A sample processing and detection device

Abstract

The application provides a sample processing and detection device, which can efficiently complete the whole process detection of sample extraction and amplification of pathogen nucleic acid in a short time and has good safety and convenience.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to a sample processing and detection device. Background Technology

[0002] Infectious diseases are caused by infectious pathogens infecting the human body. Because these pathogens are contagious, some infectious diseases can lead to serious consequences (high mortality rates, severe complications, etc.), making early diagnosis particularly important. Since early infection with infectious pathogens often presents with no specific symptoms, early diagnosis of infectious diseases mainly relies on the detection of the pathogen.

[0003] Pathogens mainly include viruses, bacteria, and mycoplasma, which are primarily composed of nucleic acid molecules and protein molecules (antigens). These two categories of molecules are the target molecules for rapid pathogen detection. The nucleic acid molecules of the pathogen are the target molecules for nucleic acid detection. Different pathogens have different nucleic acid molecules. By designing specific nucleic acid fragments that specifically bind to pathogen molecules, various methods such as PCR, RT-PCR, LAMP, and nucleic acid hybridization can be used to detect pathogen nucleic acid molecules. Molecular detection plays an important role in infectious disease diagnosis, genetic disease diagnosis, cancer diagnosis, biochemical indicator detection, and scientific research services. Conventional laboratory testing methods are generally time-consuming and involve many steps, typically requiring sample pretreatment, detection reactions, and result interpretation. These steps require sophisticated instruments, regulated experimental spaces, and experienced operators. In many situations, rapid detection of target molecules is necessary, such as the rapid detection of cardiac markers in cardiovascular emergency departments and the rapid identification of pathogenic microorganisms causing disease in fever clinics. These situations often lack the necessary equipment and personnel, making complex experimental operations difficult. Therefore, there is an urgent need for an integrated solution that is easy to operate and integrates multiple steps such as sample processing, detection, and result interpretation.

[0004] Currently, rapid pathogen identification methods mainly include nucleic acid detection and immunological detection (antigens and / or antibodies). Nucleic acid detection generally includes sample pretreatment, nucleic acid amplification, and result interpretation. Sample pretreatment includes four main steps: lysing the pathogen, adsorbing nucleic acids onto a solid phase, washing away impurities, and eluting the nucleic acids from the adsorbed solid phase. The entire nucleic acid detection process requires not only a series of reagents (nucleic acid extraction reagents such as centrifugal salts, pH buffers, inorganic salts, and ethanol, and amplification reagents) and supporting equipment (centrifuges, PCR instruments, automated workstations, etc.), but also a specific experimental environment (a strictly divided nucleic acid amplification laboratory, such as a specially designed PCR operating room) to address the possibility of nucleic acid contamination. Furthermore, due to the infectivity of the pathogen, biosafety issues must be considered (personnel safety and potential environmental hazards). In addition, a complete nucleic acid test based on PCR / RT-PCR generally takes more than 1 hour.

[0005] To improve testing efficiency, integrated testing solutions have been developed. A common integrated solution involves adding different liquids (for example, in nucleic acid amplification: sample lysis buffer, magnetic beads, washing buffer, elution buffer, and amplification reaction solution) to a series of small test tubes. An electromagnetic rod is used to move the liquids between the tubes to purify the nucleic acid before amplification. This method is an open system (the tube caps are open), which is prone to contamination. Furthermore, due to the large size of the instrument and the need for biosafety laboratories, it is difficult to achieve rapid on-site testing in emergency clinics.

[0006] Biomicrofluidic chips are created by etching channels and sample cells on materials such as glass or plexiglass to allow liquid flow and storage. Liquid is then propelled through these tiny channels using mechanical, magnetic, or voltage methods to achieve functions such as nucleic acid amplification, cell culture, and even chemical synthesis. Biomicrofluidic chips offer advantages such as high integration and small size, making them suitable for solving the challenges of rapid on-site detection.

[0007] However, a drawback of bio-microfluidic chips is that liquid flow within tiny channels (mm or even smaller) is significantly affected by surface tension, making them difficult to control. Furthermore, microfluidic chips require an external pump for power, thus remaining an open system. Miniaturization is challenging when attempting to create an integrated device that also incorporates a drive mechanism.

[0008] In 2004, Iquum Corporation applied for a patent (US6,780,617B2) for PCR using a tubing. This patent used mechanical compression of different sections of the tube to move the liquid inside, allowing it to move between different temperature modules for nucleic acid amplification. Subsequently, Iquum applied for a series of related patents, all utilizing compressible membranes to move liquid between different functional blocks under external force. The core of this technology is a compressible tubing divided into several sections, each containing a different solution. Thermal adhesion separates each section. Mechanical compression of the sections achieves the flow of liquid. The disadvantage of this technology is that one tube can only perform 4-5 types of PCR. If multiplex quantitative PCR is required, the number of tubes must be increased, or the tubes must be branched.

[0009] How to simplify the experimental procedures for pathogen nucleic acid detection, reduce detection time and biosafety risks, and achieve "sample in, result out" remains a technical problem that those skilled in the art need to solve, and it is also the core problem that this invention addresses. Summary of the Invention

[0010] To address the aforementioned problems, the present invention provides a sample processing and testing device.

[0011] In some embodiments of the present invention, the device is a nucleic acid extraction and detection device, the device including a nucleic acid extraction unit and a nucleic acid amplification unit, wherein the nucleic acid extraction unit includes a microchannel structure, and a piston drives the liquid flow in the microchannel; the nucleic acid amplification unit includes an amplification zone that can be pushed back and forth and a temperature control module.

[0012] In some embodiments of the present invention, the nucleic acid extraction unit is a cartridge with multiple channels for accommodating solution bottles containing different solutions; the cartridge has a valve to guide liquids to different microchannels; the bottom of the liquid bottle is a piston that can move up and down, and by squeezing the piston, the solution can be discharged into the channels of the cartridge; the bottom of the cartridge has a groove for placing an adsorbable nucleic acid matrix; the matrix and the solution bottle are interconnected through a pathway system consisting of the groove of the cartridge and a membrane covering the groove.

[0013] In some embodiments of the present invention, the card holder has two or two sets of holes, the holes are connected by a channel, and the holes are covered with silicone caps. Liquid can flow between the two holes by sequentially squeezing the silicone caps.

[0014] Preferably, the cap of the liquid bottle is made of a puncture-resistant material; more preferably, the material is silicone.

[0015] In some embodiments of the present invention, the bottom of the tube of the cartridge has a hollow thin tube for piercing the cap of the solution bottle.

[0016] In some embodiments of the present invention, the solution includes a lysis buffer, a washing buffer, and an elution buffer, and the different solutions are located in different solution bottles.

[0017] In some embodiments of the present invention, the nucleic acid amplification unit includes PCR reaction wells containing PCR reaction components, preferably, the PCR reaction components being powders.

[0018] In some embodiments of the present invention, the temperature control module includes one or more temperature modules for setting different temperatures to achieve the thermal cycling process of PCR.

[0019] In some embodiments of the present invention, the device further includes a detection unit that can collect signals from the nucleic acid amplification unit. Preferably, the detection unit includes a fluorescence excitation and collection device.

[0020] In some embodiments of the present invention, the detection unit further includes a data processing module, which can process the collected data and display it directly on a display screen.

