Nucleic acid extraction and detection device and use method

Abstract

According to the nucleic acid extraction and detection device and the use method, by forming a plurality of communicating holes and taking a rotary valve as a communicating medium, communication among functional chambers is achieved, then sample transfer is achieved, the whole transfer process is completed in an internally-closed environment, sample pollution is not caused, and the detection efficiency is improved. The rotating cover and the rotating valve rotate synchronously, and the rotating cover is also closed when the rotating valve is closed, so that the sealing performance of the cavity in the uniform mixing and reaction process is ensured, and the stability of a pre-stored reagent and the safety of the reaction process are ensured; the sample transfer step is reduced, the pollution risk is reduced, the whole process is completed only through manual operation without a power supply or other equipment, and the method can be used for household self-inspection or in areas with limited resources or even in extreme environments without power supply.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nucleic acid detection, and relates to a nucleic acid extraction and detection device and a use method. BACKGROUND

[0002] In the past few years, nucleic acid-based detection methods have been accepted by clinical diagnostics because of their high specificity, ease of configuration to detect almost any target, requirement for minimal sample size (high sensitivity), and automation capability. Polymerase chain reaction (PCR) has become the method of choice for rapid diagnosis of infectious agents, which can be used to detect pathogenic microorganisms with high sensitivity and high specificity. However, nucleic acid amplification detection requires a series of complex processing steps, including collection of clinical samples and transportation to a central laboratory, lysis / digestion, nucleic acid binding, buffer washing, and nucleic acid elution to extract pathogenic DNA / RNA, RNA reverse transcription, and then PCR [5]. This method usually requires specialized equipment and trained technicians to perform tests in a laboratory environment. In addition, during manual operation, especially the multiple steps of reopening the test tube and pipetting reagents, accidental laboratory-acquired infections or aerosol contamination between samples can occur.

[0003] To meet the needs of nucleic acid rapid detection in non-central laboratory environments, small integrated (Point of Care, POC) nucleic acid detection systems have become an important development direction. The current small integrated nucleic acid detection systems generally have performance limitations in order to simplify the nucleic acid detection process and reduce costs and operation thresholds. Many products rely on in situ thermal lysis steps, but can only target specific pathogens and sample sources, omitting key nucleic acid extraction, purification, and enrichment steps, resulting in insufficient detection sensitivity, poor result stability, and repeatability.

[0004] In order to improve the sensitivity of nucleic acid detection and detect more complex samples, sample pretreatment is required before detection. Current manual nucleic acid extraction methods have complicated operation steps, which can easily cause aerosol leakage and contamination during uncapping and pipetting. Most of the nucleic acid extraction equipment on the market is in an open or semi-closed system, such as the commercial full-automatic nucleic acid extraction and purification instrument Molecision MP-32. The high-speed rotation of the magnetic rod during nucleic acid extraction can easily generate target nucleic acid aerosols, posing a risk to personnel and laboratories.

[0005] The whole-process nucleic acid detection device integrates the complete nucleic acid detection process in a single small device, integrates nucleic acid extraction and detection steps, broadens the range of detectable pathogens, improves detection sensitivity and stability, and reduces dependence on professional personnel and special laboratory environment. Currently, there are many mature whole-process nucleic acid detection schemes on the market, such as the Atlas Genetics io nucleic acid detection chip disclosed in patent US2016158746. The instrument drives the transfer of liquid reagents in the chip through a pneumatic drive module and a mechanical drive module. The nucleic acid extraction reagent inside the chip is stored in liquid form, and the reagents required for nucleic acid amplification and detection are pre-stored in freeze-dried solid form. The GeneXpert molecular diagnostic microfluidic kit disclosed in patent US8673238B2 and the special test instrument for the whole automatic analysis of the kit use microfluidic chip technology to achieve rapid nucleic acid detection from sample to result. The GeneXpert molecular diagnostic system uses column extraction technology, uses a disposable independent module to avoid contamination risk, and the whole extraction and QPCR real-time monitoring of amplification signals can effectively ensure the sensitivity and specificity of the product. However, these chips and instruments are expensive, for example, the price of a single GeneXpert chip is about 50-150 US dollars, and the cost of purchasing an instrument is about 20-50 thousand US dollars, and the chip can only be used with the corresponding matching instrument, the cost of single detection is high, which may limit its application in resource-limited areas.

[0006] To solve the problem of high detection cost of the whole integrated nucleic acid detection device, the palm-sized nucleic acid detection device, system and method disclosed in Chinese patent CN202211204476.8 simplify the operation process and shorten the detection reaction time on the basis of realizing the miniaturization of the nucleic acid detection device, and the miniaturized system matched therewith can be produced at low cost. The palm-sized base station is opened in the patent, which is used to assist sample preparation and provide heat control for isothermal nucleic acid amplification without plug-in. Magnetic bead nucleic acid extraction can be performed multiple times clockwise and counterclockwise, loop-mediated isothermal amplification reaction can be performed by colorimetric method, and can be directly observed by naked eye or analyzed by mobile phone APP. However, the device will produce a certain volume of liquid residue during nucleic acid extraction, which will make the washing liquid enter the eluent, finally participate in the nucleic acid amplification reaction and produce an inhibitory effect, affecting the sensitivity and / or specificity of the detection.

[0007] In summary, the main shortcomings of existing miniaturized nucleic acid detection products are:

[0008] The nucleic acid detection performance is insufficient, which is the biggest problem of the current micro nucleic acid detection system. In order to simplify the whole nucleic acid detection step and reduce the size of the instrument, many products only have simple in-situ thermal cracking without the process of nucleic acid extraction purification enrichment, which can cause limited types of detectable pathogens, insufficient detection sensitivity, and unstable experimental results and poor repeatability. The detection of "false positive" and "false negative" results is not reliable.

