Reaction tube and its detection method

Abstract

This invention discloses a reaction tube and its detection method. The reaction tube has a first reaction chamber and a second reaction chamber, and includes: a tube body, an air insulation structure, a flow guiding structure, and a sample introduction structure. The reaction tube of this invention achieves heat insulation and full sealing, avoiding the steps of frequently opening the cap to add reagents while preventing contamination, thus improving detection accuracy. The reaction tube of this invention has broad application prospects and will have great application potential in academic research and industrial R&D fields. It can be applied to simple biochemical or chemical reactions, and even to IVD products for rapid pathogen diagnosis based on nucleic acid or protein detection, possessing enormous market value.

Description

Technical Field

[0001] This invention relates to the field of testing products, specifically a reaction tube and its testing method. Background Technology

[0002] The in vitro diagnostics (IVD) market has been booming in recent years, with the Chinese market reaching 70 billion RMB in 2017, a year-on-year increase of approximately 15%. A significant downstream segment of IVD products is point-of-care testing (POCT). In 2018, POCT accounted for 11% of the entire IVD market in my country, and the Chinese POCT market is growing rapidly, with an annual growth rate of 20%–30%, far exceeding the global annual growth rate of 7%–8%. As for molecular diagnostic products, the technology is highly complex, requiring high accuracy. Currently, the relatively mature molecular testing products are all from overseas. These products are relatively mature and of high quality, but expensive. Domestically, there is currently no high-quality molecular testing platform on the market that can rival the performance of star products in Europe and the United States.

[0003] The CRISPR system has been widely used in academia, primarily for its gene-editing function. In recent years, with the continuous development of this technology and the creation of new CRISPR systems, a number of application-oriented products utilizing this technology have emerged. Currently, the majority of these applications utilize Cas9, Cas12, Cas13, and Cas14 systems. Cas12, Cas13, and Cas14 have been previously reported; for example, Zhang Feng's team developed the SHERLOCK test strip using Cas13 combined with colloidal gold technology, which can rapidly detect Zika and dengue viruses with similar symptoms. Doudna's team developed DETECTOR using Cas12, which can also be used for rapid pathogen detection. Cas14 has been reported for applications in SNP detection. The detection principles of the Cas12, Cas13, and Cas14 systems mentioned above are similar. Cas12, Cas13, and Cas14 combine with specific gRNA to form a complex. Guided by the gRNA, they can specifically recognize target nucleic acid sequences (Cas12 mainly recognizes double-stranded DNA, Cas13 mainly recognizes RNA, and Cas14 mainly recognizes single-stranded DNA) and cleave the target nucleic acid sequence. The recognition of the target nucleic acid sequence also triggers its non-specific cleavage of surrounding single-stranded DNA or RNA. Using this method, researchers have designed reporter probes for single-stranded DNA or RNA, with one end modified with a quencher group and the other end modified with a fluorescent group. Taking Cas12 as an example, the target double-stranded DNA is first enriched by PCR amplification or isothermal amplification using RPA, LAMP, or RCA, and then mixed with Cas12, gRNA, and the single-stranded DNA detection probe. When the Cas12 / gRNA complex specifically cleaves the target double-stranded DNA sequence, it begins to randomly and non-specifically cleave the single-stranded DNA probe. After the single-stranded DNA probe is cleaved, it emits fluorescence. Subsequently, a fluorescence detection instrument is used to read the results, thereby enabling qualitative and even quantitative detection of the target nucleic acid sequence. Alternatively, the CRISPR system can be combined with colloidal gold lateral chromatography for color development of the test strips, allowing for visual qualitative analysis. Conventional detection methods are all non-closed systems. A few products combine this technology with microfluidic systems to create a fully closed system, but this is costly. This technology has wide applications in rapid pathogen detection, characterized by high sensitivity and specificity.