[0021] The present invention also provides a method for detecting nucleic acids using the device, comprising:

[0022] Add the sample containing nucleic acid to a solution bottle containing lysis buffer;

[0023] Adsorbing nucleic acids onto the matrix;

[0024] The nucleic acids adsorbed on the matrix were washed with a washing solution;

[0025] The nucleic acids adsorbed on the matrix were eluted with elution buffer and introduced into the PCR reaction wells; and

[0026] The PCR reaction wells are brought into direct contact with the temperature module to perform the PCR reaction.

[0027] In some embodiments of the present invention, the method further includes detecting the signal of the PCR reaction well and analyzing the signal; preferably, the signal is a fluorescence signal.

[0028] In some embodiments of the present invention, the sample containing nucleic acid is a sample containing pathogens, preferably a sample containing viruses, chlamydia, rickettsia, mycoplasma, bacteria, spirochetes and fungi.

[0029] In some embodiments of the present invention, the matrix is ​​a membrane capable of adsorbing nucleic acids, preferably a silica membrane or a glass fiber membrane.

[0030] In some embodiments of the present invention, the temperature module comprises two parts, each capable of being set to a first temperature and a second temperature; preferably, the nucleic acid is denatured at the first temperature and annealed and extended at the second temperature. In other embodiments of the present invention, the temperature module comprises one part, capable of performing isothermal amplification reactions.

[0031] In other embodiments of the present invention, the device mainly comprises two parts: a sample processing unit and a detection unit. The sample processing unit and the detection unit can be an integrated structure or an operable connection structure.

[0032] Sample processing unit

[0033] Nucleic acid extraction is performed via a microchannel-guided process, where a piston propels the solution flow and membranes / magnetic beads adsorb nucleic acid molecules. First, a sample processing solution is pre-stored in the integrated device. This solution can be a lysis buffer for nucleic acid purification or a buffer solution for immunological detection. Clinical samples are added to the sample processing solution, and following a pre-programmed sequence, the processing solution and different buffer solutions are sequentially propelled through the piston into the membrane / magnetic bead area. Waste liquid flows through a conduit into the waste liquid area, completing the sample processing. During nucleic acid purification, the sample processing solution contains guanidine hydrochloride or guanidine isothiocyanate plasma salts, a low-pH buffer system such as Tris.Cl, and surfactants to release nucleic acids and promote their binding to the nucleic acid adsorption material. The buffer solution serves to clean impurities from the nucleic acid adsorption material and to elute the nucleic acids from it. The eluted nucleic acids then flow through a conduit into the nucleic acid amplification area.

[0034] Detection unit

[0035] It includes a reciprocating mixing zone and a temperature control module. During nucleic acid detection, eluted nucleic acid molecules enter the amplification zone, mix and dissolve thoroughly with pre-placed amplification components (which can be in dry powder or solution form), and then undergo an amplification reaction according to a predetermined program. The temperature control module provides the appropriate temperature and reaction time. During immunological detection, magnetic beads adsorbed with target molecules and labels enter the detection zone, and detection is performed through fluorescence, luminescence, or color development.

[0036] In other embodiments of the invention

[0037] The device is used to process immunological test samples. The sample processing solution is a solution that dilutes the sample and provides the conditions for the immunoreaction; the buffer solution includes a washing solution, labeled buffer, substrate, and reactants. The purpose of the washing solution is to wash away impurities from the target molecule adsorption material, such as immunomagnetic beads. After washing, the immunomagnetic beads are propelled into the detection unit by the liquid.

[0038] When using magnetic beads for nucleic acid testing and immunological testing, the magnetic beads can be placed in the sample processing solution.

[0039] The device of the present invention may further include a signal acquisition and processing unit, which acquires signals by the detection module according to a predetermined program, and the acquired data can be uploaded wirelessly. The signal detection unit may include a data processing module, which processes the acquired data and displays it directly on a display screen.

[0040] Nucleic acid adsorption materials can be any material that can adsorb nucleic acids, such as large-pore silica gel membranes / filter cartridges, glass fiber membranes, or particles containing silica.

[0041] Nucleic acid testing can be performed using any conventional sampling method, such as amplification reactions, which can be isothermal amplification, PCR, or quantitative real-time PCR.

[0042] Detection can be single-channel or multi-target, or multiple channels can be detected simultaneously.

[0043] Nucleic acid purification and amplification methods can be combined arbitrarily.

[0044] In other embodiments of the present invention, the sample processing unit includes a syringe (1) and a body (2).

[0045] The main body (2) is provided with one or more wheel-shaped solution chambers (21), the solution chambers (21) surround and form a syringe movement cavity (25), the bottom of the solution chambers (21) has a channel (22), the contact surface between the channel (22) and the syringe movement cavity (25) is provided with a silicone sealing plug (23), and the channel (22) is arranged radially with the center of the bottom of the main body as the center;

[0046] The syringe (1) is provided with a puncture needle (16) at one end facing the syringe movement cavity (25) and a push-pull rod (11) at the other end. The syringe (1) also includes a shell (12).

[0047] The syringe (1) is located at the central axis of the body (2) and can rotate relative to the body (2); the puncture needle (16) is not located at the geometric center of the bottom surface of the syringe.

[0048] In some other embodiments of the present invention, the bottom of the main body (2) is provided with a sample dispensing channel (26) which is connected to the detection unit.

[0049] In some other embodiments of the present invention, a target adsorption material (15) is fixed at the needle tube of the syringe (1) near the puncture needle (16). The target adsorption material (15) allows liquid to pass through under pressure conditions, and there is no gap between the target adsorption material (15) and the syringe cavity (17), so that the liquid cannot flow freely.

[0050] When an external force is applied to the bottom of the push-pull rod (11), the syringe (1) moves toward the bottom of the body (2), and the puncture needle (16) punctures the silicone sealing plug (23); by pushing and pulling the push-pull rod (11), the solution in the solution chamber (21) passes through the target adsorption material (15), and the solution can pass through in both directions depending on the direction of the push and pull;

[0051] Pull the syringe (1) away from the main body (2) to make the puncture needle (16) leave the silicone sealing plug (23); rotate the syringe (1) to move the puncture needle (16) to the corresponding target silicone sealing plug (23) position, and repeat the operation.

[0052] In other embodiments of the invention, the solution chambers (21) are one or more, preferably 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. The solution chambers (21) are used to contain the same or different solutions.

[0053] In some other embodiments of the present invention, a silicone rubber plug (24) is provided on the top of the solution tank (21). Preferably, if there is more than one silicone rubber plug (24), the more than one silicone rubber plug (24) can be provided on a silicone rubber cap (29), the silicone rubber cap (29) being more than one silicone rubber plug arranged in a ring on a silicone plane.

[0054] In other embodiments of the invention, the syringe (1) further includes a return spring (13) and a sealing ring (14).

[0055] In other embodiments of the present invention, the target adsorption material (15) is a material that can adsorb nucleic acids, preferably a silica membrane or a glass fiber membrane.

[0056] In other embodiments of the present invention, the target adsorption material (15) is a material capable of adsorbing proteins.

[0057] In other embodiments of the present invention, the target adsorption material (15) is in the form of a filter cartridge or in the form of particles.

[0058] In some other embodiments of the present invention, the target adsorption material (15) is in the form of particles, and a filter (18) is provided on the syringe (1) near the puncture needle (16), the filter (18) can prevent the particle adsorption material from entering the puncture needle (16).