[0009] The nucleic acid extraction is difficult to integrate. At present, most micro devices rely on simplified nucleic acid extraction process (such as magnetic bead adsorption or membrane filtration), but the mature magnetic bead method nucleic acid extraction device is a semi-closed system, which is easy to cause aerosol pollution and difficult to integrate the nucleic acid extraction and detection system into a closed system.

[0010] The integrated nucleic acid detection cost is high, the cost of the instrument and the chip is too high, and the closed system causes the third party reagent to be incompatible, forming a "device-consumable" binding business model, so that the single detection cost is much higher than the traditional PCR reagent cost. At present, the micro nucleic acid detection system abroad is more mature, but due to the high cost, it does not have strong attraction and promotion prospect. SUMMARY

[0011] The purpose of the present application is to solve the problems of sample pollution, complex sample transfer process and the inability to provide a full-closed environment for sample transfer in the prior art nucleic acid detection device, and to provide a nucleic acid extraction and detection device and a use method.

[0012] In order to achieve the above purpose, the following technical scheme is adopted in the present application:

[0013] A nucleic acid extraction and detection device comprising an extraction card box and an amplification tube connected to each other;

[0014] The extraction card box comprises a chamber main body, a rotary valve cavity is arranged in the chamber main body, sample chambers, lysis chambers, reaction chambers, elution chambers, gas chambers, waste liquid chambers and washing chambers are distributed on the periphery of the rotary valve cavity, and liquid transfer holes are formed in the bottoms of the sample chambers, the lysis chambers, the reaction chambers, the elution chambers, the gas chambers, the waste liquid chambers and the washing chambers;

[0015] A cover assembly is arranged on the chamber main body, and air holes corresponding to the rotary valve cavity, the sample chambers, the lysis chambers, the reaction chambers, the elution chambers, the gas chambers, the waste liquid chambers and the washing chambers are formed in the cover assembly;

[0016] A rotary valve is arranged in the rotary valve cavity, a rotating cover is sleeved on the rotary valve, the rotating cover is located in the cover assembly, the rotating cover rotates synchronously with the rotary valve, the rotating cover is provided with an air hole, and the air hole of the rotating cover can communicate with any air hole of the cover assembly when the rotary valve is rotated;

[0017] The rotating valve is internally hollow, and a proximal flow channel port and a distal flow channel port are arranged in communication with each other at the lower end of the rotating valve, and the distal flow channel port can be in communication with any one of the liquid transfer holes at the bottom of the chamber body when the rotating valve is rotated.

[0018] An amplification tube connecting port is arranged at one side of the chamber body, the amplification tube is installed in the amplification tube connecting port, and the amplification tube connecting port is in communication with the distal flow channel port and the air chamber respectively.

[0019] Further improvements of the present application are as follows:

[0020] The cover assembly comprises an inner cover and an outer cover connected in sequence in the axial direction.

[0021] A rotating cover and a sealing gasket are arranged in sequence from top to bottom between the inner cover and the outer cover, and air vents corresponding to the rotating valve cavity, the sample chamber, the lysis chamber, the reaction chamber, the elution chamber, the air chamber, the waste liquid chamber and the washing chamber are arranged on the outer cover, the sealing gasket and the inner cover.

[0022] A waterproof and breathable film is arranged at each air vent on the inner cover.

[0023] A toothed clamping groove is arranged at the upper end of the rotating valve, a toothed protrusion corresponding to the toothed clamping groove is arranged on the rotating cover, and the toothed protrusion is embedded into the toothed clamping groove.

[0024] A piston rod is further included, the piston rod penetrates through the outer cover, and the piston rod extends into the interior of the rotating valve.

[0025] The rotating valve comprises a plunger chamber, a disc-shaped valve body is arranged at the bottom of the plunger chamber, the plunger chamber is located in the interior of the rotating valve cavity, the disc-shaped valve body is located at the lower end of the rotating valve cavity, and the lower end of the rotating valve cavity is attached to the upper end of the disc-shaped valve body.

[0026] The proximal flow channel port is arranged at the center of the disc-shaped valve body, and the distal flow channel port is arranged at one side of the proximal flow channel port.

[0027] A boss is arranged at one side of the chamber body, and the amplification tube connecting port is arranged on the boss.

[0028] The boss and the shell of the chamber body are in an integral structure.

[0029] An air flow channel and a transfer flow channel are arranged in the interior of the boss.

[0030] One end of the transfer flow channel is in communication with the amplification transfer hole, and the other end is in communication with the amplification tube connecting port.

[0031] One end of the air flow channel is in communication with the air vent, and the other end is in communication with the amplification tube connecting port.

[0032] When the rotating valve is rotated, the distal flow channel port can be in communication with the amplification transfer hole.

[0033] The vent hole communicates with the air chamber.

[0034] A method for using a nucleic acid extraction and detection device, comprising the following steps:

[0035] Sample injection: inject the sample into the sample chamber, rotate the rotary valve to make the distal end of the flow channel communicate with the liquid transfer hole at the bottom of the sample chamber, and transfer the sample from the sample chamber to the reaction chamber through the rotary valve;

[0036] Nucleic acid lysis: rotate the rotary valve to make the distal end of the flow channel communicate with the liquid transfer hole at the bottom of the lysis chamber, transfer the lysis liquid in the lysis chamber to the reaction chamber through the rotary valve, close the rotary valve, rotate the cover synchronously with the rotary valve, at this time all the chambers are closed, mix the extraction card box, start nucleic acid adsorption, after nucleic acid adsorption is completed, remove the waste liquid through the rotary valve, make the distal end of the flow channel communicate with the liquid transfer hole at the bottom of the waste liquid chamber, and transfer the liquid to the waste liquid chamber;

[0037] Nucleic acid washing: rotate the rotary valve to transfer the washing liquid in the washing chamber to the reaction chamber for nucleic acid washing, and after washing is completed, transfer the liquid to the waste liquid chamber through the rotary valve;

[0038] Nucleic acid elution: rotate the rotary valve to transfer the elution liquid to the reaction chamber for nucleic acid elution;

[0039] Nucleic acid amplification: after elution is completed, transfer the liquid to the inside of the amplification tube through the rotary valve, and start the amplification reaction.