[0004] However, as described above, the biggest drawback of this series of detection technologies for developing rapid testing products is that the amplification and enrichment process and the CRISPR detection process are two independent systems. First, the amplification and enrichment of the target fragment is carried out in one system. After amplification, 1-2 μL of the amplification product is added to the CRISPR detection system for signal detection. This process requires opening the cap to sample the amplified and enriched product, which easily generates aerosol contamination. For example, in professional laboratories that conduct long-term pathogen detection, the long-term amplification reaction and the generated aerosols can cause significant environmental pollution, leading to false positive results. Even using the CRISPR test strips developed by Zhang Feng and Doudna cannot avoid the step of opening the cap after amplification, thus preventing aerosol contamination. Furthermore, after the target fragment is amplified and enriched, samples are added separately to the CRISPR detection system. In laboratories with limited experimental conditions or in field operations, it is impossible to guarantee the availability of multi-channel pipettes. When multiple samples are co-detected, different samples cannot simultaneously begin the CRISPR cutting and detection reaction, rendering the initial fluorescence values ​​unreliable for comparison. Even if multiple pipettes can be used to simultaneously add different test samples, the test system must be placed on ice before being transferred to the fluorescence reading device to avoid a cutting reaction before the test, which greatly limits its convenience.

[0005] Furthermore, the two reaction systems typically operate at different temperatures. For example, the conventional PCR used for enrichment and amplification of the target fragment in CRISPR rapid detection is a temperature-variable process, with a temperature range generally between 50-95℃. Isothermal amplification, on the other hand, typically operates between 37-65℃. The CRISPR detection system, however, operates at 37-48℃. To achieve the goal of single-tube detection without opening the caps of the two reaction processes, simply physically isolating the two reaction systems cannot effectively prevent the impact of the high-temperature reaction system on the performance of the low-temperature detection system due to temperature conduction.

[0006] Currently, molecular, biochemical, and even chemical reactions often involve two or more reaction systems, sometimes even requiring the mixing of two independent reaction systems for a third step, as described above. Another application example is RNA reverse transcription, which typically involves RNA denaturation, reverse transcription of the first and second strands of RNA. The first step involves mixing the RNA template with primers, cooling at 65°C for 5 minutes, then on ice for 2 minutes. The reverse transcription reaction system (buffer, dNTPs, reverse transcriptase, RNase inhibitor) is then added for the second step. Besides the CRISPR detection system and RNA reverse transcription process mentioned above, chemical reactions often involve two-part processes where the two parts need to be fully or partially fused, or where additional reagents need to be added during the experiment. These situations all present challenges such as long operation times, sample contamination, and inconvenience.

[0007] For the aforementioned CRISPR rapid detection technology, a one-tube detection system has been developed using mineral oil for sealing and insulation. The target fragment amplification and enrichment reaction one reagent is placed at the bottom of the tube, covered with mineral oil for insulation, while the CRISPR detection reagent two is embedded at the top. After reaction one is completed, the tube is inverted to mix, combining reaction one and reaction two reagents, thus achieving one-tube detection without opening the tube. Test results show that using mineral oil for insulation achieves a certain insulation effect, but it is not optimal. Furthermore, this insulation method doubles both the volume of the CRISPR detection system and the volume of the detection sample, presumably to compensate for the heat-induced loss of CRISPR detection system performance and the impact of mineral oil on the system's performance during the mixing of reactions one and two. Summary of the Invention

[0008] The technical problem this invention aims to solve is to overcome the shortcomings of existing stepwise detection techniques, such as cumbersome steps, easy contamination, and long time consumption when mixing reaction phases of two reaction systems with different reaction temperatures. This invention provides a reaction tube and its detection method. The reaction tube of this invention is heat-insulated and fully sealed, avoiding the need for frequent opening of the cap to add reagents while preventing contamination and improving detection accuracy.