[0059] In other embodiments of the present invention, the detection unit includes a reaction tube (3), and the sample processing unit is connected to the reaction tube (3) by a channel.

[0060] In other embodiments of the present invention, the reaction tube (3) is pre-loaded with reaction reagents, preferably, the reaction reagents contain reagents that can be used for PCR reactions or isothermal reactions.

[0061] In other embodiments of the present invention, the detection unit further includes a signal acquisition section.

[0062] In other embodiments of the present invention, the detection unit further includes a data processing section, which can process the data obtained by the signal acquisition section and display it on a display screen.

[0063] Features of the device of the present invention:

[0064] Reagents are stored and moved within pipelines using piston-type liquid bottles;

[0065] A hollow needle is used to pierce the mouth of a liquid bottle under piston pressure to connect the liquid bottle and the pipeline.

[0066] The adsorption, washing, and elution of nucleic acids, as well as immunological reactions, are achieved by using membranes or magnetic beads pre-placed at specific locations within the pipeline.

[0067] The reaction solution is moved between different temperature ranges by pushing the solution.

[0068] In addition, the arrangement of the liquid tubes can be changed. For example, a revolver-style magazine can be used, arranging the liquid chambers (sample, washing solution 1, washing solution 2, eluent, etc.) in a cylinder, placing the magnetic bead unit in the barrel, and switching between different liquid flow paths by rotating the sample chamber. Another approach is to place the piston in the center of the reagent-containing cylinder, with the piston moving up and down to generate positive and negative pressure, allowing for unidirectional or bidirectional liquid flow between the central piston and the reagent cylinder.

[0069] The advantages of the device of this invention are: 1. The use of a liquid bottle with a piston structure solves the problems of storing and moving nucleic acid extraction reagents within the pipeline. 2. The use of etched pipelines to achieve liquid movement solves the problem of difficult liquid flow in long-distance microchannels. 3. Liquid flow control is achieved through preset valves on the pipeline, allowing the eluted nucleic acid solution to be distributed to different reaction wells, thereby integrating multiple PCR reaction units. 4. The matching cartridges of the integrated device are made of polymer, metal, or silicone materials, making them easy to process. 5. The design of dual temperature control zones allows the amplification mixture to complete the amplification reaction by moving back and forth between the two temperature control zones, reducing amplification time compared to traditional single-temperature-controlled heating and cooling methods. 6. Signal acquisition devices, such as fluorescence acquisition devices, can be placed close to the PCR reaction zone to fully utilize fluorescence intensity.

[0070] The above design achieves fully automated, enclosed nucleic acid extraction while optimizing the nucleic acid amplification process, reducing amplification time to some extent and increasing the number of detectable signal channels, thereby increasing the number of detectable samples. Overall, it integrates nucleic acid extraction and amplification. Attached Figure Description

[0071] To more clearly illustrate the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other embodiments based on these descriptions without any creative effort.

[0072] Figure 1 This is a schematic diagram of a sample processing and detection device provided by the present invention.

[0073] Figure 2 shows another sample processing and detection device provided by the present invention. Figure 2A This is a schematic diagram of the cross-sectional structure. Figure 2B This is a 3D image.

[0074] Figure 3 is a schematic diagram of another sample processing and detection device provided by the present invention, wherein... Figure 3A This is a schematic diagram of the cross-sectional structure. Figure 3B This is a 3D image.

[0075] Figure 4 is a schematic diagram of another sample processing and detection device provided by the present invention. Figure 4A This is a schematic diagram of the cross-sectional structure; Figure 4B This is a structural schematic diagram and a liquid flow diagram of the valve body (015); Figure 4C It is a 3D image; Figure 4D This is a perspective view.

[0076] Figure 1 The captions for Figure 4 are as follows:

[0077] I: Card Box

[0078] II: Nucleic Acid Extraction Section

[0079] III: Reaction Section

[0080] 001: Solution bottle

[0081] 0011: Piston

[0082] 0012: Separated Space

[0083] 0013: Silicone rubber cap

[0084] 004: Waste Liquid Tank

[0085] 005: Three-way valve

[0086] 006: PCR Low Temperature Zone

[0087] 0061: Silicone rubber cover

[0088] 007: High-temperature zone for PCR

[0089] 0071: Silicone rubber cover

[0090] 008: Target Adsorption Materials

[0091] 009: Puncture needle

[0092] 010: Liquid flow channel (a transparent biofilm is attached to the bottom of the reaction cartridge, which forms a liquid flow channel with the bottom of the cartridge. The channel is connected to each puncture needle and a three-way valve. A filter membrane is installed between the three-way valve and the channel of the elution solution for filtering, washing and eluting nucleic acids.)

[0093] 011: Three-way valve connecting to waste liquid tank

[0094] 012: Three-way valve connecting to the PCR low-temperature zone

[0095] 013: Connection channel between the PCR low-temperature zone and the PCR high-temperature zone

[0096] 014: Reaction Chamber

[0097] 015: Valve body

[0098] 016: Magnet

[0099] Figure 5 This is a schematic diagram of another sample processing and detection device provided by the present invention.

[0100] Figure 6 This invention provides an internal top view of the bottom surface of a card box body.

[0101] Figure 7This is a bottom view of the main body of a card box provided by the present invention.

[0102] Figure 8 shows a planar view (A) and a side view (B) of a silicone material provided by the present invention.

[0103] Figure 9 This is a half-sectional schematic diagram of a sample processing and detection device provided by the present invention.

[0104] Figure 10 This is a schematic diagram showing the insertion of a puncture needle into the channel, connecting the syringe chamber to the solution reservoir channel.

[0105] Figure 11 A schematic diagram of the syringe head mechanism when using target adsorption material in the form of magnetic beads.

[0106] Figures 5 to 11 The captions in the figure are as follows:

[0107] 1: Syringe

[0108] 11: Push-pull rod

[0109] 12: Syringe casing

[0110] 13: Return spring

[0111] 14: Sealing ring

[0112] 15: Target Adsorption Materials

[0113] 16: Puncture needle

[0114] 17: Syringe cavity

[0115] 18: Filter

[0116] 19: Particulate adsorbent materials

[0117] 2: Card box body

[0118] 21: Solution Chamber

[0119] 211: Bottom surface of the solution tank

[0120] 212: Round hole

[0121] 22: Channel

[0122] 23: Silicone rubber sealing plug

[0123] 24: Silicone rubber stopper

[0124] 25: Syringe movement cavity

[0125] 26: Sample Addition Channel

[0126] 27, 28: Fasteners

[0127] 29: Silicone rubber cap

[0128] 3: Reaction tube Detailed Implementation

[0129] Unless otherwise stated, the terms used herein have the meanings commonly understood by one of ordinary skill in the art. For terms explicitly defined herein, their meanings shall be as defined herein.

[0130] Pathogens: A collective term for microorganisms and parasites that can cause disease. Microorganisms include viruses, chlamydia, rickettsiae, mycoplasma, bacteria, spirochetes, and fungi. This article primarily refers to pathogens that cause human diseases.

[0131] Nucleic acid: It is the general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It is a type of biopolymer and the most important substance among all biological molecules. It is widely present in all animal and plant cells and microorganisms.