[0040] Compared with the prior art, the nucleic acid extraction and detection device has the following beneficial effects:

[0041] The nucleic acid extraction and detection device is disclosed, a plurality of communication holes are arranged, a rotary valve is used as a communication medium, communication between various functional chambers is realized, and then sample transfer is realized, the whole transfer process is completed in an internally closed environment, sample pollution is avoided, the cover is synchronously rotated with the rotary valve, the rotary cover is closed when the rotary valve is closed, the sealing performance of the mixing and reaction process chambers is ensured, the stability of the pre-stored reagent and the safety of the reaction process are ensured, the extraction card box and the amplification tube are packaged into one body, colorimetric method is used for naked eye identification after extraction is completed, sample transfer steps are reduced, pollution risk is reduced, and the whole process is completed through manual operation without power supply or other equipment, and the nucleic acid extraction and detection device can be used for family self-checking or in resource-limited areas or even in extreme environments without power supply.

[0042] Further, in the present application, a waterproof and breathable film is arranged at each vent hole on the inner cover to form a secondary pollution prevention mechanism, which can prevent reagent leakage and nucleic acid diffusion pollution when the rotary valve is opened, so that the present application is in a sealed state during the entire nucleic acid extraction and detection process, and can effectively prevent nucleic acid from causing pollution to personnel and the environment. BRIEF DESCRIPTION OF DRAWINGS

[0043] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0044] Figure 1 is a schematic diagram of the overall structure of the present application;

[0045] Figure 2 is an exploded view of the overall structure of the present application;

[0046] Figure 3 is a schematic diagram of the structure of the chamber body in the present application (wherein a is an overall structure diagram; b is an enlarged layout diagram of figure a);

[0047] Figure 4 is a front view of the chamber body structure in the present application;

[0048] Figure 5 is a structural diagram of the chamber body in the present application (wherein a is a top view of the chamber body; b is an A-A cross-sectional view of figure a; c is a B-B cross-sectional view of figure a);

[0049] Figure 6 is a front view of the structure of the rotary valve in the present application;

[0050] Figure 7 is a top view of the structure of the rotary valve in the present application (wherein a is a top view of the rotary valve; b is an A-A cross-sectional view of figure a);

[0051] Figure 8 is a top view of the present application after removing the top cover, rotating cover, sealing gasket, waterproof and breathable film and inner cover (wherein a is a top view; b is an A-A cross-sectional view of figure a; c is a B-B cross-sectional view of figure a; d is a C-C cross-sectional view of figure a);

[0052] Figure 9 is a schematic diagram of the sealing cover installed on the interface boss (wherein a is a structure diagram from view one; b is a structure diagram from view two);

[0053] Figure 10Figure 2 is a schematic view of the interface boss and the hand-held portion cooperating with the rotary valve (wherein a is a bottom view; b is an assembled isometric view; c is an assembled exploded view).

[0054] Wherein: 1 - outer cover, 2 - piston rod, 3 - rotating cover, 4 - sealing gasket, 5 - waterproof air permeable membrane, 6 - inner cover, 7 - chamber body, 8 - sealing piece, 9 - rotary valve, 10 - positioning snap ring, 11 - sealing cover, 12 - amplification tube, 13 - hand-held portion; 71 - sample chamber, 72 - lysis chamber, 73 - reaction chamber, 74 - elution chamber, 75 - gas chamber, 76 - third washing chamber, 77 - second washing chamber, 78 - first washing chamber, 79 - waste liquid chamber, 711 - sample chamber through hole, 721 - lysis chamber through hole, 731 - reaction chamber through hole, 741 - elution chamber through hole, 761 - third washing chamber through hole, 771 - second washing chamber through hole, 781 - first washing chamber through hole, 791 - waste liquid chamber through hole, 750 - boss, 751 - air vent, 752 - air vent flow channel, 753 - amplification tube connecting port, 754 - transfer flow channel, 755 - amplification transfer hole, 756 - interface boss, 757 - sealing cover mounting hole, 91 - plunger chamber, 92 - proximal end flow channel port, 93 - valve body transfer flow channel, 94 - distal end flow channel port, 95 - clamping groove. DETAILED DESCRIPTION

[0055] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. The components of the embodiments of the present application described and shown in the drawings herein can be arranged and designed in various different configurations.

[0056] Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed application, but only represents selected embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative labor are within the scope of protection of the present application.

[0057] It should be noted that: similar reference numerals and letters represent similar items in the following drawings, therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0058] In the description of the embodiments of the present application, it should be noted that if the terms "upper", "lower", "horizontal", "inner" and the like indicating the orientation or position relationship are based on the orientation or position relationship shown in the drawings, or the orientation or position relationship when the product of the present application is usually placed, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second" and the like are only used for differentiation and cannot be understood as indicating or implying relative importance.

[0059] In addition, if the term "horizontal" appears, it does not mean that the component must be absolutely horizontal, but can be slightly inclined. For example, "horizontal" only means that its direction is relatively more horizontal than "vertical", and does not mean that the structure must be completely horizontal, but can be slightly inclined.

[0060] In the description of the embodiments of the present application, it should be noted that unless otherwise explicitly specified and limited, if the terms "arrangement", "installation", "connection", "connection" appear, they should be understood in a broad sense, for example, they can be fixedly connected, or can be detachably connected, or integrally connected; can be mechanically connected, or can be electrically connected; can be directly connected, or can be indirectly connected through an intermediate medium; can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0061] The present application will be described in further detail below with reference to the accompanying drawings:

[0062] Referring to Figures 1 to 10 The embodiments of the present application disclose a nucleic acid extraction and detection device, which highly integrates a complete nucleic acid detection process in a single small device, improves the nucleic acid extraction efficiency and enhances the detection performance, increases the use scene to create conditions for popular application (including home scene), and adopts a closed system design to reduce the risk of cross contamination. The device comprises an extraction card box and an amplification tube packaged into one body. The extraction card box is composed of a plurality of chambers and a rotatable rotary valve. The opening and closing of the flow channel is controlled by rotating the rotary valve, and the liquid is driven by a plunger. The directional transfer of the liquid between the chambers is realized in a closed system, so as to realize the nucleic acid extraction based on the magnetic bead method. The extraction card box is connected to the amplification tube in a sealed manner to realize fluid transmission, and the amplification and detection of the nucleic acid are realized in the amplification tube.