[0009] The present invention solves the above problems through the following technical solutions:

[0010] A first aspect of the present invention provides a reaction tube having a first reaction chamber and a second reaction chamber, the reaction tube comprising: a tube body, an air insulation structure, a flow guiding structure, and a sample introduction structure; wherein:

[0011] The tube has a closed bottom and an open top;

[0012] The air insulation structure is disposed inside the tube, and the first reaction tank is at least enclosed by the air insulation structure and the bottom of the tube;

[0013] The flow guiding structure is disposed inside the pipe body and penetrates the air insulation structure;

[0014] The injection structure is detachably disposed at the top of the tube or the top of the flow guiding structure. The injection structure and the flow guiding structure are separated by a membrane. The membrane is disposed at the bottom of the injection structure or inside the flow guiding structure. The second reaction cell is at least surrounded by the injection structure and the membrane. The injection structure can change the internal volume or pressure of the second reaction cell so that the second reaction cell is unidirectionally connected to the first reaction cell through the flow guiding structure.

[0015] In this invention, when the thin film is disposed at the bottom of the sample introduction structure, the second reaction cell is a closed cavity formed by the thin film and the sample introduction structure; when the thin film is disposed within the flow guiding structure, the second reaction cell is a closed cavity formed by the thin film, the sample introduction structure, and the flow guiding structure. The closed cavity can be sealed to prevent liquid in the second reaction cell from flowing out.

[0016] In this invention, the air insulation mechanism is used to reduce heat conduction between the first reaction tank and the second reaction tank, which can effectively block the reaction of the reagents in the first reaction tank at possible high temperatures, and keep the reagents stored in the second reaction tank within an effective temperature range.

[0017] In some embodiments of the present invention, the air insulation structure includes at least one closed cavity, which is not in communication with either the first reaction tank or the second reaction tank.

[0018] In some embodiments of the present invention, the air insulation structure includes a closed cavity.

[0019] In other embodiments of the invention, the air insulation structure comprises two enclosed cavities.

[0020] In some specific embodiments of the present invention, the air insulation structure includes at least two layers of partitions arranged vertically, the periphery of each partition is connected to the inner wall of the tube, the partitions form cavities with the inner wall of the tube, and each partition is provided with an opening that cooperates with the flow guiding structure.

[0021] In this invention, the air insulation structure can effectively block the heat generated in the first reaction tank through the cavity between one or more of the partitions, preventing the heat from being conducted to the second reaction tank and affecting the activity of the reagents stored therein.

[0022] In some embodiments of the present invention, a one-way valve is provided at one end of the flow guiding structure that is connected to the first reaction tank, and the one-way valve is flush with or lower than the bottom of the air insulation structure.

[0023] In this invention, by setting a one-way valve, the reagents in the first reaction tank and the reagents in the second reaction tank can be mixed in one direction, eliminating the need to open the lid to add reagents and preventing the aerosols generated when the reagents in the first reaction tank react at high temperature from entering the second reaction tank and causing cross-contamination.

[0024] In some embodiments of the present invention, the one-way valve is flush with the bottom of the air insulation structure.

[0025] In other embodiments of the invention, the one-way valve is located below the bottom of the air insulation structure.

[0026] In some embodiments of the present invention, the film is made of one of the following materials: brittle resin, glass, polytetrafluoroethylene or derivatives thereof.

[0027] In some preferred embodiments of the present invention, the polytetrafluoroethylene derivatives may be conventional in the art, and are preferably derivatives that retain the hydrophobic and oleophobic film properties of polytetrafluoroethylene; for example, expanded polytetrafluoroethylene.

[0028] In some embodiments of the present invention, the air insulation structure, the air guiding structure, and the pipe body form an integrated structure.

[0029] In some specific embodiments of the present invention, the air insulation structure, the flow guiding structure and the tube body form an integrated structure, and the air insulation structure and the flow guiding structure are detachably disposed within the tube body.

[0030] In other embodiments of the present invention, the air insulation structure and the airflow guiding structure are separate structures.

[0031] In some specific embodiments of the present invention, the air insulation structure, the flow guiding structure, and the pipe body are separate structures.

[0032] In other embodiments of the present invention, the tube body, the air insulation structure, and the flow guiding structure form an integral structure.

[0033] In this invention, the partition of the air insulation structure can be a rubber gasket.

[0034] In this invention, the flow guiding structure can be a rigid conduit.

[0035] In this invention, the shape of the flow guiding structure can be funnel-shaped.

[0036] In this invention, the injection structure can be a press valve.

