Molecule detection device and detection method

Abstract

The invention relates to a molecular detection device and method, and the device comprises a detection card which comprises at least one detection group, and each detection group comprises a reaction cavity, a release cavity and a chromatography detection module which are in one-to-one correspondence; a second reagent is stored in the reaction cavity, and one end of the chromatography detection module extends to the release cavity; the sample to be detected can react with the second reagent to generate a first intermediate sample; a first pretreatment container for providing a first reagent; the first pretreatment container is detachably and hermetically communicated with the reaction cavity; and the fluid control assembly is used for selectively separating or communicating the reaction cavity and the release cavity. The molecule detection device and the detection method provided by the embodiment of the invention have the advantages of simple structure and high detection precision.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, and in particular to a molecular detection device and detection method. Background Technology

[0002] Since the beginning of the 21st century, various infectious diseases have experienced intermittent outbreaks. Coupled with the rapid advancement of globalization, disease prevention and control have faced immense pressure. Firstly, the diagnosis and treatment system has shifted from a traditional hospital- and laboratory-centric model to a decentralized one. Secondly, testing scenarios have shifted from hospital-based testing to community or home testing. Thirdly, higher demands are being placed on testing technologies, with large-scale, automated, and rapid testing becoming the trend.

[0003] Currently, most home testing products on the market are developed based on LAMP or RPA isothermal amplification technologies. However, due to the difficulty in designing primers and probes for these traditional detection technologies, false positives are often produced, affecting the reliability of the test results.

[0004] Next-generation molecular diagnostic technology (CRISPR detection technology) effectively solves the false positive problem caused by traditional isothermal amplification processes. Large medical testing institutions combine the CRISPR / Cas system with various isothermal nucleic acid amplification strategies (most commonly RPA and LAMP). This involves pre-amplifying the target to be tested to enrich low-abundance molecular targets, thereby improving the specificity and sensitivity of the analysis, and then introducing the CRISPR / Cas system. This approach is very suitable for the development of rapid nucleic acid detection products.

[0005] However, this testing method is cumbersome to operate, has high sealing requirements, and the testing device has a complex structure, making it unsuitable for home use. Summary of the Invention

[0006] Therefore, it is necessary to provide a molecular detection device and detection method to address the problems of complex structure and low detection accuracy of existing detection devices.

[0007] A first aspect of this application provides a molecular detection device, comprising: a detection card including at least one detection group, each detection group including a corresponding reaction chamber, a release chamber, and a chromatography detection module; the reaction chamber storing a second reagent, and one end of the chromatography detection module extending to the release chamber; a sample to be tested capable of reacting with the second reagent to generate a first intermediate sample; a first pretreatment container for providing the first reagent; the first pretreatment container being detachably sealed and connected to the reaction chamber; the first reagent being capable of reacting with the first intermediate sample to generate a target sample; and a fluid control component for selectively isolating or connecting the reaction chamber and the release chamber; when the fluid control component controls the reaction chamber and the release chamber to be connected, the target sample can flow from the reaction chamber into the release chamber and contact the chromatography detection module.

[0008] In one embodiment, the molecular detection device further includes a second pretreatment container, which includes a bottle body, a bottle cap, and a sealing cap. The bottle cap has a second dropper on top. The bottle body is used to contain the sample to be tested. The bottle cap is placed over the mouth of the bottle body, and the sealing cap is placed over the bottle cap. The second dropper is used to drip the sample to be tested into the reaction chamber.

[0009] In one embodiment, the detection card includes an upper shell and a lower shell; the chromatography detection module is laid on the lower shell, and the reaction chamber and the release chamber are disposed on the lower shell; the upper shell covers the lower shell; the upper shell is provided with a first mounting platform in the area corresponding to the reaction chamber, and the first mounting platform forms a sample dispensing port communicating with the reaction chamber; the first pretreatment container is detachably assembled on the first mounting platform.

[0010] In one embodiment, the molecular detection device further includes an aerosol barrier plug; the aerosol barrier plug includes a plug body and a one-way duckbill valve, the plug body having a through-hole, the one-way duckbill valve standing vertically below the plug body and communicating with the through-hole; the plug body is placed on the first mounting platform, the one-way duckbill valve is sealed and inserted into the sample dispensing port; the first pretreatment container is sealed and installed on the through-hole.

[0011] In one embodiment, the first pretreatment container includes a tube body, a sealing aluminum film, and a tube cap. The sealing aluminum film seals and covers the opening of the tube body. The top of the tube cap is provided with a first drip head. The tube cap is placed over the opening of the tube body, and the first drip head is sealed and inserted into the docking hole.

[0012] In one embodiment, the molecular detection device includes a thermostatic incubator for heating the reaction chamber.

[0013] In one embodiment, the upper shell is provided with a second mounting platform in the region corresponding to the release chamber, and the second mounting platform forms a mounting port communicating with the release chamber; the fluid control assembly includes a limiting member, a first linkage rod, a liquid release plug, and a release button; the first linkage rod is vertically movable through the mounting port, and the release button is located at the end of the first linkage rod protruding from the mounting port; the liquid release plug is located at the end of the first linkage rod away from the release button; the release button has a first release position and a first initial position; when the release button is held in the first initial position, the liquid release plug is located in the flow channel of the reaction chamber and the release chamber to isolate the reaction chamber and the release chamber; when the release button is held in the first release position, the liquid release plug is located in the release chamber, and the reaction chamber and the release chamber remain in communication; the limiting member limits the release button to keep the release button in the first initial position.

[0014] In one embodiment, the limiting member is a U-shaped limiting clamp; the limiting member is horizontally embedded between the release button and the second mounting platform.

[0015] In one embodiment, the fluid control assembly includes a sealing plug, a second linkage rod, and a knob; the sealing plug passes through the second linkage rod and is disposed on the opening of the release chamber; the second linkage rod is rotatably mounted on the lower housing about its own axis, and the knob is disposed at the end of the second linkage rod away from the sealing plug; the knob has a second release position and a second initial position; when the knob is rotated to the second initial position, the sealing plug seals and covers the opening of the release chamber to isolate the reaction chamber and the release chamber; when the knob is rotated to the second release position, there is a gap between the sealing plug and the opening of the release chamber, and the reaction chamber and the release chamber remain in communication.

[0016] In one embodiment, the sealing plug is a cylinder with a semi-circular cross-section, and the periphery of the sealing plug includes an arc surface and a plane connecting the arc surface; when the knob is rotated to the second initial position, the arc surface seals and covers the opening of the release cavity; when the knob is rotated to the second release position, a gap is formed between the plane and the opening of the release cavity.

[0017] In one embodiment, the test sample is reacted with the second reagent in at least one of loop-mediated isothermal amplification, recombinase polymerase amplification, cross-primer amplification, and nuclease-mediated isothermal amplification; the first reagent is reacted with the first intermediate sample in a CRISPR / Cas system; the test sample is an extract containing nucleic acid components; the second reagent includes RPA reaction reagent or LAMP reaction reagent; the first reagent includes CRISPR / Cas reagent.

[0018] A second aspect of this application provides a detection method applied to the aforementioned molecular detection device; the detection method includes:

[0019] The sample to be tested is injected into the reaction chamber;

[0020] The isothermal amplification reaction was completed, and the first intermediate sample was obtained.

[0021] The first pretreatment container is controlled to seal and inject the first reagent into the reaction chamber; the first intermediate sample and the first reagent continue to react for a first preset time to obtain the target sample.

[0022] The fluid control component is controlled to connect the reaction chamber and the release chamber, and the target sample enters the release chamber from the reaction chamber and comes into contact with the chromatography detection module;

[0023] Observe the results of the chromatography detection module.

[0024] In one embodiment, the step of injecting the sample to be tested into the reaction chamber specifically includes: adding an original sample into a second pretreatment container, the second pretreatment container storing nucleic acid extraction solution, the original sample reacting with the nucleic acid extraction solution to generate the sample to be tested, the original sample including blood; and dripping the sample to be tested from the second dropper of the second pretreatment container into the sample dispensing port on the first mounting stage so that the sample to be tested enters the reaction chamber.

[0025] In one embodiment, before the step of completing the isothermal amplification reaction and obtaining the first intermediate sample, the method further includes: installing an aerosol barrier plug on the first mounting stage to seal the sample application port; and controlling the isothermal incubator to vibrate the reaction chamber for 3 to 10 minutes.

[0026] In one embodiment, the step of completing the isothermal amplification reaction and obtaining the first intermediate sample specifically includes: heating the reaction chamber using a controlled isothermal incubator for a duration of 1 min to 10 min.