[0063] Further, the chamber body 7 is provided with a vent hole for balancing the air pressure inside and outside the chamber to ensure smooth liquid transfer. A waterproof and breathable film 5 is arranged at the position of the vent hole, which allows gas to pass through but blocks liquid and biomolecules, so that the extraction cartridge is in a closed state during nucleic acid extraction. When the vent hole is opened, the waterproof and breathable film 5 effectively prevents reagent splashing and environmental pollution during liquid transfer, while avoiding the diffusion of nucleic acids to the external environment, ensuring the safety of the operation.

[0064] Further, the extraction cartridge is designed to be linked with the rotary valve 9 through the rotating cover 3, realizing dynamic sealing of the chamber. A rotating cover 3 is arranged above the vent hole of the chamber, and the position state of the rotating cover 3 is synchronously controlled by rotating the rotary valve 9 to realize the opening and closing control of the vent hole. The rotating cover 3 is provided with an air hole, and when the rotary valve 9 is opened and connected with the chamber, the air hole of the rotating cover 3 is aligned with the vent hole above the corresponding chamber, the chamber is connected with the atmosphere, and the liquid is smoothly transferred; when the rotary valve 9 is closed and connected with any chamber, the air hole of the rotating cover 3 is misaligned with the air hole of any chamber, and the vent holes of all chambers are in a completely closed state, so that the reagent cannot be transferred or leaked, and is isolated from the outside. The rotating cover 3 is clamped with the rotary valve 9, and the rotating cover 3 is linked and controlled when the rotary valve 9 is actively rotated, and the rotating cover 3 is opened when the rotary valve 9 is opened. During transportation, storage and mixing, the rotary valve 9 is in a closed state, and the reagent chamber is mechanically sealed by the rotating cover 3 and protected by the waterproof and breathable film 5, forming a double leakage prevention mechanism to ensure the stability of the reagent and prevent aerosol pollution during the extraction process. The present application realizes the goals of full-process leakage prevention and pollution prevention while ensuring the flexibility of operation, and is especially suitable for high-sensitivity and high-specificity nucleic acid detection scenarios.

[0065] In the extraction process, the rotary valve 9 is oriented to open, and the reagent can be transferred to the plunger chamber by the suction action of the piston rod 2 in the plunger chamber, and the rotary valve 9 is oriented to connect the plunger chamber with the reaction chamber 73, so that the reagent can be transferred to the reaction chamber 73. After the rotary valve 9 is closed, the closed extraction cartridge is manually shaken or inverted to realize the full mixing of the reaction liquid and the magnetic beads, and after the reaction is completed, the waste liquid is precisely introduced into the waste liquid chamber 79 for centralized storage to avoid cross contamination. Sequential operation can complete the lysis, washing and elution reactions.

[0066] The LAMP isothermal amplification reagent is pre-embedded in the amplification tube 12, and the amplification tube is separately sealed and stored before the experiment. The amplification tube 12 needs to be installed on the extraction box during the experiment. After the nucleic acid extraction is completed, the rotary valve 9 is oriented to open, and the sample extraction liquid is quantitatively transferred to the amplification tube 12 through the sealed flow channel, so as to reach the scale line on the amplification tube; the amplification is carried out by providing a constant reaction temperature from the outside, and the detection is completed by naked eye identification by colorimetric method.

[0067] Further, the extraction cartridge can be manually operated throughout the process without the need for automation equipment. The rotation valve 9 and the piston rod 2 can be manually operated. The mixing method can be manual shaking or overturning mixing (no leakage occurs under the action of the double leakage prevention mechanism). After nucleic acid extraction is completed, a matching heating bag can be used to provide temperature conditions to start the amplification reaction and to be identified by the naked eye, making it possible to perform nucleic acid detection in extreme environments such as the field or disaster area without power supply. Moreover, the nucleic acid extraction cartridge can be made of plastic by injection molding. After the cartridge is processed, the reagents are pre-packaged uniformly. The heating device uses an iron powder self-heating bag, and the overall cost of nucleic acid extraction and detection is low.

[0068] In addition, when there is no nucleic acid extractor in the laboratory or there is a high requirement for nucleic acid extraction pollution protection, the nucleic acid extraction function in the device can be used alone. A laboratory standard centrifugal tube can be used instead of the amplification tube to be installed on the extraction cartridge for collecting the sample extraction liquid. After nucleic acid extraction, the centrifugal tube can be removed to set the purpose of the sample extraction liquid, for example, to set the target detection of the sample by the self-primer probe. After the extraction of the device is completed, amplification can be performed according to the needs and is suitable for any PCR analyzer or fluorescence detection system to enhance the use scenario of nucleic acid extraction and detection.

[0069] Embodiment 1

[0070] The nucleic acid extraction and detection device disclosed in the embodiment comprises an extraction cartridge and an amplification tube 12 connected to each other.