[0037] A second aspect of the present invention provides a CRISPR detection method, said CRISPR detection method being performed in a reaction tube as described in the first aspect, comprising:

[0038] The sample to be tested and the amplification system are added to the first reaction cell, the detection system is added to the second reaction cell, and the system is assembled with the air insulation structure, the flow guiding structure and the tube body into one unit, and the top opening of the tube body is sealed.

[0039] The sealed reaction tube containing the amplification system is heated to the reaction temperature of the amplification system to carry out the amplification reaction;

[0040] After the amplification reaction is completed, the temperature of the reaction tube is adjusted to the reaction temperature of the detection system. The sample introduction structure is controlled, and the detection system in the second reaction cell is added to the first reaction cell through the flow guiding structure to carry out the detection reaction and collect the detection signal.

[0041] The amplification system is a reagent combination for amplifying the nucleic acid of the sample to be tested; the detection system is a reagent combination for CRISPR detection of the nucleic acid of the sample to be tested.

[0042] In this invention, the reaction temperature of the amplification system differs from the temperature of the detection system. For example, the reaction temperature of the amplification system is 50-95℃, while the reaction temperature of the detection system is 37-65℃.

[0043] In some specific embodiments of the present invention, the amplification system includes a target fragment amplification and enrichment reagent. The amplification system and the sample to be tested are added to the first reaction chamber of the reaction tube, and the detection system is added to the second reaction chamber. Other structures are then assembled, and the tube is placed in a detection instrument to run the amplification reaction for target fragment amplification and enrichment. During this process, the air insulation structure effectively blocks the heat generated by the amplification reaction. After the amplification reaction is completed, the temperature is adjusted to the reaction temperature of the detection system. The pressure valve is manually pressed to mix the reagents contained in the detection system with the amplification enrichment product generated by the amplification reaction and perform the detection reaction. This allows two reactions with different reaction temperatures to be performed within one tube, and mixing and fluorescence result reading can be performed without opening the cap.

[0044] In other specific embodiments of the present invention, the amplification system includes a target fragment amplification and enrichment reagent. The amplification system and the sample to be tested are added to the first reaction chamber of the reaction tube via a pipette through a flow guide structure. Then, detection reagent is added to the second reaction chamber, the tube is sealed, and placed in a detection instrument to run the amplification reaction for target fragment amplification and enrichment. During this process, the air insulation structure effectively blocks the heat generated by the amplification reaction. After the amplification reaction is completed, the temperature is adjusted to the reaction temperature of the detection system. The pressure valve is manually pressed to mix the reagents contained in the detection system with the amplification enrichment product generated by the amplification reaction and perform the detection reaction. This allows two reactions with different reaction temperatures to be carried out in one tube, and mixing and fluorescence result reading can be performed without opening the cap.

[0045] When applied to CRISPR, this invention combines isothermal amplification and CRISPR technology to create a rapid molecular detection product with high sensitivity, accuracy, specificity, speed, and cost. It provides a comprehensive solution for a fully enclosed CRISPR rapid detection system with better thermal conductivity isolation, minimizing false positives caused by aerosol contamination. It also effectively addresses the impact of heat conduction from the high-temperature reaction on the performance of subsequent low-temperature reactions, increasing the portability and speed of CRISPR rapid detection. This allows for the substitution of high-quality domestically produced molecular detection products for imports. For example, for the rapid detection of the novel coronavirus, this comprehensive solution will provide a convenient and rapid diagnostic tool for epidemic diagnosis and control, and offer strong technical reserves for future outbreaks of various pathogen infections.

[0046] A third aspect of the present invention provides a method for stepwise detection in a single tube, the method being carried out in a reaction tube as described in the first aspect, comprising:

[0047] The sample to be tested and the first reaction system are added to the first reaction cell, the second reaction system is added to the second reaction cell, and the system is assembled with the flow guiding structure, the air insulation structure and the tube body into one unit, and the top opening of the tube body is sealed.