[0132] Nucleic acid extraction: In this patent, it refers to the process of extracting nucleic acid from pathogens using reagents and corresponding equipment through different methods.

[0133] Nucleic acid amplification: refers to the amplification of a specific nucleic acid sequence through the action of enzymes.

[0134] Primers are two short, artificially synthesized nucleotide sequences that specifically bind to the single-stranded target nucleic acid fragment to be amplified. In PCR, primers can be designed and synthesized based on a known nucleotide sequence of the target gene. During PCR amplification, the target nucleic acid fragment denatures upon heating and breaks down into single strands. After cooling, the primers bind to the corresponding complementary sequences of the single strands, and then extension is performed under the action of polymerase. This cycle is repeated.

[0135] Target adsorption materials refer to matrices that can specifically adsorb targets. The targets can be, for example, nucleic acids or proteins; the matrices can be in the form of membranes, magnetic beads, colloids, etc.

[0136] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0137] Figure 1This invention provides a sample processing and detection device, comprising a sample processing unit and a detection unit, for the extraction and amplification of nucleic acids. The device is a cartridge (I), the sample processing unit is a nucleic acid extraction section (II), and the detection unit is a reaction section (III). Various reagents required for the processing are concentrated in a cartridge (I) containing multiple compartments (0012). The nucleic acid extraction section (II) has multiple compartments for accommodating liquid bottles (001) containing different solutions, and a liquid pool (004) for collecting waste liquid. The cartridge has a valve body to guide the liquid to different pipes. The reaction section (III) has two (or two sets) holes connected by a channel. Silicone rubber caps cover the holes, allowing liquid to flow between the two holes by sequentially squeezing the silicone rubber caps. The liquid bottle caps inserted into the cartridge's pipes are made of a puncture-resistant material such as latex. A piston that can move up and down is located at the bottom. By squeezing the piston, the solution can be discharged and squeezed into the cartridge's pipes. At the bottom of the cartridge's tubing is a puncture needle, a hollow, thin tube with a sharp tip, used to puncture the mouth of the solution bottle. The bottom of the cartridge has grooves where a silicon-based membrane for nucleic acid adsorption is placed. The silicon-based membrane is interconnected with different solution bottles via a tubing system consisting of grooves in the cartridge itself and a membrane covering those grooves.

[0138] In the nucleic acid extraction section (II), one solution bottle (001) contains lysis buffer (also called lysis buffer cup). When the sample to be tested is added to the sample bottle, the lysis buffer mixes with the sample. Then, the piston (0011) of the sample bottle is pressed down, causing the cap to contact the puncture needle (009) at the bottom of the cartridge until the thin tube punctures the silicone rubber cap (0013). The solution passes through the puncture needle (009) and the tubing (010) to the membrane (008) that can adsorb nucleic acids, and then enters the waste liquid pool (004) through the three-way valve (005) and the tubing (011). The second solution bottle (001) contains washing solution (also called cleaning solution cup) to remove impurities adsorbed on the membrane. By pressing down the piston (0011) at the bottom of the solution bottle, the cap comes into contact with the puncture needle (009) until the cap is punctured. The solution flows through the puncture needle (009) and channel (010) through the membrane (008) adsorbed with nucleic acids, and then flows into the waste liquid pool (004) through the three-way valve (005) and channel (011). The third solution bottle (001) contains the elution buffer (also called the elution buffer cup), which is used to elute the nucleic acids adsorbed on the membrane (008). Its operation is similar to that of the previous solution bottle, with the liquid flow pushed by pressing down the piston (0011), but the direction of the three-way valve (005) is switched, and the eluted nucleic acids are introduced into the PCR reaction section (III) through channel (012).

[0139] The PCR reaction section (III) comprises two well-shaped structures: a low-temperature PCR zone (006) and a high-temperature PCR zone (007). The low-temperature PCR zone (006) stores lyophilized powder containing the components required for the PCR reaction, and the top of each well is covered with a squeezable silicone rubber cap (0061) and (0071). By repeatedly squeezing the silicone rubber caps (0061) and (0071), the elution buffer containing nucleic acids is thoroughly dissolved and mixed with the PCR reaction system through the channel (0013). The two wells containing the PCR reaction system are in direct contact with modules at different temperatures, resulting in different temperatures for the two wells. Simultaneously, the reciprocating movement of the reaction system through the squeezing of the silicone rubber caps facilitates the thermal cycling process of PCR. Furthermore, a fluorescence excitation and acquisition device is located at the low-temperature well location to enable real-time fluorescence acquisition and analysis.

[0140] The reaction cartridge (I) is a plastic injection molded structure with a transparent film sealed at the bottom. The bottom of the position where the lysis buffer cup, washing buffer cup and elution buffer cup are installed has a puncture needle (009).

[0141] The lysis buffer cup, washing buffer cup, and elution buffer cup are three independent small cups, pre-sealed with the corresponding reagents, and each has a sealing film at the bottom that can be penetrated by a puncture needle (009). Depending on the usage requirements, the three cups can be pre-installed on the reaction cartridge (inserted into the corresponding position but without puncturing), or the three small cups can be packaged independently, and the cartridge can be packaged independently, with the user combining the small cups and cartridge before use.

[0142] The PCR low-temperature zone (006) pre-encapsulates the reaction system. Its top material is a squeezeable soft material. By squeezing, the PCR reaction system after the sample is injected can move back and forth between the low-temperature zone and the high-temperature zone to carry out a rapid PCR reaction.

[0143] Depending on the experimental requirements, one or more solution cups may be used, containing the same or different reaction solutions; the capacity and order can be adjusted as needed. As an example, Figure 1 The apparatus in Figure 1 has 3 solution cups, while the apparatus in Figure 2 has 4 solution cups, which is more... Figure 1 Add a cleaning solution cup.

[0144] Figure 3 shows a sample processing and detection device according to another embodiment of the present invention. Compared with the device in Figure 2, the following changes are made: 1) The waste liquid pool (004) is set outside the solution bottle; 2) The nucleic acid adsorption area (magnetic bead, filter membrane area) is moved to the far end of the solution bottle to reserve space for the movement of the magnet (if magnetic beads are used to adsorb nucleic acid); 3) The reaction part (III) includes a reaction chamber (014), which has a sloping top design to increase the mixing efficiency when the solution is mixed.

[0145] Figure 4 shows a sample processing and detection device according to another embodiment of the present invention. (See schematic diagram below.) Figure 4A See 4C for the 3D view and 4C for the perspective view. Figure 4D The target adsorption material (008) is a magnetic bead, which is placed in the solution bottle (001), preferably in the first solution bottle. After the magnetic bead is mixed with the sample, it adsorbs the target (e.g., nucleic acid or protein). A magnet (016) is provided on the outside of the channel (010) after the end solution bottle. The position and movement of the magnetic bead are controlled by the movement of the magnet (016) so that the magnetic bead can be fully mixed with the solution. Preferably, the channel (010) is curved, such as S-shaped.

[0146] Other structural changes include: 1) placing the waste liquid tank (004) below the solution bottle; 2) positioning the reaction chamber (014) at the bottom; 3) using a valve body (015) for liquid switching. When the valve body is pulled up, the channel connects to the waste liquid tank (004), and when the valve body is pressed down, the solution enters the reaction chamber (014). A schematic diagram of the valve body (015) in one embodiment of the present invention can be found [see attached diagram]. Figure 4B .