[0071] The extraction cartridge comprises a chamber body 7, a rotary valve cavity is arranged in the chamber body 7, and sample chambers 71, lysis chambers 72, reaction chambers 73, elution chambers 74, gas chambers 75, and washing chambers are distributed on the periphery of the rotary valve cavity. Liquid transfer holes are formed in the bottoms of the sample chambers 71, the lysis chambers 72, the reaction chambers 73, the elution chambers 74, the gas chambers 75, the waste liquid chambers 79, and the washing chambers to realize liquid transfer functions. Three washing chambers are formed, which are a first washing chamber 78, a second washing chamber 77, and a third washing chamber 76. The bottom of the sample chamber 71 is provided with a sample chamber through hole 711, the bottom of the lysis chamber 72 is provided with a lysis chamber through hole 721, the bottom of the reaction chamber 73 is provided with a reaction chamber through hole 731, the bottom of the elution chamber 74 is provided with an elution chamber through hole 741, the bottom of the gas chamber 75 is provided with a gas through hole 751, the bottom of the first washing chamber 78 is provided with a first washing chamber through hole 781, the bottom of the second washing chamber 77 is provided with a second washing chamber through hole 771, and the bottom of the third washing chamber 76 is provided with a third washing chamber through hole 761.

[0072] A cover assembly is arranged on the chamber body 7, and air holes corresponding to the rotary valve cavity, the sample chambers 71, the lysis chambers 72, the reaction chambers 73, the elution chambers 74, the gas chambers 75, the waste liquid chambers 79, and the washing chambers are formed in the cover assembly.

[0073] The rotating valve 9 is arranged inside the rotating valve cavity, and a rotating cover 3 is sleeved on the rotating valve 9, the rotating cover 3 is located inside the cover body assembly, the rotating cover 3 rotates synchronously with the rotating valve 9, the rotating cover 3 is provided with a vent hole, and when the rotating valve 9 rotates, the vent hole on the rotating cover 3 can communicate with any vent hole on the cover body assembly;

[0074] The rotating valve 9 is hollow inside, the proximal flow channel port 92 and the distal flow channel port 94 that are in communication with each other are arranged at the lower end of the rotating valve 9, and when the rotating valve 9 rotates, the distal flow channel port 94 can communicate with any one of the liquid transfer holes at the bottom of the chamber main body 7.

[0075] The chamber main body 7 is provided with an amplification tube connecting port 753 on one side, the amplification tube 12 is installed at the amplification tube connecting port 753, the distal flow channel port 94 is in communication with the amplification tube connecting port 753, and the air chamber 75 is in communication with the amplification tube connecting port 753.

[0076] Further, the lysis chamber 72, the first washing chamber 78, the second washing chamber 77, the third washing chamber 76 and the elution chamber 74 are respectively pre-packaged with a lysis solution, a first washing solution, a second washing solution, a third washing solution and an elution solution. Functionalized magnetic beads are pre-embedded in the reaction chamber 73, the surface of the functionalized magnetic beads is modified with specific ligands, the specific ligands can efficiently adsorb nucleic acids during the extraction process, and pure nucleic acids can be obtained after washing and elution. In the liquid transfer stage, the magnetic beads are adsorbed and fixed on the outer wall of the chamber by an external magnet, so that the directional interception and liquid separation of the magnetic beads are realized.

[0077] In the present application, the reagents are pre-packaged in the reagent cavity, which improves the consistency of the reagents, reduces the reagent preparation and addition steps, and reduces the operation error.

[0078] Further, the lower surface of the chamber main body 7 is bonded with a sealing gasket 8, and the sealing gasket 8 is provided with a through hole corresponding to the through hole at the bottom of the chamber, so as to ensure the sealing property.

[0079] Further, the rotating valve cavity is a cylindrical cavity arranged in the center of the chamber main body 7, used for accommodating the rotating valve 9, ensuring stable operation and realizing switching and control of the fluid passage. The positioning snap ring 10 is sleeved outside the rotating valve 9 and installed below the rotating valve 9, and is fixed by cooperating with the chamber main body 7, so as to exert an axial compression force on the rotating valve 9, ensure the close contact between the rotating valve 9 and the chamber main body 7, maintain the sealing property and prevent fluid leakage. The lower surface of the chamber main body 7 is circumferentially provided with numbers and indication marks for indicating the position of the rotating valve 9.

[0080] The chamber main body 7 of the extraction cartridge is sealingly and adhesively connected with the inner cover 6 above. The inner cover 6 is provided with a vent hole above each chamber, and the vent hole can communicate with the atmosphere to balance the chamber air pressure; the inner cover 6 is provided with a flange which can be clamped with the outer cover 1.

[0081] The inner cover 6 is bonded with a sealing gasket 4, the material of the sealing gasket 4 is generally silicone rubber, and the sealing gasket 4 is provided with a through hole corresponding to the air hole of the inner cover 6. At each air hole position, a waterproof and breathable film 5 is bonded between the inner cover 6 and the sealing gasket 4. The main function of the waterproof and breathable film 5 is to effectively prevent the splashing of reagents during liquid transfer when the air hole is opened, thereby preventing environmental pollution, and also preventing reagents from leaking or nucleic acid from diffusing to the external environment.

[0082] The upper half of the rotary valve 9 is integrated with the plunger chamber 91, and the lower half is a disc-shaped valve body. The disc-shaped valve body of the lower half is located below the reagent cavity main body 7, and the upper surface thereof is in close contact with the lower surface of the reagent cavity main body 7. The valve body bottom is provided with a valve body transfer flow channel 93, which communicates the proximal flow channel port 92 and the distal flow channel port 94. The proximal flow channel port 92 is located at the center position and is in communication with the inside of the plunger chamber 91. The distal flow channel port 94 can be connected with the bottom through hole of other chambers. The rotary valve 9 can control the opening and closing of the bottom through hole of the chamber. With the rotation of the rotary valve 9, the distal flow channel port 94 communicates with one of the chambers to control the flow of liquid between different chambers. When the distal flow channel port 94 avoids the bottom through hole of the chamber, the sealing of the chamber can be achieved. The upper part of the rotary valve 9 is a hollow plunger chamber 91, and a piston rod 2 is arranged in the plunger chamber 91. When the distal flow channel port 94 of the rotary valve 9 communicates with other chambers, the reagent can be transferred from the corresponding original chamber to the plunger chamber 91 by lifting the piston rod 2. When the rotary valve 9 is rotated to another position, the reagent in the plunger chamber 91 can be transferred to the target chamber by pushing down the piston rod 2. Thus, the liquid transfer between any chambers can be realized through the rotary valve 9 and the piston device.