[0048] The closed reaction tube, which is added to the first reaction system, is heated to the reaction temperature of the first reaction system to carry out the reaction;

[0049] After the reaction is completed, the temperature of the reaction tube is adjusted to the reaction temperature of the second reaction system. The sample introduction structure is controlled to add all or part of the second reaction system in the second reaction cell to the first reaction cell through the flow guiding structure for reaction, and the detection signal is collected.

[0050] This invention enables fully enclosed rapid detection methods such as CRISPR and RNA reverse transcription, avoiding aerosol contamination and shortening detection time, thus addressing the issues of convenience and accuracy. It can be applied to any biochemical or chemical reaction consisting of two independent reaction systems at different temperatures, where the two reaction phases will be partially or completely mixed subsequently, or where additional reagents need to be added by opening the lid during the experiment. For example, this invention can develop various fully enclosed rapid nucleic acid detection products for scenarios including, but not limited to, rapid detection of clinical pathogens, detection of microorganisms or pathogens in food, detection of pathogens at import and export customs, rapid detection of pet pathogens, and rapid detection of livestock pathogens.

[0051] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0052] The reagents and raw materials used in this invention are all commercially available.

[0053] The positive and progressive effects of this invention are as follows:

[0054] (1) The reaction tube of the present invention can achieve better heat insulation effect, reduce the influence of heat conduction on the effect of reaction reagent, or use the increase of reaction system to achieve reaction effect compensation to achieve a comparable detection effect, so that the stepwise detection reaction can achieve a one-tube closed reaction, avoiding false positives that may be caused by long-term amplification, frequent opening of the cap and the aerosol generated; in multi-sample detection, it can avoid the problem that the initial detection signal has no reference value due to different sample additions.

[0055] This invention simplifies the instruments, equipment, and operating procedures required for testing, reasonably reduces costs, and makes the testing process more convenient and efficient.

[0056] (2) The reaction tube of the present invention has broad application prospects and has great application space in academic research and industrial R&D fields. It can be applied to simple biochemical or chemical reactions, and even to IVD products of rapid pathogen diagnosis detection based on nucleic acid or protein detection, which has huge market value. Attached Figure Description

[0057] Figure 1a This is a schematic diagram of the reaction tube in Embodiment 1 of the present invention.

[0058] Figure 1b This is a schematic diagram of the reaction tube in use according to Embodiment 1 of the present invention.

[0059] Figure 2 This is a schematic diagram of the reaction tube in Embodiment 3 of the present invention.

[0060] Figure 3 This is a schematic diagram of the cross-section of a 3D-printed test tube used for the heat insulation effect test in Example 4.

[0061] Figure 4 This is a schematic diagram showing the temperature changes of the inner cavities of two test tubes under different heating module temperatures in Example 4.

[0062] Figure 5 This is a schematic diagram of mineral oil-sealed heat insulation in Example 5.

[0063] Figure 6 This is a schematic diagram comparing the thermal insulation effects of mineral oil sealing insulation and air insulation interlayer in Example 5.

[0064] Explanation of reference numerals in the attached figures:

[0065] 1. Tube body; 2. Air insulation structure; 3. Flow guiding structure; 31. One-way valve structure; 4. Sample injection structure; 41. Membrane; 5. First reaction cell; 6. Second reaction cell; 7a. Pure water; 7b. Mineral oil. Detailed Implementation

[0066] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0067] Example 1

[0068] This embodiment provides a reaction tube, the specific structure of which is as follows: Figure 1a As shown, the reaction tube specifically includes: a tube body 1, an air insulation structure 2, a flow guiding structure 3, and a sample inlet structure 4; wherein:

[0069] The air insulation structure 2 is installed inside the tube body 1. The first reaction cell 5 of the reaction tube is formed by the air insulation structure 2 and the bottom of the tube body 1. The flow guiding structure 3 is also installed inside the tube body 1 and is separated from the sample injection structure 4 by the membrane 41, which is located at the bottom of the sample injection structure 4. The second reaction cell 6 of the reaction tube is formed by the sample injection structure 4 and the membrane 41, and the reagent is stored in the second reaction cell 6.