[0027] The beneficial effects are:

[0028] This application discloses a molecular detection device and method. By setting independent reaction chambers, release chambers, and chromatography detection modules on a detection card, the reaction chamber stores a second reagent. The sample to be tested can react with the second reagent beforehand to generate a first intermediate sample, thus completing the preliminary sample processing for isothermal amplification. Then, a first pretreatment container acts as a liquid addition tube, sealingly adding the first reagent into the reaction chamber of the detection card. The first reagent and the first intermediate sample then undergo a CRISPR / Cas reaction. The first pretreatment container, acting as the liquid addition tube, achieves contactless and closed-loop addition of the CRISPR / Cas reagent, completely eliminating the risk of contamination from opening and transferring the reagent. This integrates multiple steps, including isothermal amplification and the CRISPR / Cas reaction, into a closed space, effectively avoiding... This device eliminates aerosol contamination and ensures reaction specificity. The fluid control component selectively isolates or connects the reaction chamber and release chamber, allowing operators to precisely control the release of the target sample into the chromatography detection module for final detection. This integrates the CRISPR / Cas system with isothermal amplification technology into a fully enclosed molecular detection device, achieving a completely closed-loop detection process. This effectively prevents aerosol contamination and significantly improves the accuracy and reliability of the detection. Furthermore, the molecular detection device is simple and quick to operate, requiring no professional training. Users can obtain near-laboratory-level test results within minutes through simple steps, making it perfectly suitable for various scenarios such as home use, primary healthcare, and rapid on-site testing. It provides strong support for early screening of infectious diseases and personal health management. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the structure of a molecular detection device provided in some embodiments of this application.

[0030] Figure 2 This is a schematic diagram illustrating the interaction between a detection card and a fluid control component provided in some embodiments of this application.

[0031] Figure 3 for Figure 2 Exploded view of the structure shown.

[0032] Figure 4 This is a schematic diagram of the structure of a molecular detection device provided in some embodiments of this application; wherein, the first pretreatment container is omitted.

[0033] Figure 5 for Figure 4 The diagram shows the structure from another perspective.

[0034] Figure 6 This is a schematic diagram of the structure of a constant temperature incubator provided in some embodiments of this application.

[0035] Figure 7This is a schematic diagram of the structure of the aerosol barrier provided in some embodiments of this application.

[0036] Figure 8 This is a schematic diagram of the structure of a first pretreatment container provided in some embodiments of this application, wherein the tube body and the tube cap are in a state of separation.

[0037] Figure 9 This is a schematic diagram of the structure of a second pretreatment container provided in some embodiments of this application.

[0038] Figure 10 This is a partial diagram illustrating the interaction between a detection card and a fluid control component according to some embodiments of this application, wherein the release button is in a first initial position.

[0039] Figure 11 for Figure 10 The diagram shows the structure of the reaction chamber from another perspective.

[0040] Figure 12 This is a partial diagram illustrating the interaction between the detection card, aerosol barrier plug, and fluid control component provided in some embodiments of this application, wherein the release button is in a first initial position.

[0041] Figure 13 This is a partial diagram illustrating the interaction between the detection card, the first pretreatment container, the aerosol barrier plug, and the fluid control component provided in some embodiments of this application, wherein the release button is in the first initial position.

[0042] Figure 14 This is a partial diagram illustrating the interaction between the detection card, the first pretreatment container, the aerosol barrier plug, and the fluid control component provided in some embodiments of this application, wherein the release button is in the first release position.

[0043] Figure 15 This is a schematic diagram of the structure of the detection card and fluid control assembly provided in some embodiments of this application, wherein the upper shell is omitted and the knob is in the second initial position.

[0044] Figure 16 for Figure 15 A schematic diagram of a partial interaction between the detection card and the fluid control component in the illustrated embodiment.

[0045] Figure 17 This is a schematic diagram of the structure of the detection card and fluid control assembly provided in some embodiments of this application, wherein the upper shell is omitted and the knob is in the second release position.

[0046] Figure 18 for Figure 17 A schematic diagram of a partial interaction between the detection card and the fluid control component in the illustrated embodiment.

[0047] Figure 19This is a schematic diagram of the structure of a chromatography detection module provided in some embodiments of this application.

[0048] Figure 20 This is a flowchart illustrating the detection method provided in some embodiments of this application. Detailed Implementation

[0049] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0050] 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 pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0051] In the description of the embodiments of this application, if the technical terms such as "first" and "second" appear, these terms are used only for descriptive purposes to distinguish different objects, and should not be construed as indicating or implying relative importance or implicitly indicating the number, specific order or primary and secondary relationship of the indicated technical features.

[0052] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0053] In the description of the embodiments in this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0054] In the description of the embodiments of this application, if the term "multiple" appears, "multiple" means at least two (including two), such as two, three, etc., unless otherwise explicitly specified. Similarly, if the term "multiple sets" appears, "multiple sets" refers to two or more sets (including two sets), and if the term "multiple pieces" appears, "multiple pieces" refers to two or more pieces (including two pieces).

[0055] In the description of the embodiments of this application, if the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. appear, these terms indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of this application 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. Therefore, they should not be construed as limitations on the embodiments of this application.

[0056] In the description of the embodiments of this application, unless otherwise explicitly specified and limited, the technical terms "installation," "connection," "joining," "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; 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; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of this application according to the specific circumstances.

[0057] In this application, unless otherwise expressly specified and limited, the use of descriptions such as "above" or "below" the second feature indicates that the first and second features are in direct contact or indirect contact via an intermediate medium. Furthermore, "above," "on top of," and "over" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. Similarly, "below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0058] It should be noted that if an element is referred to as being "fixed to" or "set on" another element, it can be directly on the other element or there may be an intervening element. If an element is considered to be "connected to" another element, it can be directly connected to the other element or there may be an intervening element. If so, the terms "vertical," "horizontal," "upper," "lower," "left," "right," and similar expressions used in this application are for illustrative purposes only and do not represent the only possible implementation.

[0059] In the detection of certain infectious diseases, such as HIV and syphilis self-testing products, due to their high degree of privacy, most users choose to pre-test with home testing products before confirmation. However, the vast majority of self-testing products on the market are antigen or antibody products based on immunochromatography technology. These products have a significant disadvantage compared to products based on nucleic acid testing technology. During the incubation period after viral infection, the body may not have produced enough antibodies, but viral nucleic acid has already begun to replicate in the body. At this time, nucleic acid testing can detect viral RNA or DNA, enabling earlier detection of infection than immunochromatography. This allows users to understand their condition as early as possible, and if infected, to take immediate measures to block the virus and receive treatment.

[0060] qPCR is the most common method for infectious disease detection and the gold standard for nucleic acid testing, but it has many limitations. For example, in the face of public health emergencies such as avian influenza, qPCR also has certain limitations in the process of large-scale screening of infected individuals. First, the sample size is enormous, and large-scale population sampling can easily lead to infection risks. Second, since a large number of samples are tested by third-party laboratories, the time and labor costs involved in sample transportation, preservation, nucleic acid extraction and purification are relatively high. Third, to achieve its sensitivity and specificity, temperature-switching amplification is unavoidable, which leads to a large-scale replication of pathogen fragments during amplification, potentially resulting in risks of aerosol and sample cross-contamination. Therefore, it has high requirements for testing sites, making qPCR unsuitable for rapid point-of-care testing. Fourth, because the amplification process of qPCR involves temperature-switching and annealing, it has higher equipment requirements than isothermal amplification or amplification-free CRISPR technology, which has lower equipment requirements.

[0061] Isothermal amplification nucleic acid detection technologies (such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (RT-LAMP), cross-primer amplification (CPA), nuclease-mediated isothermal amplification (RHAM), multi-enzyme isothermal rapid amplification (MIRA), and recombinase-mediated isothermal amplification (RAA)) have simple instrument requirements. Unlike qPCR, they do not require complex and precise temperature control instruments; a single source of equipment capable of maintaining a constant temperature is sufficient. These isothermal detection technologies are highly sensitive, but due to the complexity of system design (e.g., insufficient primer specificity, primer dimer formation, and failure of enzymes to reach their maximum efficiency) and inherent limitations of the detection methods, non-specific amplification can easily occur under isothermal conditions, leading to false positive results. The home market urgently needs highly accurate and easy-to-use detection products based on CRISPR nucleic acid detection technology.

[0062] In related technologies, combining the CRISPR / Cas system with various isothermal nucleic acid amplification strategies (most commonly RPA and LAMP) involves pre-amplifying the target to enrich low-abundance molecular targets, improving analytical specificity and sensitivity, and then introducing the CRISPR / Cas system. This approach is well-suited for the development of rapid nucleic acid detection products. However, traditional CRISPR / Cas methods are prone to aerosol contamination due to manual operation. The CRISPR / Cas detection system involves two steps before chromatographic membrane climbing: the first step requires isothermal pre-amplification to increase the number of targets and improve sensitivity; the second step requires adding Cas system reagents to the amplified product, where the non-specific trans-cleavage ability activated by the Cas enzyme after binding to the target cleaves the fluorescent probe. These two steps are mutually exclusive; if the reagents for both steps are placed together, the cleavage activity of the Cas enzyme will reduce the yield of the amplified product. Therefore, in the routine detection process, the amplified product needs to be manually transferred into the CRISPR / Cas detection system. This step-by-step operation has a significant risk of nucleic acid aerosol leakage, which can easily lead to cross-contamination. As a result, such devices are generally cumbersome to operate and have complex structures due to cross-contamination.