[0083] An indication mark is arranged at the upper end of the rotary valve 9 for identifying the opening direction of the rotary valve 9. When the indication mark of the rotary valve 9 points to the corresponding digital mark on the chamber main body 7, the rotary valve 9 opens the corresponding chamber. When manually extracting, the rotary valve is rotated to the specified position according to the operation process, which reduces the operation complexity and enables non-professionals to complete the manual operation.

[0084] Referring to Figure 10 The bottom of the rotary valve 9 is provided with a clamping groove 95, and the hand holding part 13 can be clamped in the clamping groove 95. When the hand holding part 13 is manually rotated, the rotary valve 9 can be controlled to rotate synchronously, thereby improving the stability of the device during operation.

[0085] The rotating cover 3 is located above the inner cover 6, and the outer cover 1 is installed on the rotating cover 3. The outer cover 1 presses the rotating cover 3 and is tightly fixed on the inner cover 6, thereby ensuring the close contact between the inner cover 6 and the sealing gasket 4 on the rotating cover 3, and thus ensuring the sealing property of the system.

[0086] The rotating cover 3 is provided with a toothed protrusion which is clamped with the toothed clamping groove 96 on the upper end edge of the rotating valve 9, so that the rotating cover is synchronously rotated when the rotating valve 9 is rotated; the rotating cover 3 is provided with an air hole, when the rotating cover 3 is rotated with the rotating valve, the air hole on the rotating cover 3 is overlapped with the air hole on the inner cover 6, so that the corresponding chamber of the air hole is communicated with the atmospheric pressure, so that the chamber can smoothly realize the liquid transfer, when the air hole on the rotating cover 3 is not overlapped with any air hole on the inner cover 6, all the chambers are in a closed state, any reagent in all the chambers cannot be transferred or leaked, and cannot be communicated with the atmosphere to avoid pollution of the environment. The secondary pollution prevention is realized in a mechanical sealing manner, and the leakage caused by failure of the waterproof and air permeable film is avoided.

[0087] Further, the chamber main body 7 also integrates a part connected with the amplification tube 12, including a boss 750 for installing the amplification tube 12 and an air chamber 75 communicated with the atmosphere. Before use, the sealing cover 11 is installed on the circular interface boss 756 of the extraction card box to ensure the sealing of the extraction card box, as shown in Figure 9 After the sealing cover 11 is removed, the amplification tube 12 can be installed on the interface boss 756 of the boss 750. The boss 750 is internally provided with an air passage 752 and a transfer passage 754, which are distributed up and down. One end of the transfer passage 754 is communicated with the amplification transfer hole 755, and the other end is communicated with the amplification tube connecting port 753; one end of the air passage 752 is communicated with the air hole 751, and the other end is communicated with the amplification tube connecting port 753, and the air hole 751 is communicated with the air chamber 75; when the rotating valve 9 is rotated, the distal end passage port 94 can be communicated with the amplification transfer hole 755, so as to realize the directional transmission and control of the fluid.

[0088] The sealing cover 11 is made of rubber, and the tail of the sealing cover 11 is connected to the boss 750 through the sealing cover mounting hole 757. When the sealing cover 11 is installed in the sealing cover mounting hole 757, it can be rotated around the sealing cover mounting hole 757 to adjust the position of the sealing cover 11, so as to avoid interference with the installation of the amplification tube 12.

[0089] The colorimetric LAMP premix liquid and the reaction primer are pre-embedded in the amplification tube 12. When the experiment is carried out, the sealing cover 11 on the boss of the extraction card box is removed, and the amplification tube 12 is installed on the extraction box, as shown in Figure 10 After the nucleic acid extraction is completed, the rotating valve 9 is directionally opened, the sample extraction liquid is transferred to the amplification tube 12 to the specified scale line, and then stopped; the amplification tube is removed, the amplification tube cover is sealed, and then the amplification tube 12 is placed in a self-heating heating bag for heating. The LAMP reaction is started by providing a reaction temperature of 50-60℃ by the heating bag, and the reaction liquid in the amplification tube is changed from pink to yellow after 30-60min according to different targets by colorimetric method, which is positive. In the negative test, the color remains pink.

[0090] Example 2

[0091] The present embodiment discloses a method for using a nucleic acid extraction and detection device, comprising the following steps:

[0092] Before use, remove the sealing cover 11 and install the amplification tube 12 on the interface boss 756.

[0093] Step 1, sample injection:

[0094] Rotate the rotary valve 9 to make the distal end flow channel opening 94 communicate with the sample chamber through hole 711, as shown in Figure 8 b; pull the piston rod 2 upward to transfer the sample from the sample chamber 71 to the plunger chamber 91; rotate the rotary valve 9 to make the distal end flow channel opening 94 communicate with the reaction chamber through hole 731, as shown in Figure 8 c; press the piston rod 2 downward to transfer the reagent in the plunger chamber 91 to the reaction chamber 73.

[0095] Step 2, nucleic acid lysis:

[0096] Rotate the rotary valve 9 to open the lysis chamber 72, as shown in Figure 8 d; pull the piston rod 2 upward to transfer the lysis solution from the lysis chamber 72 to the plunger chamber 91; rotate the rotary valve 9 to open the reaction chamber, as shown in Figure 8 c; press the piston rod 2 downward to transfer the lysis solution from the plunger chamber 91 to the reaction chamber 73; close the rotary valve 9 and control the rotary cover 3 to close simultaneously, shake the extraction card box to realize mixing, promote the lysis of cells and the mixing of magnetic beads, and promote the adsorption of magnetic beads to nucleic acid; after the end, use an external magnet to adsorb and fix the magnetic beads on the outer wall of the chamber, open the rotary valve 9 to make the distal end flow channel opening 94 communicate with the reaction chamber through hole 731, pull the piston rod 2 upward to transfer the remaining liquid from the reaction chamber 73 to the plunger chamber 91, and rotate the rotary valve 9 to make the distal end flow channel opening 94 communicate with the waste liquid chamber through hole 791 to transfer the remaining liquid to the waste liquid chamber 79;