[0070] like Figure 1a As shown, in this embodiment, the air insulation structure 2 adopts a double-layer structure to improve the insulation effect. Furthermore, a through hole is provided in the middle of the air insulation structure 2 to allow the flow guiding structure 3 to pass through, enabling communication between the first reaction tank 5 and the second reaction tank 6. The flow guiding structure 3 penetrates the air insulation structure 2 from top to bottom, allowing the second reaction tank 6 to connect with the first reaction tank 5 via the flow guiding structure 3. Figure 1a The direction indicated by the middle arrow indicates unidirectional connectivity;

[0071] The sample introduction structure 4 is located at the top of the tube body 1, above the flow guiding structure 3. Specifically, in this embodiment, the top of the sample introduction structure 4 is flexible and can be deformed by external pressure from the user (see...). Figure 1b This structure allows the user to change the internal volume or pressure of the second reaction cell 6 by pressing, causing the membrane 41 to rupture under pressure, allowing the reagent to flow from the sample injection structure 4 into the flow guiding structure 3, and then into the first reaction cell 5 through the flow guiding structure 3.

[0072] In this embodiment, the shape of the injection structure 4 is as follows: Figure 1a As shown, the membrane 41 seals the bottom of the injection structure 4 to form a hollow cavity structure, and at least the top of the injection structure 4 is made of a flexible material, so that the user can press the top of the injection structure 4 inward to change the volume of the injection structure 4, causing the membrane 41 to rupture, thereby changing the pressure inside the second reaction cell 6, so that the reagents located in the second reaction cell 6 can enter the first reaction cell 5 through the guide structure 3.

[0073] In addition, in this embodiment, a one-way valve structure 31 is provided at the end of the flow guiding structure 3. Under normal circumstances (i.e., when the top of the sample injection structure 4 is not pressed), the valve plate of the one-way valve structure 31 remains attached to the opening of the flow guiding structure 3 connecting to the first reaction tank 5 (see [reference]). Figure 1a This prevents reagents in the second reaction chamber 6 from entering the first reaction chamber 5, and conversely, also prevents reagents in the first reaction chamber 5 from entering the second reaction chamber 6. When a reaction is required, pressing the top of the injection structure 4 changes the pressure inside the second reaction chamber 6, causing the valve plate of the one-way valve structure 31 to open (see...). Figure 1b This allows the reagents in the second reaction tank 6 to enter the first reaction tank 5, thus achieving a controllable reaction. Of course, this invention does not limit the specific structure of the flow guiding structure 3; in other embodiments, the flow guiding structure 3 can also employ other structures to achieve unidirectional flow.

[0074] like Figure 1a As shown, in this embodiment, the tube body 1, air insulation structure 2, flow guiding structure 3, and sample injection structure 4 are all separately configured. The upper part of the tube body 1 has an open opening. The flow guiding structure 3 is installed downward inside the tube body 1, while the sample injection structure 4 is installed at the upper opening of the flow guiding structure 3, so that the reaction tube can be obtained by assembly. The second reaction cell 6 formed by the sample injection structure 4 and the membrane 41 located at its bottom is used to contain and seal the reagent. The sample injection structure 4 and the membrane 41 are installed on the top of the flow guiding structure 3 by plugging and unplugging. This separate configuration facilitates the addition of reagents into the first reaction cell 5 and the second reaction cell 6.

[0075] Example 2

[0076] This embodiment also provides a reaction tube, whose structure is roughly the same as that of the reaction tube provided in Embodiment 1. The difference is that in this embodiment, the air insulation structure 2 and the tube body 1 are integrally formed, so that there is no seam between the air insulation structure 2 and the tube body 1, and the heat insulation and sealing effect is relatively better.

[0077] In addition, the air insulation structure 2 and the air diversion structure 3 are also integrally formed, so that there are no seams between the air insulation structure 2 and the air diversion structure 3, which further improves the heat insulation and sealing effect.

[0078] As in Example 1, the sample introduction structure 4 is installed at the opening position at the top of the flow guiding structure 3 to facilitate the addition of reagents into the first reaction cell 5 and the second reaction cell 6.