[0063] Furthermore, although immunodiagnostics has a longer window period compared to molecular detection technologies, resulting in significant differences in detection sensitivity and specificity, its advantages such as rapid detection speed, simple operation, low cost, and ease of large-scale manufacturing mean that immunochromatographic assays still account for the vast majority of current home rapid testing products. For some tests requiring high detection sensitivity, a feasible strategy is to first react the antigen / antibody, which would normally undergo a solid-liquid reaction on the chromatographic test strip, with the sample in a liquid phase (liquid-liquid reactions are homogeneous reactions), thereby improving the binding efficiency of the antigen / antibody to the analyte in the sample. The resulting immunocomplex product is then introduced into the chromatographic test strip, where it is ultimately captured by the antigen / antibody coated with the T and C lines on the nitrocellulose membrane, resulting in color development, and finally, the result is interpreted.

[0064] In summary, detection products using test strips as reaction carriers are widely used in molecular detection. For CRISPR / Cas-based test strip detection schemes, the entire process of the first step (isotropic molecular amplification) and the introduction of the Cas enzyme system for the second reaction must be completed in a liquid phase before the test strip is introduced. Furthermore, nucleic acid aerosol contamination must be prevented during both the first isothermal amplification step and the transfer to the second reaction step.

[0065] The aforementioned testing products are unsuitable for home use due to their cumbersome testing procedures, high sealing requirements, and complex testing device structures.

[0066] The first aspect of this application provides a molecular detection device that can provide high-precision detection based on a regularly clustered, interspaced short palindromic repeat sequence / CRISPR-associated protein system (hereinafter referred to as the CRISPR / Cas system).

[0067] See Figures 1 to 20 As shown, the molecular detection device includes: a detection card 100, a first pretreatment container 200, and a fluid control assembly 400.

[0068] The detection card 100 includes at least one detection group, each detection group including a corresponding reaction chamber 110, a release chamber 120, and a chromatography detection module 130. The reaction chamber 110 stores a second reagent 112, and one end of the chromatography detection module 130 extends into the release chamber 120. The sample to be tested can react with the second reagent 112 to generate a first intermediate sample. A first pretreatment container 200 can be used to provide a first reagent (not shown); the first pretreatment container 200 is detachably sealed to the reaction chamber 110; the first reagent can react with the first intermediate sample to generate a target sample. A fluid control assembly 400 is used to selectively isolate or connect the reaction chamber 110 and the release chamber 120. When the fluid control assembly 400 controls the reaction chamber 110 and the release chamber 120 to be connected, the target sample can flow from the reaction chamber 110 into the release chamber 120 and contact the chromatography detection module 130.

[0069] The fluid control component 400 may be housed within the detection card 100. The first pretreatment container 200 is detachably mounted on the detection card 100.

[0070] By setting independent reaction chambers 110, release chambers 120, and chromatography detection modules 130 in the detection card 100, the reaction chamber 110 stores a second reagent 112. The sample to be tested can react with the second reagent 112 in advance to generate a first intermediate sample, thereby completing the preliminary sample processing for isothermal amplification. Then, the first pretreatment container 200 is used as a liquid addition tube to seal and add the first reagent into the reaction chamber 110 of the detection card 100. The first reagent and the first intermediate sample then undergo a CRISPR / Cas reaction. The first pretreatment container 200, acting as a liquid addition tube, achieves contactless and closed-loop addition of CRISPR / Cas reagents, completely eliminating the risk of contamination caused by opening the cap and transferring the reagent. This integrates multiple steps such as isothermal amplification and CRISPR / Cas reaction into a closed space, effectively avoiding contamination. Aerosol contamination is eliminated while ensuring reaction specificity. The fluid control component 400 selectively isolates or connects the reaction chamber 110 and the release chamber 120, allowing the operator to precisely control the release of the target sample into the chromatography detection module 130 for final detection. This integrates the CRISPR / Cas system with isothermal amplification technology into a fully enclosed molecular detection device, achieving a completely closed-loop operation of the detection process, effectively eliminating aerosol contamination, and significantly improving the accuracy and reliability of the detection. At the same time, the molecular detection device is simple and quick to operate, requiring no professional training. Users can obtain near-laboratory-level test results within 30 minutes through simple steps, perfectly adapting to various scenarios such as home, primary healthcare, and rapid on-site testing, providing strong support for early screening of infectious diseases and personal health management.

[0071] In this embodiment of the application, the specific detection process is as follows:

[0072] The operator first injects the sample to be tested into the reaction chamber 110. The sample and the second reagent 112 complete the amplification reaction, obtaining a first intermediate sample. The sample and the second reagent 112 can undergo at least one of the following reactions: loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), cross-primer amplification (CPA), and nuclease-mediated isothermal amplification (RHAM). The sample to be tested can be an extract containing nucleic acid components. The second reagent 112 includes reagents specific to RPA, LAMP, CPA, and RHAM reactions. The second reagent 112 is typically stored in the reaction chamber 110 in the form of lyophilized bulbs / liquid.

[0073] The first pretreatment container 200 can seal and inject the first reagent into the reaction chamber 110; the first intermediate sample continues to react with the first reagent to obtain the target sample. The first reagent and the first intermediate sample undergo a CRISPR / Cas system reaction. Thus, by sealing the first reagent into the reaction chamber 110, which has already undergone isothermal amplification, through the first pretreatment container 200, and allowing the first intermediate sample to continue reacting with the first reagent, aerosol contamination can be effectively avoided, improving detection accuracy. The first reagent includes CRISPR / Cas-specific reagents.

[0074] Finally, the operator switches the fluid control component 400, allowing the target sample to enter the release chamber 120 from the reaction chamber 110 and come into contact with the chromatography detection module 130. The operator then completes the detection by reading the detection results on the chromatography detection module 130.

[0075] Optionally, the second reagent 112 may include components such as recombinase, single-stranded DNA-binding protein, and strand displacement DNA polymerase. Optionally, the second reagent 112 may include components such as strand displacement DNA polymerase and primer set.

[0076] Optionally, the first reagent includes reagents specific to the CRISPR / Cas system or LAMP reaction, consisting of regularly clustered short palindromic repeats / CRISPR-associated proteins; specifically, it may include components such as Cas protein scissors, crRNA navigation, reporter probe signal source, and buffer environment.

[0077] Optionally, the chromatography detection module 130 includes a molecular chromatography test strip.

[0078] In some possible embodiments, see Figures 1 to 20 As shown, the detection card 100 includes multiple detection groups, which are arranged side by side with intervals between them. In this embodiment, each detection group is independent of the others, that is, the reaction chamber 110, release chamber 120 and chromatography detection module 130 in each detection group are all independent and physically separated, thereby ensuring that the reaction process does not interfere with each other.

[0079] For ease of understanding, in the following embodiments, unless otherwise specified, the structures in the same detection group will be used for description, such as reaction chamber 110, release chamber 120, and chromatography detection module 130.

[0080] In some possible embodiments, see Figures 1 to 20 As shown, the lower shell 150 includes an inclined transition structure 160.

[0081] The transition structure 160 may be a groove or a ramp connecting the release chamber 120 and the reaction chamber 110.

[0082] The bottom of the release chamber 120 is at a lower level than the bottom of the reaction chamber 110; the release chamber 120 and the reaction chamber 110 are connected by an inclined transition structure 160; thus, when the reaction chamber 110 and the release chamber 120 are connected, the target sample can enter the release chamber 120 from the reaction chamber 110 through the transition structure 160 and finally come into contact with the chromatography detection module 130 to complete the detection process.

[0083] In some possible embodiments, see Figures 1 to 20 As shown, the molecular detection device also includes a second pretreatment container 500, which includes a bottle body 510, a bottle cap 520, and a sealing cap 530. The bottle cap 520 is provided with a second dropper (not shown) on its top. The bottle body 510 is used to hold the sample to be tested. The bottle cap 520 is placed on the bottle mouth of the bottle body 510, and the sealing cap 530 is placed on the bottle cap 520. The second dropper is used to drop the sample to be tested into the reaction chamber 110.

[0084] Combination Figure 3 and Figure 9 As shown, the second pretreatment container 500 is sealed by the bottle body 510, the bottle cap 520 and the sealing cap 530. The bottle body 510 and the bottle cap 520 can be connected by threads, and the sealing cap 530 and the bottle body 510 can be fastened by threaded connection.

[0085] The second pretreatment container 500 can be used to contain the extraction solution and the original sample for extraction reaction to generate the sample to be tested. In this embodiment, the original sample can be blood, urine, feces, or other possible biological samples. The extraction solution can be a nucleic acid extraction solution.

[0086] The second pretreatment container 500 enables the pretreatment reaction of the original sample and nucleic acid extraction solution in a closed space, effectively preventing sample leakage and external contamination. The second pretreatment container 500 is equipped with a second dropper at the top, which facilitates the dripping of the sample to be tested into the sample application port 142 of the test card 100, ensuring that the operator can inject the sample to be tested into the reaction chamber 110.

