Portable multiple nucleic acid rapid detection integrated test tube, matched detection system and detection method

Abstract

The invention relates to the technical field of nucleic acid detection, in particular to a portable integrated test tube for multiple rapid nucleic acid detection, a matched detection system and a detection method.The portable integrated test tube comprises a tube body, a nut and a plug-in card; wherein the magnet adjusting device is arranged on the outer side wall of the pipe body; external threads are arranged on the outer side wall of the top of the pipe body, internal threads are arranged on the inner side wall of the nut, and the nut is matched with the threads of the pipe body; the middle of the nut extends downwards to form a convex column, an elastic rubber head is fixedly arranged at the bottom of the convex column, and the elastic rubber head is in sealing fit with a pipe cavity of the pipe body, so that when the nut moves downwards, the elastic rubber head can compress liquid in the pipe cavity; a third partition plug, a second partition plug and a first partition plug are sequentially arranged at the bottom of a tube cavity of the tube body from bottom to top; in the aspects of detection efficiency and safety, integration of sample cracking, purification and liquid transfer is realized through the integrated test tube.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, and in particular to a portable integrated test tube, a matching detection system, and a detection method for rapid multiplex nucleic acid detection. Background Technology

[0002] Nucleic acid testing, as a core technology for accurately identifying pathogens, diagnosing diseases, and monitoring specific nucleic acid sequences in biological samples, has been widely applied in clinical disease diagnosis, emergency testing for public health emergencies, food and drug safety monitoring, and animal and plant quarantine. With the continuous upgrading of testing needs, especially in point-of-care testing (POCT) scenarios such as primary healthcare institutions, community testing sites, port quarantine, and field operations, there is an urgent need for nucleic acid testing technology to be "portable, rapid, integrated, and multi-functional." The limitations of traditional nucleic acid testing technologies are gradually becoming apparent, becoming a key bottleneck restricting their widespread application.

[0003] Traditional testing relies on multiple large-scale devices and specialized laboratories, making it inconvenient to use in grassroots settings and ports of entry. The testing process involves multiple steps, including sample lysis, nucleic acid purification, amplification, and detection, requiring repeated manual sample transfer, which is time-consuming and prone to cross-contamination. Furthermore, it has weak multi-target detection capabilities, often targeting only a single target, resulting in low efficiency and high cost.

[0004] In the sample processing stage, the sample lysis process requires specialized lysis equipment or high temperature and long-term incubation, and the lysis buffer formulation often has problems such as complex components and low lysis efficiency. To address the above technical pain points, we have developed a portable technical solution that can realize "processing of multiple sample types such as blood, urine and swabs - nucleic acid extraction - nucleic acid amplification - result detection" and support rapid multiplex nucleic acid detection. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a portable integrated test tube for rapid multiplex nucleic acid detection, a matching detection system, and a detection method. To achieve this objective, the technical solution adopted by this invention is as follows: The first aspect of this invention provides a portable integrated test tube for rapid multiplex nucleic acid detection, comprising: a tube body, a nut, a clip, and a magnetic adjustment device; wherein, the magnetic adjustment device is disposed on the outer wall of the tube body; an external thread is provided on the top outer wall of the tube body, and an internal thread is provided on the inner wall of the nut, the nut engaging with the threaded tube body; a protruding post extends downward from the middle of the nut, and an elastic rubber head is fixedly disposed at the bottom of the protruding post, the elastic rubber head sealingly engaging with the tube body's cavity, so that when the nut moves downward, the elastic rubber head can compress the liquid inside the cavity; the bottom of the tube body's cavity extends downward from... The tube is provided with a third septum, a second septum, and a first septum in sequence. The sidewalls of the first septum, the second septum, and the third septum all form a first microchannel with the inner wall of the tube. The first septum has a first groove communicating with the first microchannel, the second septum has a second groove communicating with the first microchannel, and the third septum has a third groove communicating with the first microchannel. The bottom of the third septum has a central hole communicating with the third groove. A detachable insert is provided at the bottom of the tube. The insert has several second microchannels communicating with the central hole. Several rubber sealing plugs are detachably provided at the bottom of the insert. A hollow steel needle is fixedly provided in each rubber sealing plug. A test tube is sealed outside each rubber sealing plug. The needle cavity of the hollow steel needle communicates with the cavity of the second microchannel and the test tube.

[0006] Furthermore, the magnet adjustment device includes: a first fixing block fixedly disposed on the outer wall of the tube body, a guide post fixedly disposed at the bottom of the first fixing block, a second fixing block fixedly disposed at the bottom of the guide post, a magnetic slider, a threaded rod, and an adjustment component slidably disposed on the outer wall of the guide post; wherein, the magnetic slider has a threaded hole along its thickness direction that matches the threaded rod, one end of the threaded rod is rotatably connected to the first fixing block, the other end of the threaded rod passes through the threaded hole and is rotatably connected to the second fixing block, and the bottom of the adjustment component is fixedly connected to one end of the threaded rod.

[0007] Furthermore, the width of the first microchannel is 2μm-4μm, and the diameter of the central hole is 0.5mm-1mm.

[0008] Furthermore, the second groove contains mineral oil, the third groove contains sterile water containing magnesium ions, and the test tube 10 contains CRISPR / Cas13a-RPA lyophilized powder reagent.