[0147] The amplification reaction can be either PCR or isothermal amplification. The reagent bottles contain, in order: lysis buffer (containing purification magnetic beads), wash buffer 1 (for cleaning impurities), wash buffer 2 (for cleaning impurities), nucleic acid elution buffer (TE), and isothermal amplification reconstitution solution. After nucleic acid elution, the eluted nucleic acid is injected into the reaction zone by pressing down the stopper of the elution buffer bottle. Then, the reconstitution solution is injected into the reaction zone by pressing down the stopper of the reconstitution solution bottle. In the mixing zone, the isothermal amplification powder, nucleic acid template, and reconstitution solution are mixed.

[0148] In one embodiment of the present invention, the device can sequentially complete the extraction and detection of pathogen nucleic acids within the device and provide the detection results. The detection results can be uploaded via Bluetooth. The entire nucleic acid extraction and detection process requires no manual operation, no additional equipment or reagents, and the entire detection process is completed within 1 hour.

[0149] In other embodiments of the present invention, the sample processing and detection device has a wheel-shaped structure.

[0150] Figure 5 This invention illustrates a sample processing and detection apparatus according to an embodiment of the present invention. The apparatus is a cartridge comprising a sample processing unit and a detection unit, which can be used for nucleic acid extraction and PCR detection. In this embodiment, the cartridge (I) includes a nucleic acid extraction portion (II) as the sample processing unit and a reaction portion (III) as the detection unit, which can be used for nucleic acid purification and amplification.

[0151] The nucleic acid extraction section (II) consists of a syringe (1), a main body (2) with one or more wheel-shaped solution chambers (21) and channels (22), a silicone sealing plug (23) for sealing the contact surface between the channel (22) and the syringe movement cavity (25), a silicone rubber plug (24) for sealing the top of the solution chamber (21), a sample dispensing channel (26), a reaction tube (3), and other parts.

[0152] The syringe consists of a push-pull rod (11) capable of piston movement, a syringe housing (12), a syringe return spring (13), a syringe sealing ring (14), a target adsorption material (15), and a syringe puncture needle (16). The puncture needle (16) is not located at the geometric center of the syringe bottom surface, but rather on a radial line with the geometric center as the origin. When the syringe rotates relative to the cartridge body (2), the syringe puncture needle (16) will move in an arc on the bottom surface of the cartridge body. The cartridge body (2) includes one or more hollow cylindrical solution chambers (21), which are axially parallel and parallel to the long axis of the cartridge, arranged in a wheel-like pattern, with a hollow center forming a syringe movement cavity (25). The syringe movement cavity (25) is used to insert the syringe (1). The syringe is fitted with a silicone sealing ring (14), which seals the syringe tip (including the puncture needle (16)) and the hollow structure of the cartridge, isolating the syringe tip from the outside environment to prevent contamination.

[0153] The solution chamber (21) can be one or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, used to hold solutions such as lysis buffer, washing buffer, and elution buffer for nucleic acid purification and amplification reactions. The bottom of the solution chamber (21) is conical and has a channel (22) that connects downward to the transverse channel, which is arranged radially with the center of the bottom of the cartridge as the center. The channel is vertically upward in the centripetal direction and opens at the bottom contact surface with the syringe movement cavity (25), with the center of the opening coinciding with the rotation trajectory of the syringe puncture needle (16). The channel opening is sealed with a silicone rubber stopper (23).

[0154] Taking a cartridge body with 6 solution compartments as an example, see the top view of the bottom surface of the cartridge body. Figure 6 On the bottom surface of the solution compartment (211), corresponding to the reagent container, there are six circular holes (212). These holes point vertically downwards and connect to six horizontally distributed radial channels. As the channels extend below the reagent container, they point vertically upwards and connect to the reagent container. Five of the six channels connect to the reagent compartment, and one connects to a central, vertically downward-facing hole, which serves as the sample loading port for connecting the amplification tube.

[0155] See the bottom view of the card box body. Figure 7The bottom of the card holder has two short cylindrical retainers (27, 28) of different diameters, used for positioning the card holder on the all-in-one machine. At the card holder fixing position on the all-in-one machine, there are two circular recesses of different diameters, corresponding to the two positioning cylinders on the card holder.

[0156] The solution chamber (21) has an opening at the top, and a silicone rubber plug (24) is provided at the opening. Different silicone rubber plugs corresponding to different solution chambers can be set on a silicone rubber cap (29) (Figure 8). The silicone rubber cap is a structure of a flat surface with six hollow silicone rubber pillars. The positions of the hollow pillars correspond to the six openings on the bottom surface of the card box body. The silicone rubber cap serves to seal the solution chamber. In one embodiment of the present invention, the opening at the top of the solution chamber (21) can be blocked with a filter element that can filter aerosols. The function of the filter element is to ensure that air can circulate and that the air pressure inside and outside the chamber is consistent, but not to allow aerosols to pass through, so as to avoid contamination.

[0157] Figure 9 This is a half-section view of a sample processing and testing device according to an embodiment of the present invention. When the syringe is rotated to a certain angle, the puncture needle (16) is aligned with the lower circular hole. By pressing down the syringe, the puncture needle (16) pierces the silicone cap, and the syringe is connected to the reagent chamber. By pulling and pressing down the syringe piston rod, negative pressure and positive pressure are generated, driving the solution in the reagent chamber, or the solution in the syringe flows between the syringe and the solution chamber. The flow can be unidirectional or reciprocating.

[0158] The nucleic acid extraction section consists of a syringe for aspirating liquid and several solution chambers containing different solution components. The syringe comprises a barrel, piston, and lever. At the bottom of the syringe, there is a material that can adsorb nucleic acids, such as a filter membrane or filter element containing silica; or a filter membrane that blocks magnetic beads or glass beads used for nucleic acid purification. The syringe tip can be aligned with the sealed cap of the solution chamber by rotating or swinging. The piston in the syringe moves up and down to aspirate and expel liquid. The syringe can move parallel to its axis, and the syringe tip punctures the sealed caps of different solution chambers to establish communication between the syringe and the solution chambers. The aspiration and expulsion of liquid between the syringe and the solution chambers achieves functions such as solution mixing, filtration, and washing. During nucleic acid purification, the syringe first punctures the solution chamber containing lysis buffer and the sample. By moving the piston up and down, the solution is guided through the nucleic acid adsorption material, simultaneously mixing the solution. The other solution chambers contain washing buffer, nucleic acid elution buffer, and solutions required for nucleic acid amplification, respectively. The solution chambers can be arranged around the syringe, sequentially on a plane, or in groups. When the solution chambers are arranged around the syringe, the solution outlets are arranged in a ring and sealed with materials such as silicone. The syringe is placed on the eccentric shaft of the inner cylinder of the cartridge. By rotating the syringe on the eccentric shaft, the puncture needle (16) is positioned at the outlet of different solution chambers; or the syringe puncture needle (16) is placed outside the center of the bottom plane of the syringe, and the syringe is coaxial inside the cartridge. When the syringe is rotated, the puncture needle (16) is positioned at the outlet of different solution chambers.