[0087] In some possible embodiments, see Figures 1 to 20 As shown, the detection card 100 includes an upper shell 140 and a lower shell 150; the chromatography detection module 130 is laid on the lower shell 150, and the reaction chamber 110 and the release chamber 120 are disposed on the lower shell 150; the upper shell 140 covers the lower shell 150; a first mounting platform 141 is provided on the area of ​​the upper shell 140 corresponding to the reaction chamber 110, and the first mounting platform 141 forms a sample dispensing port 142 communicating with the reaction chamber 110. The first pretreatment container 200 is detachably assembled on the first mounting platform 141.

[0088] The detection card 100, through the cooperation of the upper shell 140 and the lower shell 150, provides a stable, enclosed space for the chromatography detection module 130, the reaction chamber 110, and the release chamber 120. The first mounting platform 141 and its sample dispensing port 142 set in the upper shell 140 provide a precise positioning and sealed assembly basis for the first pretreatment container 200, ensuring that there is no leakage or contamination during the addition of the first reagent. By adopting the above-mentioned shell structure, not only is a compact layout of each functional component achieved, but also the user operation steps are simplified through the detachable assembly method. While ensuring the airtightness of the reaction system, the overall structural strength and ease of operation of the device are significantly improved, providing a guarantee for the reliability of home testing environments.

[0089] In some possible embodiments, see Figures 1 to 20 As shown, the molecular detection device also includes an aerosol barrier plug 600; the aerosol barrier plug 600 includes a plug body 610 and a one-way duckbill valve 620, the plug body 610 has a through docking hole 630, the one-way duckbill valve 620 is erected below the plug body 610 and communicates with the docking hole 630; the plug body 610 is covered on the first mounting platform 141, and the one-way duckbill valve 620 is sealed and inserted into the sample application port 142; the first pretreatment container 200 is sealed and installed on the docking hole 630.

[0090] Aerosol barrier plug 600 can be made of rubber.

[0091] The one-way duckbill valve 620 has a cylindrical upper part and a duckbill lower part. The diameter of the cylindrical part is slightly larger than that of the sample inlet 142, so that the one-way duckbill valve 620 is interference-fitted into the sample inlet 142 to achieve a seal. The duckbill structure opens under pressure and closes when there is no pressure, effectively improving the sealing performance. The overall shape of the plug body 610 can be the same as or match the mounting surface at the top of the first mounting platform 141.

[0092] By tightly sealing the plug body 610 with the first mounting platform 141, the one-way duckbill valve 620 seals the sample inlet 142, and the first pretreatment container 200 is sealed and installed on the docking hole 630; after the sample to be tested in the reaction chamber 110 completes the amplification reaction with the second reagent 112 and obtains the first intermediate sample, the aerosol barrier plug 600 constructs a reliable aerosol physical barrier. By operating and squeezing the first pretreatment container 200 to add liquid, the pressure of the first reagent can open the one-way duckbill valve 620 and enter the reaction chamber 110; after the pressure is removed, the duckbill valve automatically closes, thereby, the first reagent is injected from the first pretreatment container 200 through the docking hole 630, the sample inlet 142 and finally sealed into the reaction chamber 110, and the first intermediate sample continues to react with the first reagent to obtain the target sample. The aerosol barrier plug 600 maintains the airtightness of the entire reaction system and eliminates the risk of false positives caused by nucleic acid aerosol contamination, ensuring high reliability of test results while maintaining the airtightness of the entire reaction system.

[0093] Optionally, combined Figure 2 and Figure 3 As shown, a sealing gasket 145 can be provided on the first mounting platform 141. When the test card 100 is not equipped with the aerosol barrier plug 600, it can be produced and transported separately. The sealing gasket 145 covers the first mounting platform 141 to seal the sample inlet 142 and prevent the reaction chamber 110 from contacting the external environment.

[0094] When the molecular detection device is used for detection, the operator can open the sealing gasket 145, drop the sample to be tested into the sample application port 142 through the second drop head of the second pretreatment container 500, and then install the aerosol barrier plug 600 to complete the subsequent operation and ensure airtightness.

[0095] In some possible embodiments, see Figures 1 to 20 As shown, the first pretreatment container 200 includes a tube body 210, a sealing aluminum film (not shown), and a tube cap 220. The sealing aluminum film seals and covers the opening of the tube body 210. A first drip head 230 is provided on the top of the tube cap 220. The tube cap 220 covers the opening of the tube body 210, and the first drip head 230 is sealed and inserted into the docking hole 630.

[0096] Since the first reagent often has an expiration date, it can be divided into liquid reagent and lyophilized bulb components.

[0097] Combination Figure 8 As shown, tube 210 is used to generate the first reagent. Tube 210 contains a reconstituted solution, and its opening is sealed with a removable aluminum foil. The lyophilized bulbs can be stored in the retaining cage 221 of the cap 220. In use, the aluminum foil on the opening of tube 210 is removed, the connecting arm 240 is rotated, and the cap 220 is pressed into place, aligning with the opening of tube 210. The cap 220 and the inner wall of the tube opening are sealed due to an interference fit. The operator can then invert the first pretreatment container 200 times. The reconstituted solution inside tube 210 will pass through the gap in the retaining cage 221 into the cap 220, coming into contact with the lyophilized bulbs. The lyophilized bulbs are fully reconstituted, ultimately generating the first reagent.

[0098] In some possible embodiments, see Figures 1 to 20 As shown, the top side of the plug body 610 away from the one-way duckbill valve 620 is a mounting plane 611, and a slot 612 is provided on the mounting plane 611 for fixing the first pretreatment container 200.

[0099] The first dropper head 230 of the first pretreatment container 200 is aligned with the docking hole 630 of the aerosol barrier plug 600 and inserted. The edge of the cap 220 of the first pretreatment container 200 has a protrusion that corresponds to the slot 612 on the mounting plane 611, thereby fixing the first pretreatment container 200 onto the aerosol barrier plug 600. At this time, the operator can squeeze the tube body 210, and the squeezed first reagent will open the one-way duckbill valve 620 on the aerosol barrier plug 600, ensuring that the first reagent can enter the reaction chamber 110 of the test card 100. When the squeezing pressure on the tube body 210 is removed, the one-way duckbill valve 620 closes again, restoring the internal and external isolation state of the test card 100; forming a complete closed flow path, which not only realizes the integration and sealing of the first reagent reconstitution and liquid addition operation, but also effectively prevents the leakage of the first reagent and environmental pollution, and avoids the risk of aerosol contamination caused by manual transfer operation, ensuring the reliability of the test results.

[0100] In some other embodiments, the first pretreatment container 200 may pre-store the complete first reagent; this application does not limit this.

[0101] In some possible embodiments, see Figures 1 to 20 As shown, the molecular detection device includes a constant temperature incubator 300, which is positioned below the detection card 100 and is used to heat the reaction chamber 110.

[0102] The constant temperature incubator 300 is usually located below the detection card 100 and can be detached and fitted with the lower shell 150.

[0103] The sample to be tested is injected into the reaction chamber 110. The sample to be tested reacts with the second reagent 112 in the reaction chamber 110 to obtain a first intermediate sample. The constant temperature incubator 300 can heat the reaction chamber 110, providing a stable and precise temperature environment to ensure that the sample to be tested and the second reagent 112 can complete the isothermal amplification reaction in the reaction chamber 110. This overcomes the influence of ambient temperature fluctuations on the detection results and ultimately improves the detection accuracy of the molecular detection device.

[0104] In some possible embodiments, see Figures 1 to 20 As shown, the constant temperature incubator 300 includes a constant temperature body 340 with a mounting groove 330, and the bottom side of the lower shell 150 facing away from the upper shell 140 is fitted into the mounting groove 330; the bottom of the mounting groove 330 facing the lower shell 150 is provided with a heating boss 310 corresponding to the reaction chamber 110.

[0105] The heating boss 310 has a heating element inside, which can be either resistance heating or ceramic heating.

[0106] By fitting the mounting slot 330 into the lower shell 150 of the test card 100, precise positioning and tight fit between the test card 100 and the constant temperature incubator 300 are achieved. The bottom of the mounting slot 330 is equipped with heating protrusions 310 that correspond one-to-one with the positions of the reaction chamber 110, thus forming a directional heat conduction path. This ensures that heat is efficiently and centrally transferred to the reaction system within the reaction chamber 110. This not only effectively improves heating efficiency and temperature uniformity, preventing heat loss, but also ensures that the reaction chamber 110 receives consistent heating conditions during each test, providing reliable temperature protection for the isothermal amplification reaction of the sample and the second reagent 112 within the reaction chamber 110.

[0107] In some possible embodiments, see Figures 1 to 20 As shown, the bottom of the release chamber 120 protrudes from the side of the lower shell 150 away from the upper shell 140; the mounting groove 330 faces the bottom of the lower shell 150 and is recessed downward to form a clearance groove 320 corresponding to the release chamber 120.

[0108] By setting the bottom of the release cavity 120 to protrude downwards and interlock with the clearance groove 320, a precise mechanical clearance structure is formed between the constant temperature incubator 300 and the detection card 100. On the one hand, this can effectively avoid structural interference that may occur between the release cavity 120 and the heating boss 310 area, ensuring that the lower shell 150 of the detection card 100 can be fully embedded in the mounting groove 330 and tightly fitted with the heating boss 310, thereby ensuring the heat transfer efficiency between the heating boss 310 and the reaction chamber 110. On the other hand, the clearance treatment of the release cavity 120 enables the rapid and accurate positioning of the detection card 100 and the constant temperature incubator 300, improving the smoothness of device assembly and the reliability of use.