[0097] Step 3, nucleic acid washing:

[0098] Rotate the rotary valve 9 to make the distal end flow channel opening 94 communicate with the first washing chamber through hole 781, pull the piston rod 2 upward to transfer the washing liquid from the first washing chamber 78 to the plunger chamber 91; rotate the rotary valve 9 to make the distal end flow channel opening 94 communicate with the reaction chamber through hole 731, press the piston rod 2 downward to transfer the washing liquid from the plunger chamber 91 to the reaction chamber 73; close the rotary valve 9 and control the rotary cover 3 to close simultaneously, shake the extraction card box to realize the uniform mixing of magnetic beads and washing liquid, and accelerate the washing of impurities on the magnetic beads; use an external magnet to adsorb and fix the magnetic beads on the outer wall of the chamber, open the rotary valve 9 to make the distal end flow channel opening 94 communicate with the reaction chamber through hole 731, pull the piston rod 2 upward to transfer the remaining liquid from the reaction chamber 73 to the plunger chamber 91, and rotate the rotary valve 9 to make the distal end flow channel opening 94 communicate with the waste liquid chamber through hole 791 to transfer the remaining liquid to the waste liquid chamber 79;

[0099] The washing step can be repeated two to three times according to sample requirements.

[0100] Step 4, nucleic acid elution:

[0101] The rotary valve 9 is rotated to make the distal flow channel port 94 communicate with the elution chamber through hole 741, the piston rod 2 is pulled upward, and the eluent is transferred from the elution chamber 74 to the plunger chamber 91; the rotary valve 9 is rotated to make the distal flow channel port 94 communicate with the reaction chamber through hole 731, the piston rod 2 is pressed downward, and the eluent is transferred from the plunger chamber 91 to the reaction chamber; the rotary valve 9 is closed, and the rotating cover 3 is closed in linkage control, the extraction card box is shaken to mix the magnetic beads and the eluent, and the nucleic acid on the magnetic beads is eluted; the external magnet is used to adsorb and fix the magnetic beads on the outer wall of the chamber, the rotary valve 9 is opened to make the distal flow channel port 94 communicate with the reaction chamber through hole 731, and the eluent is transferred to the plunger chamber 91.

[0102] Step 5, nucleic acid amplification:

[0103] The rotary valve 9 is rotated to make the distal flow channel port 94 communicate with the amplification transfer hole 755, and the sample extraction liquid in the plunger chamber 91 is quantitatively transferred to a specified scale in the amplification tube 12, and amplification and detection reagents are pre-embedded in the amplification tube 12; the rotary valve 9 is rotated to close the corresponding air hole on the upper end of the air chamber 75 through the rotating cover 3; the amplification tube 12 is removed and covered with an amplification tube cover, the amplification tube is placed in a heating bag, the heating bag is started, and after reaching the specified temperature, the reaction is automatically started, and the reaction is judged by colorimetry.

[0104] Through the above steps, the nucleic acid extraction and amplification integrated card box of the application can efficiently and conveniently complete the whole process operation of nucleic acid extraction and detection without automatic equipment, is suitable for various sample types and detection requirements, and increases the flexibility and applicability of the nucleic acid extraction card box.

[0105] In addition, after manual extraction is completed, a clean centrifugal tube can be installed on the interface boss 756 to transfer the sample extraction liquid to the clean centrifugal tube for standby, and under laboratory conditions, a nucleic acid detection reaction liquid can be configured by oneself and a fluorescence PCR instrument is used to complete detection of multiple targets in multiple methodologies. The application can extract and purify a large sample of 400-1000 μL to obtain 300 μL of pure nucleic acid, which can be used for detection of multiple targets.

[0106] The rotating cover is synchronously rotated with the rotating valve in the application, and the rotating cover is closed when the rotating valve is closed, so as to ensure the mixing and the sealing of the reaction process chamber, and to ensure the stability of the pre-stored reagent and the safety of the reaction process. In the application, the air vent is sealed by a waterproof air-permeable film to form a secondary anti-pollution mechanism, and the reagent leakage and nucleic acid diffusion pollution can be prevented when the rotating valve is opened. Therefore, the application is in a sealed state in the whole nucleic acid extraction and detection process, and can effectively prevent the nucleic acid from causing pollution to personnel and environment.

[0107] In the application, the amplification tube can be configured with an amplification reaction liquid according to the needs to realize the detection of target nucleic acid, and the amplification tube can be detached and compatible with other general PCR instruments, so as to avoid the equipment binding problem and enhance the flexibility of the laboratory.

[0108] The application can extract and purify 400-1000 μL large samples to obtain 300 μL large amount of pure nucleic acid, and can meet the needs of subsequent multiple detections through one-time extraction. Manual operation can be used to complete the extraction under limited conditions to increase the use scenarios of the device, and the cost can be flexibly selected.

[0109] The extraction card box and the amplification tube are packaged into one body in the application, and the extraction is identified by colorimetric method with naked eyes after the extraction is completed, so as to reduce the sample transfer steps and reduce the pollution risk. The whole process is completed by manual operation without power supply or other equipment, and can be used for home self-detection or in resource-limited areas or even in extreme environments without power supply.