[0009] A second aspect of the present invention is to provide a portable multiplex nucleic acid rapid detection system, comprising: an integrated test tube for portable multiplex nucleic acid rapid detection as described above, a base, a control component fixedly disposed in the base, a mounting frame fixedly disposed on one side of the upper part of the base, a heating module fixedly disposed in the mounting frame, a laser fixedly disposed in the mounting frame, a photodiode fixedly disposed on one side of the mounting frame, and a thermostat electrically connected to the heating module; wherein, the top of the heating module has a plurality of receiving slots for accommodating test tubes in the integrated test tube for portable multiplex nucleic acid rapid detection, the emitting end of the laser is located in the receiving slot, a plurality of light-transmitting holes are opened on the side wall of the heating module, and the receiving slots communicate with the light-transmitting holes; a filter is detachably disposed on the light-transmitting hole; the photodiode is disposed on the side of the mounting frame near the light-transmitting hole, and the receiving end of the photodiode is coplanar with the central axis of the light-transmitting hole; wherein, the control component is electrically connected to the thermostat, the heating module, the photodiode, and the laser respectively.

[0010] Furthermore, the control components include: a controller, indicator lights, and a battery; wherein the controller has a Bluetooth module and is electrically connected to the indicator lights and the battery respectively.

[0011] The third aspect of this invention provides a rapid multiplex nucleic acid detection method, employing the portable rapid multiplex nucleic acid detection system described above, comprising the following steps: Step 0, assembling the insert card, hollow steel needle, rubber sealing plug, and test tube; Step 1, taking the sample to be processed and diluting it with phosphate buffer to obtain a diluted sample; Step 2, transferring the diluted sample into the tube, and then sequentially adding lysis buffer and magnetic bead solution into the tube; Step 3, assembling the tube with the nut, allowing the mixture inside the tube to stand and lyse at room temperature to obtain the lysed sample; Step 4, rotating the adjusting component to rotate the threaded rod, causing the magnetic slider to move downwards along the tube, thereby driving the magnetic beads in the magnetic bead solution in the first groove through the first microchannel, through the second groove, and into the third groove, thus obtaining sterile water containing magnesium ions and nucleic acids; Step 5, rotating the nut to compress the tube through the elastic rubber head. The liquid in the tube cavity allows the sterile water containing magnesium ions and nucleic acids in the third groove to drip sequentially into the tube cavity through the second microchannel and the hollow steel needle. Step six: Place the test tube containing the sample into the receiving tank of the heating module. Start the portable multiplex nucleic acid rapid detection system through the control component, and control the heating module through the constant temperature controller to adjust the temperature in the receiving tank to the constant temperature required for nucleic acid amplification, and perform the amplification reaction on the sample in the test tube. During or after amplification, the detection light emitted by the laser passes through the test tube, the light-transmitting hole, and the light signal generated after being filtered by the filter, and is transmitted to the receiving end of the photodiode. The photodiode converts the light signal into an electrical signal and transmits it to the controller. The controller processes and analyzes the electrical signal, transmits the detection data to the external terminal through the built-in Bluetooth module, and the indicator light displays the corresponding color according to the detection status, completing the multiplex nucleic acid rapid detection.

[0012] Furthermore, the sample to be processed is either EDTA-anticoagulated whole blood or a nasopharyngeal swab.

[0013] Furthermore, in step two, the volume ratio of the sample to be processed to phosphate buffer, lysis buffer, and magnetic bead solution is 1:1:4:1; the lysis time at room temperature in step two is 5-7 minutes.

[0014] Furthermore, the components in the lysis buffer include: 5M guanidine thiocyanate, 10% Tween-20, 2M sodium chloride, 2.5mM ethylenediaminetetraacetic acid, and 20mM tris(hydroxymethyl)aminomethane-hydrochloric acid.

[0015] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: In terms of detection efficiency and safety, the present invention integrates sample processing, pathogen lysis, nucleic acid purification and liquid transfer at room temperature by integrating test tubes, eliminating the need for repeated manual liquid transfer, which not only greatly shortens the detection time, but also avoids the risk of cross-contamination caused by sample exposure; at the same time, it supports simultaneous detection of multiple targets, which improves efficiency several times compared with the traditional single target detection mode, and reduces the cost of reagents and consumables.