[0079] In this embodiment, the tube body 1, the air insulation structure 2, and the flow guiding structure 3 can be integrally manufactured using 3D printing. Of course, in other embodiments, the integral structure formed by the tube body 1, the air insulation structure 2, and the flow guiding structure 3 can also be manufactured using other existing manufacturing processes.

[0080] Example 3

[0081] This embodiment also provides a reaction tube, the structure of which is substantially the same as that of the reaction tube provided in Embodiment 1, such as... Figure 2 As shown, the difference lies in this embodiment: the thin film is disposed within the flow guiding structure, and the thin film, the upper part of the flow guiding structure, and the sample injection structure together form the second reaction cell 6. The thin film is located within the flow guiding structure to enhance the heat insulation effect. After changing the volume of the sample injection structure by pressing it, both thin films will rupture, allowing the reagents in the first reaction cell to enter the second reaction cell.

[0082] Example 4: Verification of the Air Barrier Effect

[0083] Two reaction tubes, 2 cm long, as described in Examples 1 and 3, were 3D printed (structure as follows). Figure 1a and Figure 2 As shown, the cross-section is as follows Figure 3 As shown in the figure, pure water was injected into the second reaction chamber 6 located at the bottom of the reaction tube. The tube was heated at 35℃, 45℃, 55℃, and 65℃ for five minutes on the PCR instrument heating module, and the temperature of the inner cavity of the second reaction chamber 6 was measured. The room temperature was 24.6℃. The results are shown in Table 1 and... Figure 4 As shown, when the module heating temperature is 35℃, which is the temperature of the pure water in the first reaction tank 5, the internal temperature is approximately 32℃. As the module heating temperature increases (in increments of 10℃), the rate of temperature increase in the internal cavity of the second reaction tank 6 gradually becomes much lower than the rate of temperature increase in the module. When the module temperature reaches 75℃, the internal temperature in the second reaction tank 6 is only about 40℃. This demonstrates that the air insulation structure has a good effect on heat insulation and heat conduction resistance.

[0084] Table 1 Module heating temperature and measurement area temperature

[0085] Module temperature (°C) 35 45 55 65 75 The inner cavity of the reaction tube in Example 1 32.1 33 34 36.6 39.5 The inner cavity of the reaction tube in Example 3 32.6 33.9 37.8 38.2 40.2

[0086] Example 5: Comparison Experiment of Thermal Insulation Effect of Air Insulation Structure and Mineral Oil

[0087] As mentioned above, current CRISPR detection tube methods utilize mineral oil-sealed reaction for insulation, but this has certain drawbacks, resulting in significant losses in sample and reagent usage. To compare the insulation effect of the air-insulated structure of this invention with that of mineral oil-sealed insulation, the following tests were conducted to obtain corresponding data on mineral oil-sealed insulation, which were then compared with the insulation effect data of the air-insulated structure in Example 4.

[0088] The test procedure for the sealing and heat insulation effect of mineral oil is as follows: Figure 5 As shown, in two reaction tubes, each 2 cm long, as described in Examples 1 and 3 ( Figure 5 The reaction tube used in Example 1 was added, along with the same volume of pure water 7a as in Example 4. 25 μL of mineral oil 7b was added to the pure water 7a. The tube was then heated at 35°C, 45°C, 55°C, and 65°C for five minutes in the heating module. The temperature of the second reaction chamber 6 (inner cavity) was then measured. The room temperature was 23.2°C. The results are shown in Table 2. Figure 6 As shown, under the same experimental conditions, mineral oil sealing insulation also has the effect of blocking heat conduction, but the insulation effect of air insulation structure is relatively better.

