Integrated pathogen nucleic acid detection chip and nucleic acid detector having the same

Abstract

The present invention provides an integrated pathogen nucleic acid detection chip and a nucleic acid detector having the same. The detection chip includes: a housing, in which a plurality of tube valves each sealed are installed, and a sample tube pre-filled with a nucleic acid lysis solution is installed outside the housing; a flow channel plate, in which flow channels are configured, a reconstitution chamber and a reaction chamber are configured, and a nucleic acid capture filter paper is provided in the reaction chamber; the content in the sample tube can be controlled to enter the reaction chamber through the flow channels, and nucleic acid capture is achieved at the nucleic acid capture filter paper; the content in the cleaning solution tube can be controlled to enter the reaction chamber through the flow channels to clean the captured nucleic acid; the content in the reaction solution tube can be controlled to enter the reconstitution chamber and enter the reaction chamber through the flow channels under the control of a switching valve; the content in the reaction chamber can be controlled to be stored in the waste liquid pool tube. The present invention realizes the integrated design of nucleic acid sample pretreatment, nucleic acid lysis, nucleic acid capture, nucleic acid cleaning, and nucleic acid amplification.

Description

Technical Field

[0001] The present invention belongs to the technical field of digital nucleic acid detection chip design, and particularly relates to an integrated pathogen nucleic acid detection chip and a nucleic acid detector having the same. Background Art

[0002] Nucleic acid detection generally includes three processes: sample pretreatment, nucleic acid extraction, and nucleic acid amplification. Traditional nucleic acid detection needs to be carried out in a central laboratory. Generally, chemical lysis is used to release nucleic acids, a magnetic bead method nucleic acid extractor is used to extract nucleic acids, a certain amount of eluate nucleic acid is taken out after extraction, and then reaction reagents and nucleic acids are manually prepared and mixed in a PCR instrument for amplification, and PCR amplification often takes 1 hour. From the above traditional nucleic acid detection process, the existing technologies have the following disadvantages: the process is cumbersome and time-consuming, which is likely to cause nucleic acid loss; the required equipment is precise and there are many types; often professional trained personnel are needed for operation; the detection location is often in the central laboratory and separate rooms are required for operation. This results in the inability of conventional detection methods to give nucleic acid detection results in a timely, effective, and rapid manner. In addition, due to multiple processes and long time consumption, traditional detection methods have the risk of nucleic acid degradation, especially for samples with low virus copy numbers, which may lead to the inability to provide ultra-high sensitive detection results, and thus false negative results may occur, so multiple rounds of detection are often required to detect positive. In addition, the low detection sensitivity of conventional methods also makes the current methods inapplicable to nucleic acid detection in some low-copy detection scenarios, such as for air aerosol, object surface, etc. Conventional methods cannot meet the detection requirements of their low virus copy numbers.

[0003] After nearly 20 years of development, microfluidic chips have begun to be applied and promoted on a certain scale in the industrial field. It uses structures such as microchannels, micropumps, and microvalves to concentrate the biochemical detection process of a traditional laboratory in a tiny device. Especially for the sample preparation, nucleic acid extraction, and nucleic acid amplification processes of nucleic acid detection, microfluidic chips have unique advantages and can achieve fully integrated nucleic acid detection. In addition, in recent years, in addition to polymerase chain reaction (PCR), a variety of isothermal amplification methods have emerged. Compared with the temperature-variable amplification of PCR, isothermal amplification can make the detection instrument supporting the microfluidic chip more simple, without the use of precise temperature control cycles and complex optical detection modules, which can further reduce the complexity and cost of the supporting instrument and is conducive to promoting the further popularization of microfluidic nucleic acid detection equipment and its application in on-site detection.

[0004] However, although there are existing microfluidic chip technologies on the market currently, there are no devices and systems that can achieve the full process of integrated nucleic acid sample pretreatment, nucleic acid capture, nucleic acid amplification, and nucleic acid detection, and can provide fast and highly sensitive detection capabilities. Many systems cannot store reagents for a long time during long-distance transportation and thus cannot be actually used. SUMMARY OF THE INVENTION

[0005] Therefore, the technical problem to be solved by the present invention is to provide an integrated pathogen nucleic acid detection chip and a nucleic acid detector having the same, so as to realize fully integrated, ultra-fast and ultra-sensitive pathogen nucleic acid detection.