[0159] The detection unit of the device of this invention can be a nucleic acid amplification section, including a reciprocating amplification zone, a temperature control module, and a fluorescence detection module. The eluted nucleic acid molecules enter the amplification zone, mix with and fully dissolve the pre-placed amplification powder in this zone, and then undergo an amplification reaction according to a predetermined program. The temperature control module provides a suitable temperature and reaction time, and the fluorescence detection module collects fluorescence signals according to a predetermined program. The collected data is displayed in a concise manner and can be uploaded via Bluetooth.

[0160] In one embodiment, the sample loading channel (26) and the amplification tube (3) correspond to the integrated instrument's temperature control module. When the cartridge is placed on the integrated instrument, the amplification tube makes full contact with the temperature control module. Each sample compartment opening is covered with a silicone membrane with a central opening, and the opening on the silicone membrane is blocked with a filter cartridge that can filter aerosols. The left and right sides of the filter cartridge ensure airflow and consistent air pressure inside and outside the sample compartment, but do not allow aerosols to pass through to avoid contamination.

[0161] Advantages of the above devices:

[0162] 1. By using a syringe to rotate and position, different solution chambers can be punctured to achieve the switching of different solutions, avoiding the use of tiny flow paths and valves. The structure is simple and reliable.

[0163] 2. By utilizing the positive and negative pressure generated by the pulling of a syringe to move liquid, the problem of difficult liquid flow in long-distance microchannels is solved.

[0164] 3. The nucleic acid adsorption material is fixed to the bottom of the syringe, allowing the liquid flow to be completed simultaneously with the nucleic acid adsorption and impurity washing processes. The design is simple and reliable.

[0165] 4. The syringe can also be used as a pipetting device for precise solution aspiration and transfer, and as a mixing device for mixing and preparing incompatible components.

[0166] 5. The cartridge arranges the solution compartments in a circular shape, making full use of space and resulting in a small overall volume.

[0167] 6. The above design achieves fully automated, enclosed nucleic acid extraction while optimizing the nucleic acid amplification process, reducing amplification time to some extent, and increasing the number of detectable fluorescent channels, thereby increasing the number of detectable pathogens. Overall, it integrates pathogen nucleic acid extraction and amplification.

[0168] Example 1: Real-time PCR

[0169] Taking a cartridge with 6 solution compartments (21) as an example, compartments 1-3 contain 500 μL of lysis buffer, 800 μL of wash buffer 1, and 800 μL of wash buffer 2, respectively. Compartment 4 contains no reagents or solutions, and compartment 5 contains 200 μL of elution buffer. The amplification tube is then filled with a real-time PCR reaction system containing the novel coronavirus.

[0170] Experimental process

[0171] 1) Sample addition: Insert the nasopharyngeal swab containing the sample into solution compartment 1, stir for 10 seconds, then break it and close the lid. Alternatively, remove the nasopharyngeal swab and discard it in a waste bag, disposing of it according to biosafety procedures. Or add 200 μL of sample solution and close the lid.

[0172] 2) Adsorption of nucleic acid: Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to the No. 1 solution chamber. Press down the syringe (1) to pierce the silicone rubber cap. Pull and press down the push rod (11). Under pressure, the lysis solution repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1). After pulling 10 times, press down the syringe (1) to return all the liquid to the No. 1 solution chamber.

[0173] 3) Washing impurities: Rotate the syringe (1) and align the puncture needle (16) with the corresponding silicone rubber cap of tube 2. Press down the syringe (1) to pierce the silicone rubber cap. Pull and press down the push rod (11). Under pressure, the washing solution 1 repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1). After pulling 10 times, press down the syringe (1) to return all the liquid to the solution chamber 2.

[0174] 4) Washing impurities: Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to tube 3. Press down the syringe (1) to pierce the silicone rubber cap. Pull and press down the push rod (11). Under pressure, the washing solution 2 repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1). After pulling 10 times, press down the syringe (1) to return all the liquid to the solution chamber 3.

[0175] 5) Drying the nucleic acid adsorbent material: Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to the No. 4 solution chamber. Press down the syringe (1) to pierce the silicone rubber cap. Pull and press down the push rod (11). The No. 4 solution chamber is an empty tube. Under pressure, the air repeatedly passes through the nucleic acid adsorbent material fixed at the bottom of the syringe (1). The purpose is to evaporate the residual washing solution 2 into the air and dry the nucleic acid adsorbent material.

[0176] 6) Eluting nucleic acid: Rotate the syringe (1), align the puncture needle (16) with the silicone rubber cap corresponding to solution compartment 5, press down the syringe (1) to puncture the silicone rubber cap, pull and press down the push rod (11), and the TE solution will repeatedly pass through the nucleic acid adsorption material fixed at the bottom of the syringe (1) under pressure. The nucleic acid on the adsorption material will be eluted into the TE solution. After pulling 10 times, the syringe (1) will not be pressed down, and all liquid will remain in the syringe (1).

[0177] 7) Sample addition: Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to solution compartment 6. Press down the syringe (1) to pierce the silicone rubber cap. Press down the push-pull rod (11). The TE solution containing nucleic acid enters the nucleic acid amplification tube through the central channel under pressure. The TE solution is mixed with the prepared fluorescent PCR reaction system to complete the sample addition.

[0178] 8) Amplification detection: Control the temperature module and execute the following program: reverse transcription (50 degrees Celsius for 10 minutes); pre-denaturation (95 degrees Celsius for 60 seconds); amplification reaction (95 degrees Celsius for 10 seconds, 65 degrees Celsius for 10 seconds, fluorescence acquisition at 65 degrees Celsius, 40 cycles).

[0179] Example 2: Premixed SARS-CoV-2 nucleic acid real-time PCR

[0180] 500 μL of lysis buffer (component), 800 μL of washing buffer 1, and 800 μL of washing buffer 2 were respectively contained in solution compartments 1-3 of the cartridge. 200 μL of elution buffer was placed in solution compartment 4, and lyophilized powder of the COVID-19 amplification reaction system was placed in solution compartment 5. The amplification tubes were empty.

[0181] Experimental process

[0182] 1) To add the sample, insert the nasopharyngeal swab containing the sample into solution compartment 1, stir for 10 seconds, then break it off and close the lid. Alternatively, remove the nasopharyngeal swab and discard it in a waste bag, following relevant biosafety procedures. Or, add 200 μL of sample solution and close the lid.

[0183] 2) Adsorb nucleic acid. Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to the No. 1 solution chamber. Press down the syringe (1) to pierce the silicone rubber cap. Pull and press down the push rod (11). Under pressure, the lysis solution repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1). After pulling 10 times, press down the syringe (1) to return all the liquid to the No. 1 solution chamber.

[0184] 3) Wash impurities, rotate the syringe (1), align the puncture needle (16) with the silicone rubber cap corresponding to the No. 2 solution chamber, press down the syringe (1) to pierce the silicone rubber cap, pull and press down the push rod (11), the washing liquid 1 repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1) under pressure; after pulling 10 times, press down the syringe (1) to return all the liquid to the No. 2 solution chamber.

[0185] 4) Wash impurities, rotate the syringe (1), align the puncture needle (16) with the silicone rubber cap corresponding to the No. 3 solution chamber, press down the syringe (1) to pierce the silicone rubber cap, pull and press down the push rod (11), the washing liquid 2 repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1) under pressure; after pulling 10 times, press down the syringe (1) to return all the liquid to the No. 3 solution chamber.