[0089] Table 2 Module heating temperature and measurement area temperature

[0090] Module temperature (°C) 35 45 55 65 75 The inner cavity of Example 1 (mineral oil seal) 31.2 35.4 40 41.1 44.3 The inner cavity of Example 3 (mineral oil seal) 31.5 35.8 40 41.4 44.7 The inner cavity (air-insulated structure sealed) of Example 1 32.1 33 34 36.6 39.5 The inner cavity (air-insulated structure sealed) of Example 3 32.6 33.9 37.8 38.2 40.2

Claims

1. A reaction tube comprising a first reaction chamber and a second reaction chamber, characterized in that, include: The tube body, air insulation structure, flow guiding structure, and sample inlet structure; among which: The tube has a closed bottom and an open top; The air insulation structure is disposed inside the tube, and the first reaction tank is at least enclosed by the air insulation structure and the bottom of the tube; The flow guiding structure is disposed inside the pipe body and penetrates the air insulation structure; The injection structure is disposed at the top of the tube or the top of the flow guiding structure. The injection structure and the flow guiding structure are separated by a membrane. The membrane is disposed at the bottom of the injection structure or inside the flow guiding structure. The second reaction cell is at least surrounded by the injection structure and the membrane. The injection structure can change the internal volume or pressure of the second reaction cell so that the second reaction cell is unidirectionally connected to the first reaction cell through the flow guiding structure.

2. The reaction tube as described in claim 1, characterized in that, The air insulation structure includes at least one closed cavity, which is not connected to either the first reaction tank or the second reaction tank.

3. The reaction tube as described in claim 1, characterized in that, A one-way valve is provided at one end of the flow guiding structure that is connected to the first reaction tank; The one-way valve is flush with or lower than the bottom of the air insulation structure.

4. The reaction tube as described in claim 1, characterized in that, The film is made of one of the following materials: brittle resin, glass, and polytetrafluoroethylene.

5. The reaction tube as described in claim 1, characterized in that, The air insulation structure and the airflow guiding structure form an integrated structure.

6. The reaction tube as described in claim 5, characterized in that, The air insulation structure and the airflow guiding structure are detachably installed inside the pipe.

7. The reaction tube as described in claim 1, characterized in that, The air insulation structure and the airflow guiding structure are separate structures.

8. The reaction tube as described in claim 7, characterized in that, The air insulation structure, the air guiding structure, and the pipe body are separate structures.

9. The reaction tube as described in claim 1, characterized in that, The tube body, the air insulation structure, and the air guiding structure form an integrated structure.

10. The reaction tube according to any one of claims 1 to 7, 9, characterized in that, The sample injection mechanism is a press valve.

11. A CRISPR detection method, characterized in that, The CRISPR detection method is performed in a reaction tube as described in any one of claims 1-10, comprising: The sample to be tested and the amplification system are added to the first reaction cell, the detection system is added to the second reaction cell, and the system is assembled with the air insulation structure, the flow guiding structure and the tube body into one unit, and the top opening of the tube body is sealed. The sealed reaction tube containing the amplification system is heated to the reaction temperature of the amplification system to carry out the amplification reaction; After the amplification reaction is completed, the temperature of the reaction tube is adjusted to the reaction temperature of the detection system. The sample introduction structure is controlled, and the detection system in the second reaction cell is added to the first reaction cell through the flow guiding structure to carry out the detection reaction and collect the detection signal. The amplification system is a reagent combination for amplifying the nucleic acid of the sample to be tested; the detection system is a reagent combination for CRISPR detection of the nucleic acid of the sample to be tested.

12. The CRISPR detection method as described in claim 11, characterized in that, The reaction temperature of the amplification system is 50-95℃, and the reaction temperature of the detection system is 37-65℃.

13. A method for stepwise detection using a single tube, characterized in that, The method is carried out in a reaction tube as described in any one of claims 1-10, comprising: The sample to be tested and the first reaction system are added to the first reaction cell, the second reaction system is added to the second reaction cell, and the system is assembled with the flow guiding structure, the air insulation structure and the tube body into one unit, and the top opening of the tube body is sealed. The closed reaction tube, which is added to the first reaction system, is heated to the reaction temperature of the first reaction system to carry out the reaction; After the reaction is completed, the temperature of the reaction tube is adjusted to the reaction temperature of the second reaction system. The sample introduction structure is controlled to add all or part of the second reaction system in the second reaction cell to the first reaction cell through the flow guiding structure for reaction, and the detection signal is collected.

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