom of the eluent tube 243, connecting the eluent tube 243 with the magnetic bead extraction chamber tube 245.

[0110] Next, the external air pump hollow needle (existing technology) is inserted into the air source needle channel 201 of the pneumatic rubber stopper 200 at the top of the elution tube 243, and air is continuously blown in. At this time, the solution inside enters the reuse chamber of the magnetic bead extraction chamber 245. Then, the magnetic suction module of the fully integrated instrument is removed. As air continues to be blown, the magnetic beads will be fully resuspended in the elution solution. Air is blown in for a period of time to fully elute. Air blowing is stopped, the elution tube 243 is pressed down, so that the tip of the third hollow needle is inserted into the first rubber stopper 2021. The third hollow needle is closed, thus completing the entire elution process of magnetic beads capturing nucleic acid.

[0111] Step g, Nucleic acid enrichment: Press down the enrichment solution tube 244 to connect it with the magnetic bead extraction chamber tube 245, blow air into the enrichment solution tube 244 so that the solution inside enters the reuse chamber of the magnetic bead extraction chamber tube 245 (the enrichment solution and the elution solution are mixed, and the nucleic acid contained in the elution solution is dispersed in the enrichment solution for subsequent capture and enrichment by filter paper); press down the switch valve 246 to blow air into the magnetic bead extraction chamber tube 245. The purified nucleic acid sample solution in the magnetic bead extraction chamber tube 245 will pass through the switch valve 246 and flow through the fluid pipe 36 to the reaction chamber 38. The nucleic acid sample solution flows through the nucleic acid enrichment filter paper 35 and is enriched by the filter paper. The enriched solution enters the waste liquid pool tube 248.

[0112] Specifically, the enrichment tube 244 is pressed down a certain distance, and the top of the fourth hollow pin passes through the first lower rubber plug 2031 at the bottom of the enrichment tube 244, connecting the enrichment tube 244 with the magnetic bead extraction chamber 245.

[0113] Next, insert the external air pump hollow needle (existing technology) into the air source needle channel 201 of the pneumatic rubber stopper 200 at the top of the enrichment liquid tube 244, and continuously blow air into it. At this time, the solution inside enters the reuse chamber of the magnetic bead extraction chamber 245. At this time, with the magnetic attraction module of the fully integrated instrument, as air is slowly blown, the magnetic beads will be slowly attracted to the two side walls of the magnetic bead extraction chamber. Stop blowing air, press down the enrichment liquid tube 244, so that the top of the fourth hollow needle is inserted into the first rubber stopper 2021, and close the fourth hollow needle.

[0114] The pneumatic rubber stopper of the magnetic bead extraction chamber 245 is sealed to seal the reuse chamber of the magnetic bead extraction chamber 245; the switch valve 246 is pressed down a certain distance, and the sixth hollow needle and the seventh hollow needle are connected to the inner cavity of the switch valve, so the switch valve 246 is in the open flow state; then, the hollow needle of the external air pump (existing technology) is inserted into the air source needle channel 201 of the pneumatic rubber stopper 200 at the top of the magnetic bead extraction chamber 245, and air is continuously blown in. At this time, the purified nucleic acid sample solution in the magnetic bead extraction chamber 245 will pass through the switch valve 246 and flow into the reaction chamber through the second flow channel 362. The nucleic acid sample solution flows through the nucleic acid enrichment filter paper 35 and is enriched by the filter paper. The enriched solution enters the waste liquid pool pipe 248 through the third flow channel 363 and the ninth hollow needle.