[0016] In terms of accuracy and ease of operation, the detection system precisely controls the amplification temperature through a constant temperature controller, reduces optical interference through filters, and efficiently converts signals through photodiodes, ensuring accurate detection results. Operation requires only simple assembly, reagent addition, and human-machine interaction to start the system. In addition, the Bluetooth module enables real-time transmission of detection data, indicator lights provide intuitive feedback on status, and smartphones display the instrument status, detection process, and results in real time, further enhancing operational convenience and providing efficient and reliable technical support for rapid on-site testing. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the upper axial side structure of an integrated test tube for portable rapid detection of multiplex nucleic acids according to the present invention; Figure 2 This is a schematic diagram of the left axial side structure of an integrated test tube for portable rapid detection of multiplex nucleic acids according to the present invention; Figure 3 This is a schematic diagram of the axial cross-sectional structure of an integrated test tube for portable rapid detection of multiplex nucleic acids according to the present invention; Figure 4 This is a schematic diagram of the axonal structure of a portable multiplex nucleic acid rapid detection system according to the present invention; Figure 5 This is a schematic diagram of the process structure of a portable multiplex nucleic acid rapid detection system according to the present invention; Figure 6 This is a schematic diagram of the axial cross-sectional structure of the nut in an integrated test tube for portable multiplex nucleic acid rapid detection according to the present invention; Figure 7 This is a schematic diagram of the elastic rubber head in an integrated test tube for portable rapid multiplex nucleic acid detection according to the present invention; Figure 8 This is a schematic diagram of the nut structure in an integrated test tube for portable rapid multiplex nucleic acid detection according to the present invention; Figure 9 The present invention provides the results of detecting postoperative plasma-related viruses in liver transplant recipients using a nucleic acid detection instrument. Figure 9 The left image in the image is image A. Figure 9 The right image in the image is image B; Figure 10The results of the nucleic acid detection instrument of the present invention for detecting respiratory infection-related pathogens, Figure 10 The left image in the image is image A. Figure 10 The right image in the image is image B; Figure 11 The present invention provides the results of nucleic acid detection instruments for detecting mosquito-borne infectious disease-related viruses. Figure 11 The left image in the image is image A. Figure 11 The right image in the image is image B.

[0018] The reference numerals in the attached drawings are as follows: 1. Nut; 2. Sheet; 3. Tube body; 4. Magnetic adjustment device; 5. First diaphragm; 6. Second diaphragm; 7. Third diaphragm; 8. Insert card; 9. Rubber sealing plug; 10. Test tube; 11. Hollow steel needle; 13. Base; 14. Fixing frame; 15. Heating module; 16. Laser; 17. Photodiode; 18. Filter; 19. Controller; 20. Thermostat; 21. Indicator light; 22. Battery; 111. Protrusion; 112. Elastic rubber head; 51. First groove; 61. Second groove; 71. Third groove; 151. Receiving groove; 41. First fixing block; 42. Guide post; 43. Second fixing block; 44. Magnetic slider; 45. Threaded rod; 46. Adjusting component. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0022] Example 1 This embodiment 1 provides a portable integrated test tube for rapid multiplex nucleic acid detection, comprising: a tube body 3, a nut 1, a clip 8, a magnetic adjustment device 4, a third septum 7, a second septum 6, a first septum 5, several rubber sealing plugs 9, several hollow steel needles 11, and several test tubes 10; wherein, the top outer wall of the tube body 3 is provided with external threads, the inner wall of the nut 1 is provided with internal threads, and the nut 1 is threadedly engaged with the tube body 3; a protrusion 111 extends downward from the middle of the nut 1, and the bottom of the protrusion 111 is fixedly disposed. The tube has an elastic rubber head 112 that is sealed to the cavity of the tube body 3. This threaded fit design allows the nut 1 to be stably mounted on the tube body 3. Rotating the nut 1 can drive the protrusion 111 and the elastic rubber head 112 to move axially along the tube body 3. The sealing fit of the elastic rubber head 112 can compress the gas in the cavity of the tube body 3 when the nut 1 moves downward, providing power for the directional transfer of liquid in the tube body 3. This avoids reliance on external pumps and other equipment during liquid transfer and improves the portability of the integrated test tube.

[0023] The bottom of the tube body 3 is provided with a third septum 7, a second septum 6, and a first septum 5 sequentially from bottom to top. The sidewalls of the first septum 5, the second septum 6, and the third septum 7 all form a first microchannel (the width of the first microchannel is 4µm) between themselves and the inner wall of the tube body 3. The first septum 5 has a first groove 51 communicating with the first microchannel, the second septum 6 has a second groove 61 communicating with the first microchannel, and the third septum 7 has a third groove 71 communicating with the first microchannel. The bottom of the third septum 7 has a center groove communicating with the third groove 71. The tube body 3 has a central hole (0.5 mm in diameter). The orderly arrangement of the three-layer septum divides the bottom of the tube into multiple functional areas. The first microchannel provides a channel for the directional flow of magnetic beads and liquid in the subsequent magnetic bead solution, while also preventing impurities generated by sample lysis from entering the downstream area. The first groove 51 can temporarily store the mixture of sample and magnetic bead solution. The second groove 61 and the third groove 71 respectively carry different functional reagents. The first microchannel enables orderly contact between reagents and samples. The central hole serves as a transition channel for liquid to enter the insert card 8 from the septum area, ensuring that the liquid flow path is precise and controllable.

[0024] The second groove 61 contains 500µl of mineral oil, and the third groove 71 contains 50µl of sterile water containing magnesium ions. The mineral oil can isolate the nucleic acid flowing in the first microchannel from air, preventing it from being oxidized and degraded due to contact with air. At the same time, it can prevent cross-contamination of liquids in different areas, ensuring the purity of the nucleic acid. The sterile water containing magnesium ions can provide the necessary magnesium ion environment for the subsequent nucleic acid amplification reaction in advance, eliminating the need to add ion reagents separately and reducing the number of sample transfers.

[0025] The insert card 8 is detachably located at the bottom of the tube body 3. The insert card 8 has several second microchannels, which are connected to the central hole. The detachable design of the insert card 8 makes it easy to replace the insert card 8 with different numbers of channels according to the detection needs, which is suitable for multiple detection scenarios of various pathogens. The connection structure between the second microchannel and the central hole can accurately divert the processed liquid in the third groove 71 to each corresponding test tube 10, realize the independent detection of multiple nucleic acids, and avoid signal interference between different detection targets.