[0109] Understandably, the bottom of the release chamber 120 protrudes from the side of the lower shell 150 away from the upper shell 140, and the bottom of the release chamber 120 is lower than the bottom of the reaction chamber 110. When the fluid control assembly 400 controls the reaction chamber 110 and the release chamber 120 to be connected, the target sample can flow from the reaction chamber 110 into the release chamber 120 without flowing in the opposite direction. The target sample comes into contact with the chromatography detection module 130, ensuring that the operator can complete the detection by reading the detection results on the chromatography detection module 130.

[0110] In some possible embodiments, see Figures 1 to 20 As shown, the thermostatic incubator 300 includes a vibrator (not shown) installed within the thermostatic body 340; the vibrator is used to vibrate the lower shell 150.

[0111] By incorporating a vibrator within the isothermal incubator 300, the vibrator applies periodic mechanical vibration to the lower shell 150 of the test card 100. This vibration is transmitted through the shell to the reaction chamber 110 during the initial incubation stage before isothermal amplification, ensuring thorough mixing of the second reagent 112 with the test sample. This significantly improves the reconstitution efficiency of the second reagent 112 with the test sample and enhances the uniformity of the reaction components, effectively promoting the kinetics of the initial isothermal amplification reaction and shortening the time required to reach the plateau phase. Furthermore, the vibrator design not only avoids the risk of aerosol contamination that may be introduced by manual mixing but also further improves the sensitivity and consistency of the detection by optimizing the reaction conditions.

[0112] In addition, the constant temperature incubator 300 also includes a switch 350 and a status indicator light 360. The switch 350 is used to control the opening and closing of the vibrator, heating boss 310 and other structures; the status indicator light 360 can be used to indicate time, charging, the current status of the vibrator, and the heating temperature of the heating boss 310, so as to facilitate the operator's observation and operation.

[0113] In some possible embodiments, see Figures 1 to 14 As shown, a second mounting platform 143 is provided in the area of ​​the upper shell 140 corresponding to the release cavity 120, and the second mounting platform 143 forms a mounting port 144 that communicates with the release cavity 120.

[0114] The fluid control assembly 400 includes a limiting member 410, a first linkage rod 420, a liquid release plug 430, and a release button 440; the first linkage rod 420 is vertically movable through the mounting port 144, and the release button 440 is located at the end of the first linkage rod 420 protruding from the mounting port 144; the liquid release plug 430 is located at the end of the first linkage rod 420 away from the release button 440.

[0115] The release button 440 has a first release position and a first initial position; when the release button 440 is held in the first initial position, the liquid release plug 430 is located in the flow channel of the reaction chamber 110 and the release chamber 120 to isolate the reaction chamber 110 and the release chamber 120; when the release button 440 is held in the first release position, the liquid release plug 430 is located in the release chamber 120, and the reaction chamber 110 and the release chamber 120 remain in communication; the limiting member 410 limits the release button 440 so that the release button 440 is held in the first initial position.

[0116] The first linkage rod 420 and the mounting port 144 are integrally interference-fitted to ensure a seal. The first linkage rod 420 can move along its own axial direction within the mounting port 144 (that is... Figure 10 (Up and down direction) movement.

[0117] In this embodiment, the fluid control component 400 includes a limiting member 410, a first linkage rod 420, a liquid release plug 430, and a release button 440. The limiting member 410 reliably limits the release button 440 to maintain its first initial position, ensuring that the liquid release plug 430 can stably isolate the flow channel between the reaction chamber 110 and the release chamber 120 during the reaction phase. This facilitates the isothermal amplification reaction of the second reagent 112 and the test sample, as well as the CRISPR / C reaction of the first intermediate sample and the first reagent. The reaction provides the necessary independent sealed space. When the result needs to be interpreted, the user presses the release button 440 to the first release position, which drives the first linkage rod 420 to push the liquid release plug 430 completely into the release chamber 120. At this time, the target sample can flow from the reaction chamber 110 into the release chamber 120 and come into contact with the chromatography detection module 130. This achieves controllable connection between the reaction system and the detection system, which not only ensures sufficient reaction time, but also simplifies the user's steps through intuitive pressing operation and effectively prevents detection failure caused by premature release.

[0118] In some possible embodiments, see Figures 1 to 14 As shown, the limiting member 410 is a U-shaped limiting clamp; the limiting member 410 is horizontally embedded between the release button 440 and the second mounting platform 143.

[0119] Thus, the limiting member 410 can act as a safety bolt to limit the release button 440, so that the release button 440 is kept in the first initial position.

[0120] Combination Figure 13 and Figure 14 As shown, when the lateral pull-out limiting member 410 (that is, Figure 3 (From the upper right to the lower left direction), the limiting member 410 disengages, the limiting is canceled, and the release button 440 can be pressed from the first initial position to the first release position under the action of external force. The release button 440 drives the first linkage rod 420 to push the liquid release plug 430 completely into the release chamber 120. At this time, the target sample can flow from the reaction chamber 110 into the release chamber 120 and come into contact with the chromatography detection module 130; thus, the controllable connection between the reaction system and the detection system is realized, which can not only ensure sufficient reaction time, but also simplify the user steps through intuitive pressing operation, and effectively prevent detection failure caused by premature release.

[0121] In this embodiment of the application, the detection process of the molecular detection device is as follows:

[0122] Unscrew the cap 520 of the second pretreatment container 500, add the original sample into the bottle 510, replace the cap 520, and shake the second pretreatment container 500 thoroughly (the shaking time and method can be adjusted according to different project types) to ensure that the original sample is fully mixed with the nucleic acid extraction solution and obtain the sample to be tested.

[0123] The test card 100 is installed onto the constant temperature incubator 300. The constant temperature incubator 300 and the lower shell 150 of the test card 100 cooperate with each other; the constant temperature incubator 300 has heating protrusions 310, which correspond one-to-one with the reaction chambers 110 of the test card 100; the constant temperature incubator 300 has clearance grooves 320, which correspond one-to-one with the bottom position of the release chamber 120 at the bottom of the test card 100, ensuring that the test card 100 and the constant temperature incubator 300 fit tightly and without interference; the vibrator inside the constant temperature incubator 300 corresponds to the vibration area 151 at the bottom of the test card 100.

[0124] Unscrew the sealing cap 530 of the second pretreatment container 500, open the sealing gasket 145 covering the test card 100, and drip the sample to be tested into the sample application port 142 through the second dropper. Each reaction chamber 110 can be precisely heated by the heating boss 310 of the isothermal incubator 300, thereby enabling the sample in the reaction chamber 110 to undergo a stable isothermal amplification reaction. It should be noted that the volume of the sample to be tested and the second reagent 112 in the isothermal amplification reaction is small, generally less than 50 μL. Due to the large surface roughness of the horizontal region at the bottom of the reaction chamber 110, which can range from 1 to 100 μm, the surface tension between the liquid and this surface is large, and the liquid can be fixed in the horizontal region without moving throughout the reaction stage.

[0125] The aerosol barrier plug 600 is installed on the first mounting stage 141, and the one-way duckbill valve 620 is sealed and inserted into the sample application port 142. The switch 350 of the isothermal incubator 300 is turned on, and the timer is automatically started to begin the isothermal amplification incubation phase, which continues until the timer automatically stops. Specifically, during one end of the incubation phase, for example, the first 5 minutes, the isothermal incubator 300 can be set to vibrate. The vibration is transmitted to the reaction chamber 110 through the vibration area 151 at the bottom of the test card 100, promoting uniform mixing of the second reagent 112 and the sample to be tested during the isothermal amplification process, thus accelerating the reaction. The aerosol barrier plug 600 ensures airtightness, ensuring that the nucleic acid aerosol in the test card 100 is sealed within the reaction chamber 110, avoiding cross-contamination. The sample to be tested and the second reagent 112 complete the isothermal amplification reaction within the reaction chamber 110, obtaining the first intermediate sample. The sample to be tested can undergo at least one of the following reactions with the second reagent 112: loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), cross-primer amplification (CPA), and nuclease-mediated isothermal amplification (RHAM). The sample to be tested can be an extract containing nucleic acid components. The second reagent 112 includes reagents specifically for RPA, LAMP, CPA, and RHAM reactions.

[0126] Remove the sealing aluminum film from the tube opening of tube body 210, rotate the connecting arm 240, and press the tube cap 220 into place, aligning it with the tube opening of tube body 210. The tube cap 220 and the inner wall of the tube opening of tube body 210 are sealed due to interference fit. At this time, the operator can invert the first pretreatment container 200 times. The reconstituted liquid in tube body 210 will enter the tube cap 220 through the gap of the fixing cage 221. The reconstituted liquid comes into contact with the lyophilized bulb inside, and the lyophilized bulb is fully reconstituted, ultimately generating the first reagent.