[0186] 5) Dry the nucleic acid adsorbent material. Lift the syringe (1) and suspend the puncture needle (16) in the moving cavity (25) of the syringe (1). Pull and press down the push rod (11). Under pressure, the air repeatedly passes through the nucleic acid adsorbent material fixed at the bottom of the syringe (1) and discharges the residual washing liquid 2 in the nucleic acid adsorbent material into the air in the moving cavity (25) of the syringe (1), so that the nucleic acid adsorbent material is dried.

[0187] 6) Elute nucleic acid. Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to solution compartment 4. Press down the syringe (1) to pierce the silicone rubber cap. Pull and press down the push rod (11). Under pressure, the TE solution repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1). The nucleic acid on the adsorption material is eluted into the TE solution. After pulling 10 times, the syringe (1) is not pressed down, and all liquid remains in the syringe (1).

[0188] 7) Mix the reaction system. Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to solution compartment 5. Press down the syringe (1) to pierce the silicone rubber cap. Press down the push-pull rod (11). The TE solution containing nucleic acid flows through the solution compartment (21) under pressure. The TE solution is mixed with the prepared fluorescent PCR reaction system. Repeatedly pull the push-pull rod (11) to fully mix the TE and the reaction system.

[0189] 8) Add sample, lift the syringe (1), rotate it, align the puncture needle (16) with the silicone rubber cap corresponding to solution compartment 6, press down the syringe (1) to pierce the silicone rubber cap, press down the push-pull rod (11), the mixed reaction system enters the nucleic acid amplification tube through the flow path channel, and the sample is added.

[0190] 9) Amplification detection, temperature control module, execute the following program: reverse transcription (50 degrees Celsius for 10 minutes); pre-denaturation (95 degrees Celsius for 60 seconds); amplification reaction (95 degrees Celsius for 10 seconds, 65 degrees Celsius for 10 seconds, fluorescence acquisition at 65 degrees Celsius, 40 cycles).

[0191] Example 3: Isothermal Amplification Experiment of Premixed SARS-CoV-2 Nucleic Acid

[0192] 500 μL of lysis buffer, 800 μL of wash buffer 1, and 800 μL of wash buffer 2 were respectively contained in solution compartments 1-3 of the cartridge. 200 μL of elution buffer was placed in solution compartment 4 (21). The reconstituted solution of the isothermal amplification reaction system of the novel coronavirus was placed in solution compartment 5 (21). The amplification tube was a lyophilized powder of the isothermal amplification system.

[0193] Experimental process

[0194] 1) To add the sample, insert the nasopharyngeal swab containing the sample into solution compartment 1, stir for 10 seconds, then break it off and close the lid. Alternatively, remove the nasopharyngeal swab and discard it in a waste bag, following relevant biosafety procedures. Or, add 200 μL of sample solution and close the lid.

[0195] 2) Adsorb nucleic acid. Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to the No. 1 solution chamber. Press down the syringe (1) to pierce the silicone rubber cap. Pull and press down the push rod (11). Under pressure, the lysis solution repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1). After pulling 10 times, press down the syringe (1) to return all the liquid to the No. 1 solution chamber.

[0196] 3) Wash impurities, rotate the syringe (1), align the puncture needle (16) with the silicone rubber cap corresponding to the No. 2 solution chamber, press down the syringe (1) to pierce the silicone rubber cap, pull and press down the push rod (11), the washing liquid 1 repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1) under pressure; after pulling 10 times, press down the syringe (1) to return all the liquid to the No. 2 solution chamber.

[0197] 4) Wash impurities, rotate the syringe (1), align the puncture needle (16) with the silicone rubber cap corresponding to the No. 3 solution chamber, press down the syringe (1) to pierce the silicone rubber cap, pull and press down the push rod (11), the washing liquid 2 repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1) under pressure; after pulling 10 times, press down the syringe (1) to return all the liquid to the No. 3 solution chamber.

[0198] 5) Dry the nucleic acid adsorbent material, lift the syringe (1), suspend the puncture needle (16) in the syringe (1) body, pull and press down the push rod (11), and the air, driven by pressure, repeatedly passes through the nucleic acid adsorbent material fixed at the bottom of the syringe (1), and discharges the residual washing liquid 2 in the nucleic acid adsorbent material into the air inside the syringe (1) shell, so that the nucleic acid adsorbent material is dried.

[0199] 6) Elute nucleic acid. Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to solution compartment 4. Press down the syringe (1) to pierce the silicone rubber cap. Pull and press down the push rod (11). Under pressure, the TE solution repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1). The nucleic acid on the adsorption material is eluted into the TE solution. After pulling 10 times, the syringe (1) is not pressed down, and all liquid remains in the syringe (1).

[0200] 7) Mix the reaction system. Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to the No. 5 solution chamber. Press down the syringe (1) to pierce the silicone rubber cap. Press down the push-pull rod (11). The TE solution containing nucleic acid flows through the solution chamber (21) under pressure. The TE solution is mixed with the prepared isothermal amplification reaction system reconstituted solution. Repeatedly pull the push-pull rod (11) to fully mix the TE and reconstituted solution.

[0201] 8) Add sample, lift the syringe (1), rotate it, align the puncture needle (16) with the silicone rubber cap corresponding to solution chamber 6, press down the syringe (1) to pierce the silicone rubber cap, press down the push-pull rod (11), the mixed complex solution system enters the amplification tube pre-loaded with isothermal expansion lyophilized powder through the flow path channel, and the sample addition is completed.

[0202] 9) Amplification detection, control the temperature module, and execute the following program: 39 degrees Celsius for 10 minutes, then collect fluorescence.

[0203] The nucleic acid adsorption material can be a large-pore silica gel filter membrane / filter cartridge or a particulate adsorption material (19), such as silica-containing particles. When using silica particles, a filter screen is used at the connection between the bottom of the syringe (1) and the puncture needle (16) to block the particles from passing through, see [reference]. Figure 11 .

[0204] Example 4: Nucleic acid purification using large-pore silica membrane / filter cartridges for SARS-CoV-2 quantitative real-time PCR detection.

[0205] 500 μL of lysis buffer, 800 μL of wash buffer 1, and 800 μL of wash buffer 2 are respectively placed in solution compartments 1-3 of the cartridge. Solution compartment 4 (21) is empty, and solution compartment 5 (21) contains 200 μL of elution buffer. The amplification tube is then filled with the fluorescence quantitative PCR reaction system containing the novel coronavirus. The bottom cylinder of the syringe (1) is filled with a large-pore silica filter cartridge or a large-pore silica filter membrane with the same diameter as the inner diameter of the cylinder. When using a filter membrane, a retaining ring for fixing the filter membrane needs to be pre-installed in the cylinder.

[0206] Reagent formulation:

[0207] Lysis buffer: 4M guanidine isothiocyanate, 0.5% NP40, 50mM Tris.Cl pH5.5, 10mM EDTA;

[0208] Washing solution 1: A buffer solution containing ethanol or isopropanol, typically consisting of 10 mM Tris-HCl, pH 7.5, or 80% ethanol.

[0209] Lotion 2: Contains a higher concentration of ethanol or isopropanol, typically 100% ethanol.

[0210] Washing solution formulations that do not use solvents such as ethanol or isopropanol:

[0211] Eluent: TE solution, consisting of 10 mM Tris and 0.1 mM EDTA, pH 8.5

[0212] 1) To add the sample, insert the nasopharyngeal swab containing the sample into solution compartment 1, stir for 10 seconds, then break it off and close the lid. Alternatively, remove the nasopharyngeal swab and discard it in a waste bag, following relevant biosafety procedures. Or, add 200 μL of sample solution and close the lid.