[0115] Step h, Nucleic acid amplification: Press down the reagent tube 247 to connect it to the reaction chamber 38 through the fluid pipe 36, blow air into the reagent tube 247 so that the solution inside enters and fills the reaction chamber 38 and the nucleic acid enrichment filter paper 35 enriched with nucleic acid samples;

[0116] Specifically, the reagent tube 247 is pressed down a certain distance, and the tip of the eighth hollow needle passes through the first lower rubber stopper 2031 at the bottom of the reagent tube 247, connecting the reagent tube 247 to the second flow channel 362; then, the external air pump hollow needle (existing technology) is inserted into the air source needle channel 201 of the pneumatic rubber stopper 200 at the top of the reagent tube 247, and air is continuously and slowly blown in. At this time, the solution inside the reagent tube 247 will enter and fill the reaction chamber 38 and the nucleic acid enrichment filter paper 35 enriched with nucleic acid samples;

[0117] Step i, Output detection results: In conjunction with the fluorescence detection module of the fully integrated instrument (existing technology), real-time fluorescence detection is performed on the reaction chamber 38 and nucleic acid enrichment filter paper 35 in the lower pipeline layer 3, and fluorescence values ​​are continuously plotted into fluorescence curves. Finally, quantitative judgment is made based on the Ct value.

[0118] As described above, the microfluidic cartridge and method for specifically detecting nucleic acids from complex samples of the present invention have the following beneficial effects:

[0119] In this invention, a sample tube, a magnetic bead extraction chamber, a reagent tube providing nucleic acid in situ amplification reaction reagents, a nucleic acid enrichment filter paper, and a reaction chamber for nucleic acid in situ amplification are integrated. This combines magnetic bead nucleic acid purification technology with chitosan-modified nucleic acid enrichment filter paper and in situ amplification detection technology for a fully integrated, specific, and highly sensitive nucleic acid detection from complex samples.

[0120] The advantages of this invention's integrated magnetic bead method for nucleic acid purification are that it can purify low-load nucleic acids from complex samples, avoiding the problems of complex procedures, long processing times, high dependence on operators, and low nucleic acid extraction efficiency associated with conventional manual nucleic acid extraction. It also avoids the shortcomings of conventional silica membranes and filter papers in handling complex samples. Furthermore, this invention integrates chitosan-modified nucleic acid enrichment filter paper for secondary nucleic acid enrichment, which avoids the problem of incomplete sample loading and low sensitivity caused by incomplete elution during conventional magnetic bead extraction.

[0121] This invention can be applied to nucleic acid detection of multiple mixed swabs, such as 50-to-1 or 100-to-1, avoiding the low sensitivity problem of conventional methods when detecting 50-to-1 or 100-to-1 samples, and also reducing costs. In addition, this invention can be further extended to the field of liquid biopsy, such as for the specific and highly sensitive detection of cancer.

[0122] The above description is merely an illustrative embodiment of the present invention and is not intended to limit the scope of the invention. Any equivalent changes and modifications made by those skilled in the art without departing from the concept and principles of the present invention should fall within the scope of protection of the present invention.

Claims

1. A microfluidic cartridge for specific detection of nucleic acids from complex samples, characterized in that, The device includes a cartridge cover, a central chamber, and a lower conduit layer. The central chamber contains a tubular structure, which includes at least a sample tube, a magnetic bead extraction tube, and a reagent tube. The sample tube is used for loading the cartridge with samples and releasing nucleic acid. The reagent tube is used to hold reaction reagents for in-situ nucleic acid amplification. The lower conduit layer includes a lower body that can be fastened to the bottom of the central chamber. The lower body has a fluid conduit and a hollow needle structure, which connects the tubular structure and the fluid conduit. The lower body also has a reaction chamber, which connects to the magnetic bead extraction tube and the reagent tube via the fluid conduit. Nucleic acid enrichment filter paper can be fixedly disposed within the reaction chamber. The reagent tube communicates with the reaction chamber to provide reaction reagents for in-situ nucleic acid amplification. The reaction chamber is used for nucleic acid enrichment and in-situ nucleic acid amplification. The cartridge cover is detachably fastened to the top of the central chamber. The magnetic bead extraction chamber is provided with a reuse chamber, and magnetic beads are pre-placed in the reuse chamber.

2. The microfluidic cartridge for specific detection of nucleic acids from complex samples as described in claim 1, characterized in that, The magnetic beads are used to magnetically capture and extract nucleic acids, and the reuse chamber is used for nucleic acid extraction, washing, and elution.