[0026] The bottom of the insert card 8 is detachably equipped with several rubber sealing plugs 9, and a hollow steel needle 11 is fixedly installed in each rubber sealing plug 9. A test tube 10 is sealed around each rubber sealing plug 9. The needle cavity of the hollow steel needle 11 connects the second microchannel with the cavity of the test tube 10. The rubber sealing plugs 9 ensure a sealed connection between the insert card 8 and the test tube 10, preventing liquid leakage or external contaminants from entering the test tube 10 and ensuring a clean testing environment. The fixed installation and connection function of the hollow steel needle 11 can stably introduce the liquid in the second microchannel into the test tube 10 without manual liquid transfer, reducing the difficulty of operation and avoiding cross-contamination caused by sample exposure. The independent installation of the test tube 10 provides an independent space for the amplification reaction of each nucleic acid.

[0027] The magnet adjustment device 4 includes: a first fixing block 41 fixedly disposed on the outer wall of the tube body 3; a guide post 42 fixedly disposed at the bottom of the first fixing block 41; a second fixing block 43 fixedly disposed at the bottom of the guide post 42; a magnetic slider 44, a threaded rod 45, and an adjustment component 46 slidably disposed on the outer wall of the guide post 42; wherein, the magnetic slider 44 has a threaded hole along its thickness direction that matches the threaded rod 45; one end of the threaded rod 45 is rotatably connected to the first fixing block 41; the other end of the threaded rod 45 passes through the threaded hole and is rotatably connected to the second fixing block 43; and the bottom of the adjustment component 46 is fixedly connected to one end of the threaded rod 45; by rotating the adjustment component 46, the threaded rod 45 can be driven to rotate, thereby driving the magnetic slider 44 to slide stably along the guide post 42, realizing precise control of the magnetic force application position. Compared with the traditional method of manually moving the magnet, it can more stably drive the magnetic bead to move along the first microchannel, improving the consistency and efficiency of nucleic acid purification. At the same time, the structure is more stable, avoiding the magnet displacement during the detection process from affecting the purification effect.

[0028] In a preferred embodiment, the external thread provided on the top outer wall of the tube body 3 described in Example 1 is provided by a nut 2 (e.g., fixedly provided on the top outer wall of the tube body 3). Figure 8 As shown in the figure, the outer wall of the nut 2 is connected to the nut 1 by threads.

[0029] In one preferred embodiment, the magnetic slider 44 is a magnet.

[0030] Example 2 This embodiment 2 discloses a portable multiplex nucleic acid rapid detection system, comprising: an integrated test tube for portable multiplex nucleic acid rapid detection as described in embodiment 1, a base 13, a control component, a mounting frame 14, a heating module 15, a laser 16, a photodiode 17, a thermostat 20, and several filters 18; wherein the control component includes a controller 19, an indicator light 21, and a battery 22; wherein the base 13 provides stable support for the entire system; and the mounting frame 14 is fixedly disposed on one side of the upper part of the base 13.

[0031] The heating module 15 is fixedly mounted in the mounting frame 14. The top of the heating module 15 has several receiving slots 151, which are used to receive the test tubes 10 containing the samples to be amplified in the portable multiplex nucleic acid rapid detection integrated test tube described in Example 1, ensuring that the test tubes 10 are stably placed during amplification and detection. The side wall of the heating module 15 has several light-transmitting holes, which are connected to the receiving slots 151. A filter 18 is detachably mounted on the light-transmitting hole. The filter 18 can filter out stray light of non-target wavelengths and only allow light signals matching the nucleic acid amplification products to pass through, thereby improving the accuracy of signal acquisition. The detachable design of the filter 18 makes it convenient to replace the appropriate filter 18 according to the fluorescence signal wavelength of different detection targets.

[0032] The laser 16 is fixedly mounted in the fixture 14, and the emitting end of the laser 16 is located in the receiving groove 151, so that the detection light emitted by the laser 16 directly acts on the sample in the test tube 10 in the receiving groove 151.

[0033] The photodiode 17 is fixedly disposed on one side of the mounting bracket 14, and the receiving end of the photodiode 17 is coplanar with the central axis of the light-transmitting hole. This arrangement allows the light signal filtered by the test tube 10, the light-transmitting hole, and the filter 18 to be transmitted to the receiving end of the photodiode 17, ensuring that the photodiode 17 captures the fluorescence signal and converts it into an electrical signal.

[0034] The thermostat 20 is electrically connected to the heating module 15. Its function is to monitor the temperature of the heating module 15 in real time and adjust it according to the constant temperature conditions required for nucleic acid amplification, so as to ensure that the heating module 15 is stably maintained within the preset temperature range, avoid non-specific amplification caused by temperature fluctuations, and ensure the efficiency and specificity of nucleic acid amplification reaction.

[0035] The control components are fixedly installed in the base 13. The controller 19 is electrically connected to the thermostat 20, the heating module 15, the photodiode 17, and the laser 16, and is the core control unit of the system. It can send temperature adjustment commands to the thermostat 20 to control the start and stop of the heating module 15 and maintain the temperature. At the same time, it can activate the laser 16 to emit detection light, receive the electrical signals transmitted by the photodiode 17, process and analyze the electrical signals, and obtain the detection results. The controller 19 also has a Bluetooth module or a WiFi module, which can transmit the processed detection data to an external terminal (such as a mobile phone or computer) in real time, so that operators can remotely view and record the detection results without having to read the data on-site, thus improving the convenience of operation.