[0127] The first dropper 230 of the first pretreatment container 200 is aligned with the docking hole 630 of the aerosol barrier plug 600 and inserted. The edge of the cap 220 of the first pretreatment container 200 has a protrusion that corresponds to the slot 612 on the mounting plane 611, thereby fixing the first pretreatment container 200 onto the aerosol barrier plug 600. The operator can squeeze the tube 210, and the squeezed first reagent will open the one-way duckbill valve 620 on the aerosol barrier plug 600, ensuring that the first reagent can enter the reaction chamber 110 of the test card 100. When the squeezing pressure on the tube 210 is removed, the one-way duckbill valve 620 closes again, restoring the internal and external isolation state of the test card 100.

[0128] The first reagent is sealed and injected into the reaction chamber 110. The first intermediate sample continues to react with the first reagent to obtain the target sample. Timing begins when all the first reagent is squeezed into the reaction chamber 110 of the detection card 100, for example, 10 minutes. The first reagent in the Cas system reacts fully with the first intermediate sample after isothermal amplification to generate the target sample.

[0129] Specifically, taking nucleic acid as an example, the first reagent includes a CRISPR / Cas-specific reagent. If, during the reaction between the first reagent and the first intermediate sample, there is no target nucleic acid of the pathogen to be tested in the first intermediate sample, the Cas enzyme in the first reagent cannot be activated, and the CRISPR system probe in the first reagent will not be cleaved and will remain intact. One end of the CRISPR system probe is labeled with fluorescein, and the other end is labeled with biotin. If, during the reaction, there is target nucleic acid of the pathogen to be tested in the first intermediate sample, the Cas enzyme in the first reagent is activated, triggering non-specific trans cleavage on the target recognition. At this time, the CRISPR system probe is cleaved by the Cas enzyme.

[0130] Finally, the operator pulls the limiting piece 410 off the detection card 100 horizontally, and then presses the release button 440 to the first release position, which drives the first linkage rod 420 to push the liquid release plug 430 completely into the release chamber 120. At this time, the target sample can flow from the reaction chamber 110 into the release chamber 120 and come into contact with the chromatography detection module 130. The sample is loaded and the chromatography membrane climbing begins.

[0131] The chromatography detection module 130 may have indicator lines, combined with Figure 19 The chromatography detection module 130 includes a C-line 131, a T-line 132, and a coupling pad region 133. The C-line 131 is located downstream of the T-line 132. The target sample first flows through the T-line 132 and then to the C-line 131. The operator completes the detection by reading the detection results on the chromatography detection module 130.

[0132] The test results fall into two categories:

[0133] In the first scenario, if the target sample does not contain the target nucleic acid, the uncut CRISPR system probe (labeled with fluorescein at one end and biotin at the other) will first bind to the colloidal gold antibody in the coupling pad region 133 on the chromatography detection module 130. It will then be captured by streptomycin in the T line 132, causing the T line 132 to show color. Theoretically, all colloidal gold-biotin antibodies will be trapped here. No colloidal gold-biotin antibodies will be captured on the C line 131, so the C line 131 will not show color, indicating a negative test result.

[0134] The second scenario involves target nucleic acids in the sample. The CRISPR probe will be partially cleaved. One segment of the cleaved CRISPR probe, labeled with fluorescein, will first bind to the colloidal gold antibody in region 133 of the coupling pad. The remaining segment, along with the biotin-labeled segment, will still be captured by streptomycin at T-line 132. However, due to the partial probe breakage, the fluorescein-labeled segment will not be trapped at T-line 132 and will continue to precipitate upwards, eventually being captured by the secondary antibody at C-line 131, resulting in color development. When the nucleic acid titer in the sample is sufficiently high, all CRISPR probes will be cleaved. In this case, T-line 132 will not show color, only C-line 131 will. Both scenarios indicate a positive test result.

[0135] It should be emphasized that in the above detection process, the detection card 100 can include multiple detection groups, and correspondingly, the number of sample dispensing ports 142 can be multiple, enabling the molecular detection device to simultaneously detect multiple test samples or perform genotyping detection on the same test sample. The added test sample causes the first reagent in the reaction chamber 110, such as a lyophilized bulb, to rapidly rehydrate. In the embodiments of this application, each detection group is independent of each other, that is, the sample dispensing port 142, reaction chamber 110, release chamber 120, and chromatography detection module 130 in each detection group are all independent and physically separated, thereby ensuring that the reaction process does not interfere with each other.

[0136] In some possible embodiments, see Figures 15 to 19 As shown, the fluid control assembly 400 includes a sealing plug 450, a second linkage rod 460, and a knob 470.

[0137] A sealing plug 450 passes through the second linkage rod 460 and is located at the opening of the release chamber 120. The second linkage rod 460 is rotatably mounted on the lower housing 150 about its own axis, and a knob 470 is located at the end of the second linkage rod 460 away from the sealing plug 450.

[0138] The knob 470 has a second release position and a second initial position. When the knob 470 is rotated to the second initial position, the sealing plug 450 seals and covers the opening of the release chamber 120 to isolate the reaction chamber 110 and the release chamber 120. When the knob 470 is rotated to the second release position, there is a gap between the sealing plug 450 and the opening of the release chamber 120, and the reaction chamber 110 and the release chamber 120 remain in communication.

[0139] In this embodiment, the fluid control component 400 includes a sealing plug 450, a second linkage rod 460, and a knob 470. The knob 470 can drive the second linkage rod 460 and the sealing plug 450 to rotate to achieve flow channel control. When the knob 470 is in the second initial position, the sealing plug 450 and the opening of the release chamber 120 form a complete seal, isolating the flow channel between the reaction chamber 110 and the release chamber 120, providing the necessary independent sealed space for the isothermal amplification reaction of the second reagent 112 and the sample to be tested, as well as the CRISPR / Cas reaction of the first intermediate sample and the first reagent. When the results need to be interpreted, the knob 470 is rotated to the second release position. The specific cut of the sealing plug 450 and the opening of the release chamber 120 form a controllable flow gap. At this time, the target sample can flow from the reaction chamber 110 into the release chamber 120 and come into contact with the chromatography detection module 130. This achieves controllable connection between the reaction system and the detection system, which not only ensures sufficient reaction time, but also allows for more precise flow control through angle limiting, effectively preventing the target sample from splashing. This further enhances the reliability of the fully enclosed system and provides users with another reliable and easy-to-operate fluid control solution.

[0140] It should be noted that, compared to the fluid control assembly 400 which includes a limiting member 410, a first linkage rod 420, a liquid release plug 430, and a release button 440, the fluid control assembly 400 in this embodiment uses a sealing plug 450, a second linkage rod 460, and a knob 470. The detection process and final result determination methods are basically the same for both; the difference lies in the control method: the former involves horizontally pulling off the limiting member 410 on the detection card 100 and then pressing the release button 440 to the first release position; while the control method in this embodiment involves rotating the knob 470 to the second release position. Other identical detection processes will not be described further here.

[0141] In some possible embodiments, see Figures 15 to 19As shown, the periphery of the sealing plug 450 includes an arc surface 451 and a plane 452 connecting the arc surface.

[0142] When knob 470 is rotated to the second initial position, the arc surface 451 seals and covers the opening of the release chamber 120. When knob 470 is rotated to the second release position, a gap is formed between the plane 452 and the opening of the release chamber 120.

[0143] Specifically, the sealing plug 450 is a cylinder with a semi-circular cross-section, and the plane 452 is the structural cut.

[0144] When knob 470 is in the second initial position, the arc surface 451 seals over the opening of release chamber 120, forming a complete seal between the sealing plug 450 and the opening of release chamber 120. This isolates the flow channel between reaction chamber 110 and release chamber 120, providing the necessary independent sealed space for the isothermal amplification reaction of the second reagent 112 and the test sample, as well as the CRISPR / Cas reaction of the first intermediate sample and the first reagent. When results need to be interpreted, knob 470 is rotated to the second release position, forming a flow gap between the plane 452 and the opening of release chamber 120. At this time, the target sample can flow from reaction chamber 110 into release chamber 120 and come into contact with chromatography detection module 130.

[0145] In some possible embodiments, see Figures 1 to 20 As shown, a shallow groove 111 is formed at the bottom of the reaction chamber 110.

[0146] The reaction chamber 110 can be used to place the second reagent 112, which can be stored in the form of lyophilized bulbs, significantly extending its shelf life. The chamber wall of the reaction chamber 110 can effectively confine the second reagent 112. When the sample to be tested is added to the reaction chamber 110, the sample can quickly reconstitute the second reagent 112 and be confined within the shallow tank 111. In this way, the composite liquid component formed by the sample to be tested and the second reagent 112 can undergo an isothermal amplification reaction within the shallow tank 111.

[0147] The first pretreatment container 200 serves as the liquid addition tube, and the first reagent is sealed and added into the reaction chamber 110 of the detection card 100. The first reagent will mix with the first intermediate sample after isothermal amplification in the shallow tank to generate the target sample. The target sample can flow out through the structural gaps on the side wall of the reaction chamber 110 and flow downward along the transition structure 160. The flow channels of the reaction chamber 110 and the release chamber 120 are blocked by the fluid control component 400.