3. The microfluidic cartridge for specific detection of nucleic acids from complex samples as described in claim 2, characterized in that, The tubing structure also includes a washing liquid tube, an eluent tube, and an enrichment liquid tube. The washing liquid tube contains washing liquid and can communicate with the reuse chamber to provide washing liquid to the reuse chamber. The elution tube contains elution solution and is connected to the reuse chamber to provide elution solution to the reuse chamber; the enrichment tube contains nucleic acid resuspension solution and is connected to the reuse chamber to provide nucleic acid resuspension solution to the reuse chamber.

4. The microfluidic cartridge for specific detection of nucleic acids from complex samples as described in claim 3, characterized in that, The intermediate chamber is also equipped with a waste liquid pool pipe, which can be connected to the reaction chamber and is used to contain the waste liquid after it has flowed through the nucleic acid enrichment filter paper.

5. The microfluidic cartridge for specific detection of nucleic acids from complex samples as described in claim 4, characterized in that, The intermediate chamber is also equipped with a switching valve, which is used to connect or disconnect the fluid pipes on the reuse chamber and the lower body.

6. The microfluidic cartridge for specific detection of nucleic acids from complex samples as described in claim 5, characterized in that, The fluid conduit includes a first flow channel, a second flow channel, and a third flow channel. The lower part body is provided with a first hollow ejector pin, a second hollow ejector pin, a third hollow ejector pin, a fourth hollow ejector pin, a fifth hollow ejector pin, and a sixth hollow ejector pin that communicate with the first flow channel. The lower part body is provided with a seventh hollow ejector pin and an eighth hollow ejector pin that communicate with the second flow channel. The lower part body is provided with a ninth hollow ejector pin that communicates with the third flow channel. The sample tube is connected to the magnetic bead extraction chamber via the first hollow pin and the first flow channel; the washing solution tube is connected to the magnetic bead extraction chamber via the second hollow pin and the first flow channel; the elution solution tube is connected to the magnetic bead extraction chamber via the third hollow pin and the first flow channel; the enrichment solution tube is connected to the magnetic bead extraction chamber via the fourth hollow pin and the first flow channel; the magnetic bead extraction chamber is connected to the first flow channel via the fifth hollow pin; the switching valve is connected to the first flow channel via the sixth hollow pin; the switching valve is connected to the reaction chamber via the seventh hollow pin and the second flow channel; the reagent tube is connected to the reaction chamber via the eighth hollow pin and the second flow channel; and the waste liquid tank tube is connected to the reaction chamber via the ninth hollow pin and the third flow channel.

7. The microfluidic cartridge for specific detection of nucleic acids from complex samples as described in claim 6, characterized in that, The lower body is provided with multiple insertion holes for inserting the first, second, third, fourth, fifth, sixth, seventh, eighth, and ninth hollow ejector pins. Each insertion hole has a needle tip solution opening at its bottom end and an enlarging dispensing hole at its top end. The dispensing hole is used to dispense adhesive to fix the first, second, third, fourth, fifth, seventh, eighth, and ninth hollow ejector pins respectively.

8. The microfluidic cartridge for specific detection of nucleic acids from complex samples as described in claim 6, characterized in that, The sample tube has a first lower rubber stopper and a first upper rubber stopper spaced apart from bottom to top at its bottom end. A sample cavity is provided above the first upper rubber stopper. A first connecting cavity communicating with the sample cavity is provided between the first lower rubber stopper and the first upper rubber stopper. The tip of the first hollow ejector pin can pass through the first lower rubber stopper to communicate with the first connecting cavity, and the tip of the first hollow ejector pin can be inserted into the first upper rubber stopper to seal it. A pneumatic rubber stopper is provided at the top end of the sample tube, and an air source needle channel is provided on the pneumatic rubber stopper.