[0036] Indicator light 21 is electrically connected to controller 19. Its function is to intuitively reflect the system's working status through different colors: for example, it is white when the detection preparation stage is underway, yellow when the detection is in progress, green when the detection is completed and the result is normal, and red when the detection is abnormal (such as abnormal temperature or signal interruption). This allows operators to quickly grasp the system's operating status and deal with abnormal problems in a timely manner.

[0037] Battery 22 is electrically connected to controller 19, providing independent power to the entire system, eliminating the system's dependence on external power sources, and enabling the system to be used in field scenarios without fixed power supply, such as primary healthcare and port quarantine, greatly improving the system's portability and field applicability.

[0038] Example 3 This Example 3 describes a method for preparing a CRISPR / Cas13a-RPA lyophilized powder reagent and functionalized test tubes, comprising the following steps: I. Preparation of CRISPR / Cas13a-RPA lyophilized powder reagent: In step one, the RPA primer design needs to be adapted to the primer binding efficiency of recombinase polymerase amplification, the crRNA sequence needs to be precisely matched to the target gene specific region to ensure the targeted recognition of Cas13a protein, and the fluorescent reporter group (FAM-UUUUU-BHQ1) is used for subsequent detection of fluorescence signal induced by Cas13a cleavage activity to ensure detection specificity and sensitivity.

[0039] Step 1: Mixing and Packaging of Core Components Reagent preparation: The core components of CRISPR / Cas13a-RPA include recombinase (Wuxi Leshang Biotechnology Co., Ltd., catalog number: DNA-LS01), single-stranded DNA binding protein (SSB) (Wuxi Leshang Biotechnology Co., Ltd., catalog number: DNA-LS01), DNA polymerase (Wuxi Leshang Biotechnology Co., Ltd., catalog number: DNA-LS01), Cas13a protein (Guangzhou Megbio Biotechnology Co., Ltd., catalog number: C005S / M), NTP (nucleoside triphosphate mixture) (Shanghai Sangon Biotech Co., Ltd., catalog number: B600056), T7 polymerase (Shanghai Sangon Biotech Co., Ltd., B110085), and RNase inhibitor (…). RNaseI (Shanghai Sangon Biotech Co., Ltd., B300076); stabilizers are mannitol (Shanghai Sangon Biotech Co., Ltd., catalog number: A600335) and PEG (polyethylene glycol) (Merck Biotech Co., Ltd., catalog number: 25322-68-3); among them, the RPA primers and crRNA targeting cytomegalovirus (CMV), Epstein-Barr virus (EBV), human parvovirus B19 (B19), mycoplasma pneumoniae (Mp), influenza A virus (FluA), influenza B virus (FluB), dengue virus (DV), Zika virus (ZIKV), and chikungunya virus (CHIKV) were all synthesized by Shanghai Sangon Biotech Co., Ltd.

[0040] Mixing procedure: Add the above core components to a sterile centrifuge tube according to the preset ratio, add mannitol and PEG (the final concentrations are adapted to the stability requirements of the components respectively), place the centrifuge tube at a low temperature of 4°C and slowly invert it to mix the components evenly. Avoid violent shaking and repeated freeze-thaw cycles throughout the process to prevent protein denaturation or nucleic acid degradation.

[0041] Dispensing procedure: Dispense the mixed reagents precisely into 4 sterile reaction tubes 10 according to the single test reaction volume (25μL / reaction).

[0042] Step 3: Pre-freezing treatment: Quickly transfer the dispensed reaction tubes to an -80°C ultra-low temperature freezer and freeze for 2-4 hours to allow the reagents in the tubes to form a uniform and fine ice crystal structure, ensuring that the reagents are completely solidified.

[0043] Step 4: Freeze-drying (lyophilization): First drying: Place the pre-frozen test tube 10 into a freeze dryer, set the vacuum degree to 0.1-0.01 mBar, start the vacuum system and slowly raise the temperature from -80℃ to -20℃ to 0℃, and continue for 10-20 hours; this process removes free water from the reagent through sublimation, and the low temperature environment can prevent the active ingredients of the reagent from being deactivated.

[0044] Second drying (analytical drying): After the first drying is completed, continue to maintain the vacuum state and raise the temperature to 20℃-30℃, and continue to keep warm until the residual moisture of the reagent is controlled at 1%-3%; this further improves the stability of the freeze-dried powder and extends the storage time.

[0045] Step 5: Packaging and Storage Remove the lyophilized reagent tubes 10 from the freeze dryer and immediately place them into a sealed aluminum foil bag pre-filled with desiccant. After removing the air from the bag, seal it. After packaging, store the package in a dry, dark environment at room temperature. The desiccant can absorb trace amounts of moisture in the environment, prevent the lyophilized powder from absorbing moisture and clumping, and ensure the activity of the reagent during storage.