[0110] The above is only the preferred embodiment of the application, and is not used to limit the application. For those skilled in the art, the application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A nucleic acid extraction and detection device, characterized by, The extraction cartridge and the amplification tube (12) are connected; The extraction cartridge comprises a chamber body (7) in which a rotary valve cavity is arranged, and the rotary valve cavity is peripherally distributed with a sample chamber (71), a lysis chamber (72), a reaction chamber (73), an elution chamber (74), an air chamber (75), a waste liquid chamber (79), and a washing chamber, and the bottoms of the sample chamber (71), the lysis chamber (72), the reaction chamber (73), the elution chamber (74), the air chamber (75), the waste liquid chamber (79), and the washing chamber are each provided with a liquid transfer hole; A cover assembly is arranged on the chamber body (7), and the cover assembly is provided with air holes corresponding to the rotary valve cavity, the sample chamber (71), the lysis chamber (72), the reaction chamber (73), the elution chamber (74), the air chamber (75), the waste liquid chamber (79), and the washing chamber. A rotary valve (9) is arranged in the rotary valve cavity, and a rotating cover (3) is arranged on the rotary valve (9), the rotating cover (3) is located in the cover assembly, the rotating cover (3) rotates synchronously with the rotary valve (9), the rotating cover (3) is provided with an air hole, and the air hole of the rotating cover (3) can communicate with any air hole of the cover assembly when the rotary valve (9) rotates. The rotary valve (9) is a hollow structure, and a proximal flow channel opening (92) and a distal flow channel opening (94) are arranged at the lower end of the rotary valve (9) and are in communication with each other, and the distal flow channel opening (94) can communicate with any liquid transfer hole at the bottom of the chamber body (7) when the rotary valve (9) rotates. An amplification tube connecting port (753) is arranged on one side of the chamber body (7), the amplification tube (12) is arranged in the amplification tube connecting port, and the amplification tube connecting port (753) is in communication with the distal flow channel opening (94) and the air chamber (75), respectively.

2. The nucleic acid extraction and detection device according to claim 1, wherein The cover assembly comprises an inner cover (6) and an outer cover (1) which are connected in sequence in the axial direction. The rotating cover (3) and a sealing gasket (4) are arranged in sequence from top to bottom between the inner cover (6) and the outer cover (1), and the outer cover (1), the sealing gasket (4), and the inner cover (6) are each provided with air holes corresponding to the rotary valve cavity, the sample chamber (71), the lysis chamber (72), the reaction chamber (73), the elution chamber (74), the air chamber (75), the waste liquid chamber (79), and the washing chamber.

3. The nucleic acid extraction and detection device according to claim 2, wherein A waterproof and breathable film (5) is arranged at each air hole of the inner cover (6).

4. The nucleic acid extraction and detection device according to claim 2, wherein A toothed clamping groove (96) is arranged at the upper end of the rotary valve (9), a toothed protrusion corresponding to the toothed clamping groove (96) is arranged on the rotating cover (3), and the toothed protrusion is embedded into the toothed clamping groove (96).

5. The nucleic acid extraction and detection device according to claim 2, wherein A piston rod (2) is further arranged, the piston rod (2) penetrates through the outer cover (1), and the piston rod (2) extends into the rotary valve (9).

6. The nucleic acid extraction and detection device of claim 1, wherein The rotary valve (9) comprises a plunger chamber (91), a disc-shaped valve body is arranged at the bottom of the plunger chamber (91), the plunger chamber (91) is located in the rotary valve cavity, the disc-shaped valve body is located at the lower end of the rotary valve cavity, and the lower end of the rotary valve cavity is attached to the upper end of the disc-shaped valve body. The proximal flow channel opening (92) is arranged at the center of the disc-shaped valve body, and the distal flow channel opening (94) is arranged on one side of the proximal flow channel opening (92) in a spaced manner.

7. The nucleic acid extraction and detection device according to claim 1, wherein The chamber body (7) is provided with a boss (750) on one side, and the amplification tube connecting port (753) is arranged on the boss (750).

8. The nucleic acid extraction and detection device according to claim 7, wherein The boss (750) is in an integral structure with the shell of the chamber body (7).

9. The nucleic acid extraction and detection device according to claim 7, wherein The boss (750) is internally provided with a ventilation flow channel (752) and a transfer flow channel (754). One end of the transfer flow channel (754) is communicated with the amplification transfer hole (755), and the other end is communicated with the amplification tube connecting port (753). One end of the ventilation flow channel (752) is communicated with the ventilation hole (751), and the other end is communicated with the amplification tube connecting port (753). When the rotary valve (9) rotates, the distal end flow channel port (94) can be communicated with the amplification transfer hole (755). The ventilation hole (751) is communicated with the air chamber (75).

10. A method of using a nucleic acid extraction detection device, the method comprising: The method comprises the following steps: Sample injection: inject the sample into the sample chamber (71), rotate the rotary valve (9), and make the distal end flow channel port (94) communicated with the liquid transfer hole at the bottom of the sample chamber (71), and then transfer the sample from the sample chamber (71) to the reaction chamber (73) through the rotary valve (9); Nucleic acid lysis: rotate the rotary valve (9), and make the distal end flow channel port (94) communicated with the liquid transfer hole at the bottom of the lysis chamber (72), and then transfer the lysis liquid in the lysis chamber (72) to the reaction chamber (73) through the rotary valve (9), close the rotary valve (9), rotate the cover (3) synchronously with the rotary valve (9), at this time, all chambers are in a closed state, mix the extraction card box, start nucleic acid adsorption, after the nucleic acid adsorption is completed, remove the waste liquid through the rotary valve (9), make the distal end flow channel port (94) communicated with the liquid transfer hole at the bottom of the waste liquid chamber (79), and then transfer the liquid to the waste liquid chamber (79); Nucleic acid washing: rotate the rotary valve (9), and then transfer the washing liquid in the washing chamber to the reaction chamber (73) through the rotary valve (9) to wash the nucleic acid, and then transfer the liquid to the waste liquid chamber (79) through the rotary valve (9) after the washing is completed; Nucleic acid elution: rotate the rotary valve (9), and then transfer the elution liquid to the reaction chamber (73) through the rotary valve (9) to elute the nucleic acid; Nucleic acid amplification: after the elution is completed, transfer the liquid to the inside of the amplification tube (12) through the rotary valve (9), and start the amplification reaction. Nucleic acid amplification: after the elution is completed, transfer the liquid to the inside of the amplification tube (12) through the rotary valve (9), and start the amplification reaction.

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