[0213] 2) Adsorb nucleic acid. Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to the No. 1 solution chamber. Press down the syringe (1) to pierce the silicone rubber cap. Pull and press down the push rod (11). Under pressure, the lysis solution repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1). After pulling 10 times, press down the syringe (1) to return all the liquid to the No. 1 solution chamber.

[0214] 3) Wash impurities, rotate the syringe (1), align the puncture needle (16) with the silicone rubber cap corresponding to the No. 2 solution chamber, press down the syringe (1) to pierce the silicone rubber cap, pull and press down the push rod (11), the washing liquid 1 repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1) under pressure; after pulling 10 times, press down the syringe (1) to return all the liquid to the No. 2 solution chamber.

[0215] 4) Wash impurities, rotate the syringe (1), align the puncture needle (16) with the silicone rubber cap corresponding to the No. 3 solution chamber, press down the syringe (1) to pierce the silicone rubber cap, pull and press down the push rod (11), the washing liquid 2 repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1) under pressure; after pulling 10 times, press down the syringe (1) to return all the liquid to the No. 3 solution chamber.

[0216] 5) Dry the nucleic acid adsorbent material. Lift the syringe (1) and suspend the puncture needle (16) in the moving cavity (25) of the syringe (1). Pull and press down the push rod (11). Under pressure, the air repeatedly passes through the nucleic acid adsorbent material fixed at the bottom of the syringe (1) and discharges the residual washing liquid 2 in the nucleic acid adsorbent material into the air in the moving cavity (25) of the syringe (1), so that the nucleic acid adsorbent material is dried.

[0217] 6) Elute nucleic acid. Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to solution compartment 4. Press down the syringe (1) to pierce the silicone rubber cap. Pull and press down the push rod (11). Under pressure, the TE solution repeatedly passes through the nucleic acid adsorption material fixed at the bottom of the syringe (1). The nucleic acid on the adsorption material is eluted into the TE solution. After pulling 10 times, the syringe (1) is not pressed down, and all liquid remains in the syringe (1).

[0218] 7) Mix the reaction system. Rotate the syringe (1) and align the puncture needle (16) with the silicone rubber cap corresponding to solution compartment 5. Press down the syringe (1) to pierce the silicone rubber cap. Press down the push-pull rod (11). The TE solution containing nucleic acid flows through the solution compartment (21) under pressure. The TE solution is mixed with the prepared fluorescent PCR reaction system. Repeatedly pull the push-pull rod (11) to fully mix the TE and the reaction system.

[0219] 8) Add sample, lift the syringe (1), rotate it, align the puncture needle (16) with the silicone rubber cap corresponding to solution compartment 6, press down the syringe (1) to pierce the silicone rubber cap, press down the push-pull rod (11), the mixed reaction system enters the nucleic acid amplification tube through the flow path channel, and the sample is added.

[0220] 9) Amplification detection, temperature control module, execute the following program: reverse transcription (supplement time and temperature); pre-denaturation (supplement time and temperature); amplification reaction (95 degrees Celsius for 10 seconds, 65 degrees Celsius for 10 seconds, fluorescence acquisition at 65 degrees Celsius, 40 cycles).

[0221] Example 5: Nucleic acid purification using silica particles and quantitative real-time PCR detection of SARS-CoV-2.

[0222] In the device of this embodiment, the target adsorption material (008) is in particulate form, and a filter (18) is provided on the syringe (1) near the puncture needle (16). The filter (18) can prevent the particulate adsorption material from entering the puncture needle (16). See [reference] Figure 11 .

[0223] Other experimental procedures are similar to those in Example 4.

[0224] The sample processing and detection apparatus provided by this invention has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and core ideas of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of this invention.

Claims

1. A sample processing and detection device, the device comprising a sample processing unit and a detection unit, the sample processing unit comprising a piston structure for driving liquid flow through the piston structure, wherein the sample processing unit comprises a syringe (1) and a body (2). The main body (2) is provided with 2-20 solution chambers (21) arranged in a wheel shape. The solution chambers (21) surround and form a syringe movement cavity (25). The bottom of the solution chamber (21) has a channel (22). The contact surface between the channel (22) and the syringe movement cavity (25) is provided with a silicone sealing plug (23). The channel (22) is arranged radially with the center of the bottom of the main body as the center. The top of the solution chamber (21) is provided with a silicone rubber plug (24). The syringe (1) is provided with a puncture needle (16) at one end facing the syringe movement cavity (25) and a push-pull rod (11) at the other end. The syringe (1) also includes a shell (12). The syringe (1) is located at the central axis of the body (2) and can rotate relative to the body (2); the puncture needle (16) is not located at the geometric center of the bottom surface of the syringe; The bottom of the main body (2) is provided with a sample feeding channel (26), which is connected to the detection unit; and The syringe (1) has a target adsorption material (15) fixed at the needle tube near the puncture needle (16). The target adsorption material (15) allows liquid to pass through under pressure, and there is no gap between the target adsorption material (15) and the syringe cavity (17), so the liquid cannot flow freely. When an external force is applied to the bottom of the push-pull rod (11), the syringe (1) moves toward the bottom of the body (2), and the puncture needle (16) punctures the silicone sealing plug (23); by pushing and pulling the push-pull rod (11), the solution in the solution chamber (21) passes through the target adsorption material (15), and the solution can pass through in both directions depending on the direction of the push and pull; Pull the syringe (1) away from the main body (2) to make the puncture needle (16) leave the silicone sealing plug (23); rotate the syringe (1) to move the puncture needle (16) to the corresponding target silicone sealing plug (23) position, and repeat the operation.

2. The apparatus of claim 1, wherein the solution chamber (21) is used to contain the same or different solutions.

3. The device of claim 1, wherein the syringe (1) further comprises a return spring (13) and a sealing ring (14).

4. The apparatus according to any one of claims 1 to 3, wherein the target adsorption material (15) is a material capable of adsorbing nucleic acids.

5. The apparatus of claim 4, wherein the target adsorption material (15) is a silicone membrane or a glass fiber membrane.

6. The apparatus according to any one of claims 1 to 3, wherein the target adsorption material (15) is a protein-adsorbing material.

7. The apparatus according to any one of claims 1 to 3, wherein the target adsorption material (15) is in the form of a filter cartridge or in the form of granules.

8. The device of claim 7, wherein the target adsorption material (15) is in particulate form, and a filter (18) is provided on the syringe (1) near the puncture needle (16), wherein the filter (18) can prevent the particulate adsorption material from entering the puncture needle (16).

9. The apparatus of claim 1, wherein the detection unit includes a reaction tube (3), and the sample processing unit is connected to the reaction tube (3) by a channel.

10. The apparatus of claim 9, wherein the reaction tube (3) is pre-loaded with a reaction reagent.

11. The apparatus of claim 10, wherein the reaction reagent is a PCR reaction reagent or an isothermal reaction reagent.

12. The apparatus of claim 1, wherein the detection unit further comprises a signal acquisition section.

13. The apparatus of claim 12, wherein the detection unit further includes a data processing section, which processes the data obtained by the signal acquisition section and displays it on a display screen.

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