[0046] Example 4 This embodiment 4 uses the portable multiplex nucleic acid rapid detection integrated tube and portable multiplex nucleic acid rapid detection system described in embodiments 1-3. This embodiment 4 uses EDTA-anticoagulated whole blood processed from plasma samples of liver transplant recipients to detect cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human parvovirus B19 (B19) nucleic acids. The specific steps are as follows: Step 0: Assemble the components: Assemble the insert card 8, hollow steel needle 11, rubber sealing plug 9, and test tube 10, ensuring that the hollow steel needle 11 is firmly fixed in the rubber sealing plug 9, the rubber sealing plug 9 and the test tube 10 are sealed together, and the needle cavity of the hollow steel needle 11 is connected to the second microchannel of the insert card 8 and the cavity of the test tube 10, providing a sealed and unobstructed path for subsequent directional liquid transfer.

[0047] It should be noted that the test tube 10 mentioned in this embodiment 4 is the same as the test tube 10 containing CRISPR / Cas13a-RPA lyophilized powder reagent in embodiment 3.

[0048] Step 1, Sample dilution: Take the sample to be processed as EDTA-anticoagulated whole blood, add phosphate buffer according to the ratio to dilute it, and obtain the diluted sample.

[0049] Step 2, Reagent Addition: Transfer the diluted sample into tube 3, and then add the lysis buffer and magnetic bead solution sequentially into tube 3. The volume ratio of the sample to be treated to phosphate buffer, lysis buffer, and magnetic bead solution is 1:1:4:1. The lysis buffer consists of 5M guanidine thiocyanate, 10% Tween-20, 2M sodium chloride, 2.5mM ethylenediaminetetraacetic acid, and 20mM tris(hydroxymethyl)aminomethane-hydrochloric acid. This formula can efficiently destroy cell structure and release nucleic acids, while the magnetic beads in the magnetic bead solution can specifically adsorb nucleic acids, preparing for subsequent purification.

[0050] The volume of 10% Tween-20 contained in the lysis buffer is the percentage of the lysis buffer.

[0051] Step 3, Sample lysis: Tightly assemble tube 3 with nut 1 so that the elastic rubber head 112 seals the cavity of tube 3. Let the mixed system inside the tube stand at room temperature for 5 minutes to lyse, and obtain the lysed sample.

[0052] Step 4, Nucleic Acid Purification: Rotating the adjusting component 46 causes the threaded rod 45 to rotate, which in turn moves the magnetic slider 44 downward along the tube body 3. The magnetic force of the magnetic slider 44 then drives the magnetic beads containing nucleic acid adsorbed in the first groove 51 to pass through the first microchannel and the second groove 61. The mineral oil in the second groove 61 removes impurities adhering to the surface of the magnetic beads, and finally reaches the third groove 71. There, it mixes with the sterile water containing magnesium ions in the third groove 71 to obtain sterile water containing magnesium ions and nucleic acid adsorbed magnetic beads.

[0053] Step 5, Liquid Transfer: Rotate the nut 1 to compress the gas in the cavity of the tube body 3 through the elastic rubber head 112, generating pressure to push the liquid in the third groove 71 through the central hole of the third septum 7, the second microchannel of the insert card 8, and the cavity of the hollow steel needle 11, and drip into the cavity of the test tube 10; the sealed transfer design avoids sample exposure, reduces the risk of cross-contamination, and ensures uniform liquid distribution.

[0054] Step 6, Amplification and Detection: Place the test tube 10 containing the sample into the receiving groove 151 of the heating module 15, ensuring that the test tube 10 and the receiving groove 151 are in close contact; start the detection system through the control component, and the thermostat 20 controls the heating module 15 to adjust the temperature in the receiving groove 151 to 37℃ and maintain it for 30 minutes to amplify the sample in the test tube 10.

[0055] During or after amplification, the detection light emitted by the laser 16 irradiates the sample through the test tube 10, exciting the amplification products to generate an optical signal. The optical signal passes through the light-transmitting hole of the heating module 15 in sequence, and after being filtered out by the filter 18 on the light-transmitting hole to remove stray light, it is transmitted to the receiving end of the photodiode 17. The filter 18 can filter out the light of the target wavelength and improve the signal purity. The photodiode 17 converts the optical signal into an electrical signal and transmits it to the controller 19.

[0056] The controller 19 processes and analyzes the electrical signals to determine whether the target nucleic acid is present in the sample. The detection data is transmitted to an external terminal in real time through the built-in Bluetooth module of the controller 19. At the same time, the indicator light 21 displays the corresponding color according to the detection status (e.g., yellow for detection in progress, green for completion, and red for abnormality). The operator can view the detailed results through the external terminal and complete the rapid detection of multiplex nucleic acids.

[0057] The visualization results of the plasma samples from liver transplant recipients in Example 4 are shown below. Figure 9Figure A shows positive results for CMV and EBV, and negative results for B19. The amplification curve of the plasma sample from the liver transplant recipient in Example 4 is shown in Figure A. Figure 9 As shown in Figure B, the detection results are consistent with the visualized detection results.

[0058] Example 5 This embodiment 5 uses the portable multiplex nucleic acid rapid detection integrated tube and portable multiplex nucleic acid rapid detection system described in embodiments 1-3. The nasopharyngeal swabs of patients with respiratory tract infections are used as the test samples to detect Mycoplasma pneumoniae (Mp), influenza A virus (FluA), and influenza B virus (FluB) nucleic acids. The detection steps in this embodiment 5 are the same as those in embodiment 4 (including the operation process and parameters of component assembly, sample dilution, reagent addition, sample lysis, nucleic acid purification, liquid transfer, amplification and detection), only the type of sample to be tested and the detection target are different.