9. The microfluidic cartridge for specific detection of nucleic acids from complex samples as described in claim 6, characterized in that, The bottom end of the magnetic bead extraction tube is provided with a reusable cavity plug, and the reusable cavity is provided above the reusable cavity plug. The top end of the fifth hollow pin can pass through the reusable cavity plug and connect to the reusable cavity. The top end of the magnetic bead extraction tube is provided with a pneumatic plug, and the pneumatic plug is provided with a pneumatic needle channel.

10. The microfluidic cartridge for specific detection of nucleic acids from complex samples as described in claim 6, characterized in that, The bottom end of the waste liquid tank pipe is provided with a second lower rubber plug and a second upper rubber plug spaced apart from bottom to top. A waste liquid chamber is provided inside the waste liquid tank pipe. A second communicating cavity is provided between the second lower rubber plug and the second upper rubber plug, which communicates with the waste liquid chamber. The top end of the ninth hollow pin can pass through the second lower rubber plug to communicate with the second communicating cavity, and the top end of the ninth hollow pin can be inserted into the second upper rubber plug to seal it. A waste liquid tank pipe cover is provided at the top end of the waste liquid tank pipe, and an air vent is provided on the waste liquid tank pipe cover to allow the waste liquid chamber to communicate with the outside atmosphere.

11. A method for specifically detecting nucleic acids from complex samples for non-diagnostic purposes, characterized in that, Includes the following steps: Step a, Reagent pre-storage: Pre-store reagents in the sample tube, washing solution tube, elution solution tube, enrichment solution tube, magnetic bead extraction chamber tube and reagent tube of the microfluidic cartridge for specific detection of nucleic acids from complex samples as described in any one of claims 3 to 10; Step b, Sample collection: Place the collected sample into a sample tube; Step c, Nucleic acid release: The sample undergoes pathogen lysis within the sample tube, releasing nucleic acid; Step d, Nucleic acid capture: Prepare to use a fully integrated instrument, press down to connect the sample tube and the magnetic bead extraction chamber, blow air into the sample tube, and the solution in the sample tube flows into the reuse chamber of the magnetic bead extraction chamber and mixes with the magnetic beads. The process of magnetic beads capturing nucleic acid is completed with the help of the fully integrated instrument. Step e, Nucleic acid washing: Press down the washing solution tube to connect it with the magnetic bead extraction chamber tube, blow air into the washing solution tube, and the washing solution in the washing solution tube flows into the reuse chamber of the magnetic bead extraction chamber tube to wash the magnetic beads. With the help of the fully integrated instrument, the washing solution is withdrawn to complete the washing process of the magnetic beads capturing nucleic acid. Step f, Nucleic acid elution: Press down the elution tube to connect it with the magnetic bead extraction chamber tube, blow air into the elution tube, and the elution solution in the elution tube flows into the reuse chamber of the magnetic bead extraction chamber tube to elute the magnetic beads. The elution process of magnetic beads capturing nucleic acid is completed with the help of the fully integrated instrument. Step g, Nucleic Acid Enrichment: Press down the enrichment liquid tube to connect it with the magnetic bead extraction chamber tube, blow air into the enrichment liquid tube to allow the solution inside to enter the reuse chamber of the magnetic bead extraction chamber tube; press down the switch valve to blow air into the magnetic bead extraction chamber tube, and the purified nucleic acid sample solution in the magnetic bead extraction chamber tube will pass through the switch valve and flow through the fluid pipe to the reaction chamber. The nucleic acid sample solution flows through the nucleic acid enrichment filter paper and is enriched by the filter paper. The enriched solution enters the waste liquid tank tube. Step h, Nucleic acid amplification: Press down the reagent tube to connect it to the reaction chamber through the fluid tube, blow air into the reagent tube so that the solution inside enters and fills the reaction chamber and the nucleic acid enrichment filter paper enriched with nucleic acid samples; Step i, Output detection results: Real-time fluorescence detection is performed on the reaction chamber and nucleic acid enrichment filter paper in the lower pipeline layer, and fluorescence values ​​are continuously plotted into fluorescence curves. Finally, quantitative judgment is made based on the Ct value.

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