[0006] To solve the above problems, the present invention provides an integrated pathogen nucleic acid detection chip, comprising:

[0007] A housing, in which a cleaning liquid tube, a reaction liquid tube, a switching valve and a waste liquid pool tube are respectively sealed, and a sample tube pre-filled with nucleic acid lysis solution is installed outside the housing;

[0008] A flow channel plate, connected to the bottom of the housing, in which a flow channel is constructed, and a reconstitution chamber and a reaction chamber are also constructed on the flow channel plate, and a nucleic acid capture filter paper is provided in the reaction chamber;

[0009] The content in the sample tube can be controlled to enter the reaction chamber through the flow channel, and nucleic acid capture is achieved at the nucleic acid capture filter paper;

[0010] The content in the cleaning liquid tube can be controlled to enter the reaction chamber through the flow channel to wash the captured nucleic acid;

[0011] The content in the reaction liquid tube can be controlled to enter the reconstitution chamber and enter the reaction chamber through the flow channel under the control of the switching valve;

[0012] The content in the reaction chamber can be controlled to be stored in the waste liquid pool tube.

[0013] In some embodiments, a plurality of hollow top pins are further provided on the flow channel plate. One hollow top pin is provided at each position corresponding to the cleaning liquid tube, the reaction liquid tube and the waste liquid pool tube of the flow channel, and two hollow top pins are provided at the position corresponding to the switching valve of the flow channel. Moreover, lower sealing rubber plugs are provided at the bottoms of the cleaning liquid tube, the reaction liquid tube, the waste liquid pool tube and the switching valve. The lower sealing rubber plug has a plugging position for wrapping and sealing the hollow top pin and a communicating position for the hollow top pin to penetrate out so that the corresponding tube valve communicates with the flow channel or the chamber.

[0014] In some embodiments, the switching valve includes a valve body, an inlet chamber and an outlet chamber are constructed in the valve body, the inlet chamber and the outlet chamber are communicated through an intermediate hole, and the two hollow top pins corresponding to the switching valve are respectively an inlet top pin corresponding to the inlet chamber and an outlet top pin corresponding to the outlet chamber.

[0015] In some embodiments, a switching valve ejector rod is provided at the top of the valve body.

[0016] In some embodiments, upper sealing rubber plugs are further provided in the cleaning liquid pipe, reaction liquid pipe, waste liquid tank pipe, and switching valve. The upper sealing rubber plugs are located in the upper region of the lower sealing rubber plugs and can be controlled to move closer to the lower sealing rubber plugs.

[0017] In some embodiments, a hard layer is provided on the side of the upper sealing rubber plug facing away from the lower sealing rubber plug.

[0018] In some embodiments, a retaining ring is further provided in the reaction chamber. The retaining ring is used to fix the nucleic acid capture filter paper, and the retaining ring is a C-ring structure.

[0019] In some embodiments, a pipe rack is disposed in the housing, and the cleaning liquid pipe, reaction liquid pipe, switching valve, and waste liquid tank pipe are placed in the pipe rack.

[0020] In some embodiments, the top opening of the housing is covered with a cover plate.

[0021] The present invention further provides a nucleic acid detector, including a push rod. The push rod is used in cooperation with the above integrated pathogen nucleic acid detection chip. When the integrated pathogen nucleic acid detection chip includes an upper sealing rubber plug and a hollow top needle, the push rod can apply a force to the upper sealing rubber plug to cause the content in the corresponding pipe valve to flow out or cause the upper sealing rubber plug to wrap and block the corresponding hollow top needle.

[0022] An integrated pathogen nucleic acid detection chip and a nucleic acid detector having the same provided by the present invention realize an integrated design of nucleic acid sample pretreatment, nucleic acid lysis, nucleic acid capture, nucleic acid cleaning, and nucleic acid amplification by controlling the outflow and storage of the content in the corresponding pipe cavities in sequence according to the nucleic acid extraction process through the cleaning liquid pipe, reaction liquid pipe, waste liquid tank pipe, sample pipe, reconstitution chamber, flow channel, and switching valve. This is conducive to more rapid detection of pathogen nucleic acid and has higher sensitivity at the same time. The reconstitution chamber integrally provided in the flow channel plate enables the chip of the present invention to be applicable to long-term reagent storage along the way, and the application scenarios of the chip are more diverse. Description of the Drawings

[0023] Figure 1 is a schematic exploded view (partially exploded) of the integrated pathogen nucleic acid detection chip according to an embodiment of the present invention;

[0024] Figure 2 is another schematic exploded view (partially exploded) of the integrated pathogen nucleic acid detection chip according to an embodiment of the present invention;