[0059] The detection results of Example 5 are as follows: For the visual detection results of nasopharyngeal swabs from patients with respiratory infections, please refer to... Figure 10 Figure A shows that the sample was negative for influenza A virus (FluA) and influenza B virus (FluB), but positive for Mycoplasma pneumoniae (Mp). The amplification curve of the nasopharyngeal swab sample from this respiratory infection patient is shown in Figure A. Figure 10 As shown in Figure B, the detection results are consistent with the visualized detection results.

[0060] Example 6 This embodiment 6 uses the portable multiplex nucleic acid rapid detection integrated tube and portable multiplex nucleic acid rapid detection system described in embodiments 1-3. EDTA anticoagulated whole blood after processing plasma samples from mosquito-borne infected patients is used as the sample to be tested to detect dengue virus (DV), Zika virus (ZIKV), and chikungunya virus (CHIKV) nucleic acids. The detection steps in this embodiment 5 are the same as those in embodiment 4 (including the operation process and parameters of component assembly, sample dilution, reagent addition, sample lysis, nucleic acid purification, liquid transfer, amplification, and detection), only the type of sample to be tested and the detection target are different.

[0061] It should be noted that the test tube 10 described in this Example 6 is the same as the test tube 10 with CRISPR / Cas13a-RPA lyophilized powder reagent in Example 3.

[0062] The detection results of Example 6 are as follows: For the visualized detection results of plasma samples from patients with mosquito-borne infections, please refer to... Figure 11 Figure A shows a positive result for CHIKV and negative results for DV and ZIKV; the amplification curve of the plasma sample from this mosquito-borne infection patient is shown in Figure A. Figure 11 As shown in Figure B, the detection results are consistent with the visualized detection results.

[0063] In summary, this invention achieves efficient and safe testing by integrating sample lysis, purification, and liquid transfer into a single test tube, eliminating the need for repeated manual sample transfer. This significantly reduces the time required for the testing process and avoids the risk of cross-contamination caused by sample exposure. Furthermore, it supports simultaneous detection of multiple targets, which is several times more efficient than traditional single-target detection methods and reduces reagent and consumable costs.

[0064] In terms of accuracy and ease of operation, the detection system precisely controls the amplification temperature through a constant temperature controller, reduces optical interference through filters, and efficiently converts signals through photodiodes, ensuring accurate detection results. Operation requires only simple assembly, reagent addition, and system startup. In addition, the Bluetooth module enables real-time transmission of detection data, and indicator lights provide intuitive feedback on the status, further enhancing operational convenience and providing efficient and reliable technical support for rapid on-site testing.

[0065] The above description of the present invention is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A portable integrated test tube for rapid multiplex nucleic acid detection, characterized in that, include: The tube body (3), nut (1), insert (8), and magnet adjustment device (4) are provided. The magnet adjustment device (4) is located on the outer wall of the tube body (3). The top outer wall of the tube body (3) is provided with an external thread, and the inner wall of the nut (1) is provided with an internal thread. The nut (1) is threadedly engaged with the tube body (3). A protrusion (111) extends downward from the middle of the nut (1). An elastic rubber head (112) is fixedly provided at the bottom of the protrusion (111). The elastic rubber head (112) is sealed with the tube cavity of the tube body (3). The bottom of the tube cavity of the tube body (3) is provided with a third septum (7), a second septum (6), and a first septum (5) from bottom to top. The sidewalls of the first septum (5), the second septum (6), and the third septum (7) form a first micro-channel between them and the inner wall of the tube body (3). The first septum (5) has a first groove (51) communicating with the first microchannel, the second septum (6) has a second groove (61) communicating with the first microchannel, the third septum (7) has a third groove (71) communicating with the first microchannel, and the bottom of the third septum (7) has a central hole communicating with the third groove (71); the insert (8) is detachably disposed at the bottom of the tube body (3), the insert (8) has several second microchannels, and the second microchannels communicate with the central hole; the bottom of the insert (8) is detachably disposed with several rubber sealing plugs (9), each rubber sealing plug (9) is fixedly disposed with a hollow steel needle (11), each rubber sealing plug (9) is sealed with a test tube (10), and the needle cavity of the hollow steel needle (11) communicates with the second microchannel and the cavity of the test tube (10).

2. The integrated test tube for portable multiplex nucleic acid rapid detection according to claim 1, characterized in that, The magnet adjustment device (4) includes: a first fixing block (41) fixedly disposed on the outer side wall of the tube body (3), a guide post (42) fixedly disposed at the bottom of the first fixing block (41), a second fixing block (43) fixedly disposed at the bottom of the guide post (42), a magnetic slider (44), a threaded rod (45), and an adjustment member (46) slidably disposed on the outer side wall of the guide post (42); wherein, the magnetic slider (44) has a threaded hole along its thickness direction that matches the threaded rod (45), one end of the threaded rod (45) is rotatably connected to the first fixing block (41), the other end of the threaded rod (45) passes through the threaded hole and is rotatably connected to the second fixing block (43), and the bottom of the adjustment member (46) is fixedly connected to one end of the threaded rod (45).

3. The integrated test tube for portable multiplex nucleic acid rapid detection according to claim 2, characterized in that, The width of the first microchannel is 2μm-4μm, and the diameter of the central hole is 0.5mm-1mm.