[0148] When the limiting member 410 is pulled out laterally, it disengages, the limiting is canceled, and the release button 440 can be pressed from the first initial position to the first release position under the action of external force. The release button 440 drives the first linkage rod 420 to push the liquid release plug 430 completely into the release chamber 120. The target sample can bypass the first linkage rod 420 and contact one end of the left-side chromatography detection module 130. The target sample can flow from the reaction chamber 110 into the release chamber 120 and contact the chromatography detection module 130. The sample is loaded and the chromatography membrane climbing process begins.

[0149] A second aspect of this application provides a detection method based on a regularly clustered, interspaced short palindromic repeat sequence / CRISPR-associated protein (CRISPR / Cas) system.

[0150] The detection method is applied in the aforementioned molecular detection device, see reference. Figures 1 to 20 As shown, the detection methods include:

[0151] S10. Inject the sample to be tested into the reaction chamber 110.

[0152] S20. Complete the isothermal amplification reaction and obtain the first intermediate sample.

[0153] The sample to be tested and the second reagent 112 can undergo at least one of the following reactions: loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), cross-primer amplification (CPA), and nuclease-mediated isothermal amplification (RHAM). The sample to be tested may be an extract containing nucleic acid components. The second reagent 112 includes reagents specifically for RPA, LAMP, CPA, and RHAM reactions. The second reagent 112 is typically stored in the reaction chamber 110 in the form of lyophilized bulbs / liquid.

[0154] S30. Control the first pretreatment container 200 to seal and inject the first reagent into the reaction chamber 110; the first intermediate sample and the first reagent continue to react for a first preset time to obtain the target sample.

[0155] In this process, the first reagent reacts with the first intermediate sample in a CRISRP / Cas system. Thus, the first reagent is sealed and added to the reaction chamber 110, which has already undergone isothermal amplification, through the first pretreatment container 200. The first intermediate sample and the first reagent continue to react, effectively preventing aerosol contamination and improving detection accuracy. The first reagent includes CRISPR / Cas-specific reagents.

[0156] S40, the control fluid control component 400 connects the reaction chamber 110 and the release chamber 120, the target sample enters the release chamber 120 from the reaction chamber 110 and comes into contact with the chromatography detection module 130.

[0157] S50. Observe the results of the chromatography detection module 130. The chromatography detection module 130 may have indicator lines, combined with... Figure 19 The chromatography detection module 130 includes a C-line 131, a T-line 132, and a coupling pad region 133. The C-line 131 is located downstream of the T-line 132. The target sample first flows through the T-line 132 and then to the C-line 131. The operator completes the detection by reading the detection results on the chromatography detection module 130.

[0158] In some possible embodiments, see Figures 1 to 20 As shown, step S10, injecting the sample to be tested into reaction chamber 110, specifically includes:

[0159] S11. Add the original sample to the second pretreatment container 500. The second pretreatment container 500 contains nucleic acid extraction solution. The original sample reacts with the nucleic acid extraction solution to generate the test sample. The original sample includes blood.

[0160] S12. The sample to be tested is dripped from the second dropper of the second pretreatment container 500 into the sample inlet 142 on the first mounting stage 141 so that the sample to be tested enters the reaction chamber 110.

[0161] In this embodiment, the second pretreatment container 500 can be used to contain the extraction solution and the original sample for extraction reaction to generate the sample to be tested. In this embodiment, the original sample can be blood, urine, feces, or other possible biological samples. The extraction solution can be a nucleic acid extraction solution.

[0162] The second pretreatment container 500 enables the pretreatment reaction of the original sample and nucleic acid extraction solution in a closed space, effectively preventing sample leakage and external contamination. The second pretreatment container 500 is equipped with a second dropper at the top, which facilitates the dripping of the sample to be tested into the sample application port 142 of the test card 100, ensuring that the operator can inject the sample to be tested into the reaction chamber 110.

[0163] In some possible embodiments, see Figures 1 to 20 As shown, before step S20, which involves completing the isothermal amplification reaction and obtaining the first intermediate sample, the following steps are also included:

[0164] S21. Install the aerosol barrier plug 600 on the first mounting platform 141 to seal the sample inlet 142.

[0165] After the sample to be tested in the reaction chamber 110 completes the amplification reaction with the second reagent 112 and obtains the first intermediate sample, the aerosol barrier plug 600 constructs a reliable aerosol physical barrier, maintains the airtightness of the entire reaction system, eliminates the risk of false positives caused by nucleic acid aerosol contamination, ensures the high reliability of the test results, and maintains the airtightness of the entire reaction system.

[0166] S22. Control the constant temperature incubator 300 to vibrate the reaction chamber 110 for 3 min to 10 min.

[0167] In this embodiment, a vibrator is built into the isothermal incubator 300. The vibrator can apply periodic mechanical vibration to the lower shell 150 of the test card 100. Thus, the vibration can be conducted through the shell to the reaction chamber 110 during the initial stage of incubation before isothermal amplification, so that the second reagent 112 in the chamber is fully mixed with the sample to be tested. This significantly improves the reconstitution efficiency of the second reagent 112 and the sample to be tested and improves the uniformity of the reaction components, effectively promoting the kinetic process of the initial stage of isothermal amplification reaction and shortening the time required for the reaction to reach the plateau phase.

[0168] In some possible embodiments, see Figures 1 to 20 As shown, step S20, completing the isothermal amplification reaction and obtaining the first intermediate sample, specifically includes:

[0169] S23. The reaction chamber 110 is heated by a controlled constant temperature incubator 300 for a duration of 1 min to 10 min.

[0170] In this embodiment, the constant temperature incubator 300 has heating protrusions 310, which correspond one-to-one with the reaction chambers 110 of the test card 100, thereby ensuring that each reaction chamber 110 can be precisely heated, so that the test sample and the second reagent 112 in the reaction chamber 110 can undergo a constant temperature amplification reaction at a suitable temperature. The heating temperature provided by the constant temperature incubator 300 can be 20℃~90℃, and further, it can be 40℃~60℃.

[0171] In some possible embodiments, see Figures 1 to 20 As shown, before the steps of S30, controlling the first pretreatment container 200 to seal and inject the first reagent into the reaction chamber 110; the first intermediate sample and the first reagent continue to react for a first preset time to obtain the target sample, the method further includes:

[0172] S31. Remove the sealing aluminum film.

[0173] S32. Seal the pipe cap 220 onto the pipe opening of the pipe body 210.

[0174] S33. The freeze-dried bulbs pre-placed on the tube cap 220 are mixed with the reconstituted liquid in the tube body 210 to generate the first reagent.

[0175] In this embodiment of the application, combined with Figure 8 Remove the sealing aluminum film from the tube opening of tube body 210, rotate the connecting arm 240, and press the tube cap 220 into place, aligning it with the tube opening of tube body 210. The tube cap 220 and the inner wall of the tube opening of tube body 210 are sealed due to interference fit. At this time, the operator can invert the first pretreatment container 200 times. The reconstituted liquid in tube body 210 will enter the tube cap 220 through the gap of the fixing cage 221. The reconstituted liquid comes into contact with the lyophilized bulb inside, and the lyophilized bulb is fully reconstituted, ultimately generating the first reagent.

[0176] In some possible embodiments, see Figures 1 to 20 As shown, the steps of S30, controlling the first pretreatment container 200 to seal and inject the first reagent into the reaction chamber 110; the first intermediate sample and the first reagent continue to react for a first preset time to obtain the target sample, specifically include:

[0177] S34. Seally insert the first drop head 230 of the first pretreatment container 200 into the docking hole 630 of the aerosol barrier plug 600.

[0178] S35. Fit and fix the tube cap 220 with the slot 612.

[0179] S36. Squeeze the tube 210 of the first pretreatment container 200 to squeeze the first reagent into the reaction chamber 110.

[0180] S37. The first intermediate sample and the first reagent continue to react for a first preset time to obtain the target sample. The first preset time can usually be set to 8 min to 15 min according to the detection requirements.

[0181] In this embodiment, the first dropper 230 of the first pretreatment container 200 is inserted into the docking hole 630 of the aerosol barrier plug 600. The edge of the cap 220 of the first pretreatment container 200 has a protrusion that corresponds to the slot 612 on the mounting plane 611, thereby fixing the first pretreatment container 200 onto the aerosol barrier plug 600. The operator can squeeze the tube 210, and the squeezed first reagent will open the one-way duckbill valve 620 on the aerosol barrier plug 600, ensuring that the first reagent can enter the reaction chamber 110 of the test card 100. When the squeezing pressure on the tube 210 is removed, the one-way duckbill valve 620 closes again, restoring the internal and external isolation state of the test card 100.

[0182] The first reagent is sealed and injected into the reaction chamber 110. The first intermediate sample continues to react with the first reagent to obtain the target sample. Timing begins when all the first reagent is squeezed into the reaction chamber 110 of the detection card 100, for example, 10 minutes. The first reagent in the Cas system reacts fully with the first intermediate sample after isothermal amplification to generate the target sample.