4. The integrated test tube for portable multiplex nucleic acid rapid detection according to claim 2, characterized in that, The second groove (61) contains mineral oil, the third groove (71) contains sterile water containing magnesium ions, and the test tube (10) contains CRISPR / Cas13a-RPA lyophilized powder reagent.

5. A portable multiplex nucleic acid rapid detection system, characterized in that, include: The portable multiplex nucleic acid rapid detection integrated test tube, base (13), control component fixedly disposed in the base (13), mounting bracket (14) fixedly disposed on one side of the upper part of the base (13), heating module (15) fixedly disposed in the mounting bracket (14), laser (16) fixedly disposed in the mounting bracket (14), photodiode (17) fixedly disposed on one side of the mounting bracket (14), and thermostat (20) electrically connected to the heating module (15) as described in any one of claims 2-4; wherein the top of the heating module (15) has a top for accommodating the portable multiplex nucleic acid rapid detection integrated test tube, base (13), control component fixedly disposed in the base (13), mounting bracket (14) fixedly disposed on one side of the mounting bracket (14), and thermostat (20) electrically connected to the heating module (15); wherein the top of the heating module (15) has a top for accommodating the portable multiplex nucleic acid rapid detection integrated test tube, base (13), control component fixedly disposed in the base (13), mounting bracket (14) fixedly disposed on one side of the mounting bracket (13), and thermostat (20) electrically connected to the heating module (15); The integrated test tube (10) has several receiving slots (151), the emitting end of the laser (16) is located in the receiving slot (151), the side wall of the heating module (15) is provided with several light-transmitting holes, and the receiving slots (151) are connected to the light-transmitting holes; a filter (18) is detachably provided on the light-transmitting hole; the photodiode (17) is provided on the fixed frame (14) on the side close to the light-transmitting hole, and the receiving end of the photodiode (17) is coplanar with the central axis of the light-transmitting hole; wherein, the control component is electrically connected to the constant temperature controller (20), the heating module (15), the photodiode (17), and the laser (16) respectively.

6. The portable multiplex nucleic acid rapid detection system according to claim 5, characterized in that, The control components include: a controller (19), an indicator light (21), and a battery (22); wherein the controller (19) has a Bluetooth module and is electrically connected to the indicator light (21) and the battery (22) respectively.

7. A rapid multiplex nucleic acid detection method, employing the portable rapid multiplex nucleic acid detection system as described in any one of claims 6, characterized in that, The steps include: Step 0, assembling the insert card (8), the hollow steel needle (11), the rubber sealing plug (9), and the test tube (10); Step 1, taking the sample to be processed and diluting it with phosphate buffer to obtain a diluted sample; Step 2, transferring the diluted sample into the tube body (3), and then adding lysis buffer and magnetic bead solution to the tube body (3) in sequence; Step 3, assembling the tube body (3) with the nut (1), allowing the mixed system inside the tube to stand at room temperature for lysis, thus obtaining the lysed sample; Step 4 1. Rotate the adjusting component (46) to make the threaded rod (45) rotate, causing the magnetic slider (44) to move downward along the tube body (3), thereby driving the magnetic beads in the magnetic bead liquid in the first groove (51) through the first microchannel, through the second groove (61), and into the third groove (71), thus obtaining sterile water containing magnesium ions and nucleic acids; 2. Rotate the nut (1) to compress the liquid in the tube cavity of the tube body (3) through the elastic rubber head (112), so that the magnesium ions and nucleic acids in the third groove (71) are contained in the liquid. Acidic sterile water is dripped sequentially into the lumen of the test tube (10) through the second microchannel and the lumen of the hollow steel needle (11); Step six: The test tube (10) containing the sample is placed into the receiving tank (151) of the heating module (15); The portable multiplex nucleic acid rapid detection system is started by the control component, and the temperature controller (20) controls the heating module (15) to adjust the temperature in the receiving tank (151) to the constant temperature required for nucleic acid amplification, and the sample in the test tube (10) is amplified; Amplification process After the detection is completed, the detection light emitted by the laser (16) passes through the test tube (10), the light-transmitting hole, and is filtered by the filter (18). The resulting light signal is transmitted to the receiving end of the photodiode (17). The photodiode (17) converts the light signal into an electrical signal and transmits it to the controller (19). The controller (19) processes and analyzes the electrical signal and transmits the detection data to an external terminal through the built-in Bluetooth module. At the same time, the indicator light (21) displays the corresponding color according to the detection status, thus completing the rapid detection of multiple nucleic acids.

8. The rapid detection method for multiplex nucleic acids according to claim 7, characterized in that, The sample to be processed is either EDTA-anticoagulated whole blood or a nasopharyngeal swab.

9. The rapid detection method for multiplex nucleic acids according to claim 7, characterized in that, In step two, the volume ratio of the sample to be processed to the phosphate buffer, the lysis buffer, and the magnetic bead solution is 1:1:4:1; the lysis time at room temperature in step two is 5-7 minutes.

10. The rapid detection method for multiplex nucleic acids according to claim 7, characterized in that, The components of the lysis buffer include: 5M guanidine thiocyanate, 10% Tween-20, 2M sodium chloride, 2.5mM ethylenediaminetetraacetic acid, and 20mM tris(hydroxymethyl)aminomethane-hydrochloric acid.

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