[0183] In some possible embodiments, see Figures 1 to 20 As shown, S40, the control fluid control component 400 connects the reaction chamber 110 and the release chamber 120. The step in which the target sample enters the release chamber 120 from the reaction chamber 110 and comes into contact with the chromatography detection module 130 specifically includes:

[0184] S41, Lateral pull-out limiter 410.

[0185] S42. Press the release button 440 to enter the first release position. The first linkage rod 420 presses the liquid release plug 430 into the release chamber 120 to connect the reaction chamber 110 and the release chamber 120.

[0186] S43. The target sample enters the release chamber 120 from the reaction chamber 110 and comes into contact with the chromatography detection module 130.

[0187] In this embodiment, after the first intermediate sample reacts with the first reagent to obtain the target sample, the operator pulls off the limiting member 410 on the detection card 100 laterally, and then presses the release button 440 to the first release position, which drives the first linkage rod 420 to push the liquid release plug 430 completely into the release chamber 120. At this time, the target sample can flow from the reaction chamber 110 into the release chamber 120 and come into contact with the chromatography detection module 130. The sample is loaded and the chromatography membrane climbing begins.

[0188] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0189] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A molecular detection device, characterized by, The molecular detection device comprises: a detection card (100) comprising at least one detection group, each detection group comprising a one-to-one corresponding reaction cavity (110), a release cavity (120) and a chromatographic detection module (130); the reaction cavity (110) stores a second reagent (112) therein, and one end of the chromatographic detection module (130) extends to the release cavity (120); a sample to be detected can react with the second reagent (112) to generate a first intermediate sample; a first pretreatment container (200) for providing a first reagent; the first pretreatment container (200) is detachably sealed to communicate with the reaction cavity (110); the first reagent can react with the first intermediate sample to generate a target sample; and a fluid control assembly (400) for selectively isolating or communicating the reaction cavity (110) and the release cavity (120); when the fluid control assembly (400) controls the reaction cavity (110) and the release cavity (120) to communicate, the target sample can flow from the reaction cavity (110) into the release cavity (120) and contact the chromatographic detection module (130).

2. The molecular detection device of claim 1, wherein, The molecular detection device further comprises a second pretreatment container (500), the second pretreatment container (500) comprising a bottle body (510), a bottle cap (520) and a sealing cap (530), and the bottle cap (520) is provided with a second droplet head at the top; the bottle body (510) is used for containing the sample to be detected; the bottle cap (520) is provided on the bottle opening of the bottle body (510), the sealing cap (530) is provided on the bottle cap (520), and the second droplet head is used for dropping the sample to be detected into the reaction cavity (110).

3. The molecular detection device of claim 1, wherein, The detection card (100) comprises an upper shell (140) and a lower shell (150); the chromatographic detection module (130) is laid on the lower shell (150), and the reaction cavity (110) and the release cavity (120) are arranged on the lower shell (150); the upper shell (140) is provided on the lower shell (150); the upper shell (140) is provided with a first mounting table (141) corresponding to the region of the reaction cavity (110), and the first mounting table (141) is formed with a sample adding port (142) in communication with the reaction cavity (110); the first pretreatment container (200) is detachably assembled on the first mounting table (141).

4. The molecular detection device of claim 3, wherein, The molecular detection device further comprises an aerosol blocking plug (600); the aerosol blocking plug (600) comprises a plug body (610) and a one-way duckbill valve (620), the plug body (610) is formed with a penetrating docking hole (630), and the one-way duckbill valve (620) is erected below the plug body (610) and communicates with the docking hole (630); The plug body (610) is arranged on the first mounting platform (141), and the one-way duckbill valve (620) is sealingly arranged in the sample adding port (142); and the first pretreatment container (200) is sealingly arranged on the docking hole (630).

5. The molecular detection device of claim 4, wherein, The first pretreatment container (200) comprises a tube body (210), a sealing aluminum film and a tube cover (220), the sealing aluminum film is sealingly arranged on the tube opening of the tube body (210), the tube cover (220) is provided with a first liquid drop head (230) at the top, the tube cover (220) is arranged on the tube opening of the tube body (210), and the first liquid drop head (230) is sealingly arranged in the docking hole (630).

6. The molecular detection device of claim 3, wherein, The molecular detection device comprises a constant temperature incubator (300) for heating the reaction cavity (110).

7. The molecular detection device of claim 3, wherein, The upper shell (140) is provided with a second mounting platform (143) corresponding to the area of the release cavity (120), and the second mounting platform (143) is formed with a mounting port (144) in communication with the release cavity (120); The fluid control assembly (400) comprises a limiting piece (410), a first linkage rod (420), a liquid release plug (430) and a release button (440); The first linkage rod (420) is vertically movably arranged in the mounting port (144), and the release button (440) is arranged at the end of the first linkage rod (420) protruding from the mounting port (144); The liquid release plug (430) is arranged at the end of the first linkage rod (420) away from the release button (440); The release button (440) has a first release position and a first initial position; When the release button (440) is kept at the first initial position, the liquid release plug (430) is located in the flow channel of the reaction cavity (110) and the release cavity (120) to block the reaction cavity (110) and the release cavity (120); When the release button (440) is kept at the first release position, the liquid release plug (430) is located in the release cavity (120), and the reaction cavity (110) and the release cavity (120) are kept in communication; The limiting piece (410) limits the release button (440) to keep the release button (440) at the first initial position.

8. The molecular detection device of claim 7, wherein, The limiting piece (410) is a U-shaped limiting clip, and the limiting piece (410) is transversely arranged between the release button (440) and the second mounting platform (143).

9. The molecular detection device of claim 3, wherein, The fluid control assembly (400) comprises a sealing plug (450), a second linkage rod (460) and a knob (470); The sealing plug (450) is arranged on the second linkage rod (460), and the sealing plug (450) is arranged on the cavity opening of the release cavity (120); The second linkage rod (460) is rotatably arranged on the lower shell (150) about the axis thereof, and the knob (470) is arranged at the end of the second linkage rod (460) away from the sealing plug (450). The knob (470) has a second initial position and a second release position; When the knob (470) is rotated to the second initial position, the sealing plug (450) seals the cavity opening of the release cavity (120) to cut off the communication between the reaction cavity (110) and the release cavity (120); When the knob (470) is rotated to the second release position, the sealing plug (450) has a gap with the cavity opening of the release cavity (120), and the reaction cavity (110) and the release cavity (120) remain in communication.

10. The molecular detection device of claim 9, wherein, The sealing plug (450) is a semi-circular column in cross section, and the peripheral side of the sealing plug (450) includes an arc surface (451) and a flat surface (452) connecting the arc surface (451); When the knob (470) is rotated to the second initial position, the arc surface (451) seals the cavity opening of the release cavity (120); When the knob (470) is rotated to the second release position, a gap is formed between the flat surface (452) and the cavity opening of the release cavity (120).

11. The molecular detection device of claim 1, wherein, The sample to be tested and the second reagent (112) perform at least one of the following reactions: loop-mediated isothermal amplification reaction, recombinase polymerase amplification reaction, cross primer amplification reaction, and nuclease-mediated isothermal amplification technology; The first reagent and the first intermediate sample perform a CRISRP / Cas system reaction; The sample to be tested is an extracted liquid containing nucleic acid components; The second reagent (112) includes a special reagent for RPA reaction or a special reagent for LAMP reaction; The first reagent includes a special reagent for CRISPR / Cas.

12. A detection method applied to the molecular detection device according to any one of claims 1 to 11; characterized in that, The detection method is based on the CRISPR / Cas system; the detection method includes: Injecting a sample to be tested into the reaction cavity (110); Completing a constant-temperature amplification reaction to obtain a first intermediate sample; Controlling the first pretreatment container (200) to seal the injection of the first reagent into the reaction cavity (110); the first intermediate sample and the first reagent continue to react for a first preset time to obtain a target sample; Controlling the fluid control assembly (400) to communicate the reaction cavity (110) and the release cavity (120), so that the target sample enters the release cavity (120) from the reaction cavity (110) and contacts the chromatographic detection module (130); Observing the result of the chromatographic detection module (130).

13. The detection method of claim 12, wherein, The step of injecting a sample to be tested into the reaction cavity (110) specifically includes: Adding an original sample to a second pretreatment container (500), the second pretreatment container (500) storing a nucleic acid extraction liquid, the original sample reacting with the nucleic acid extraction liquid to generate the sample to be tested, and the original sample including blood; Dropping the sample to be tested from a second droplet head of the second pretreatment container (500) into a sample adding port (142) on a first mounting table (141) so that the sample to be tested enters the reaction cavity (110).

14. The detection method according to claim 13, characterized in that, Before the step of completing a constant-temperature amplification reaction to obtain a first intermediate sample, the method further includes: An aerosol barrier plug (600) is installed on the first mounting platform (141) to seal the sample port (142); The constant temperature incubator (300) is controlled to vibrate the reaction cavity (110) for 3-10 min.

15. The method of claim 12, wherein, The step of completing the constant temperature amplification reaction to obtain a first intermediate sample specifically comprises: controlling the constant temperature incubator (300) to heat the reaction cavity (110) for 1-10 min.

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