[0025] Figure 3 is Figure 1 the internal structure schematic diagram of the flow channel plate in

[0026] Figure 4 is Figure 1 the schematic diagram of the disassembled structure of the sample tube in

[0027] Figure 5 is Figure 2 the schematic diagram of the disassembled structure of the waste liquid tank tube in

[0028] Figure 6 is Figure 2 the schematic diagram (partial) of the structure inside the switching valve in

[0029] Figure 7 is Figure 2 the partial enlarged view (disassembled) of the structure at the reaction chamber in

[0030] The reference numerals are shown as:

[0031] 1. Outer shell; 101. Tube rack; 102. Cover plate; 11. Cleaning liquid tube; 12. Reaction liquid tube; 13. Switching valve; 131. Inlet chamber; 132. Outlet chamber; 133. Intermediate hole; 134. Switching valve push rod; 14. Waste liquid tank tube; 141. Waste liquid tank cover; 142. Waste liquid tank tube body; 15. Sample tube; 151. Upper end cap; 152. Fixed cover; 153. Sample tube body; 154. Aluminum pressing cap; 2. Flow channel plate; 21. Flow channel; 211. Sample tube inlet; 212. Cleaning liquid tube inlet; 213. Reaction liquid tube inlet; 214. Switching valve inlet; 215. Switching valve outlet; 216. Waste liquid tank tube inlet; 22. Hollow ejector pin; 221. Inlet ejector pin; 222. Outlet ejector pin; 23. Reconstitution chamber; 24. Reaction chamber; 241. Pressure ring; 242. Nucleic acid capture filter paper; 300. Lower sealing rubber plug; 301. Upper sealing rubber plug. Detailed implementation manners

[0032] Refer to in combination Figures 1 to 6As shown, according to an embodiment of the present invention, an integrated pathogen nucleic acid detection chip is provided, specifically an integrated pathogen nucleic acid microfluidic detection chip, including: a housing 1, in which a cleaning liquid tube 11, a reaction liquid tube 12, a switching valve 13, and a waste liquid pool tube 14 are respectively sealed; a sample tube 15 containing pre-set nucleic acid lysis solution is installed outside the housing 1; a flow channel plate 2 is connected to the bottom of the housing 1, in which a flow channel 21 is constructed, and a reconstitution chamber 23 and a reaction chamber 24 are also constructed on the flow channel plate 2. A nucleic acid capture filter paper 242 is provided in the reaction chamber 24; the content in the sample tube 15 can be controlled to enter the reaction chamber 24 through the flow channel 21, and nucleic acid capture is achieved at the nucleic acid capture filter paper 242; the content in the cleaning liquid tube 11 can be controlled to enter the reaction chamber 24 through the flow channel 21 to clean the captured nucleic acid; the content in the reaction liquid tube 12 can be controlled to enter the reconstitution chamber 23 and enter the reaction chamber 24 through the flow channel 21 under the control of the switching valve 13; the content in the reaction chamber 24 can be controlled to be stored in the waste liquid pool tube 14. It should be noted that by amplifying (preferably isothermal amplification) the sample extracted in the reaction chamber 24 and using a corresponding fluorescence detection device for corresponding detection, the full-process integration purpose of sample preparation, nucleic acid extraction, and nucleic acid amplification for nucleic acid detection using the chip of the present invention can be achieved.

[0033] In this technical solution, by controlling the outflow and storage of the content in the corresponding tubes in sequence according to the nucleic acid extraction process through the flow channel 21 and the switching valve 13 for the cleaning liquid tube 11, the reaction liquid tube 12, the waste liquid pool tube 14, the sample tube 15, and the reconstitution chamber 23, the integrated design of nucleic acid sample pretreatment, nucleic acid lysis, nucleic acid capture, nucleic acid cleaning, and nucleic acid amplification is realized, which is beneficial to more rapid detection of pathogen nucleic acid and has higher sensitivity. At the same time, it should be particularly noted that the reconstitution chamber 23 and the reaction liquid tube 12 integratedly arranged in the flow channel plate 2 store the freeze-dried powder reagent and the solution reagent separately, making the chip of the present invention suitable for long-term stable storage of reagents over long distances, and the application scenarios of the chip are more diverse.

[0034] The aforementioned flow channel 21 is specifically a microchannel structure with a width of 0.5 mm and a depth of 0.5 mm, which is suitable for the manipulation of microfluidics, so that the chip in the present invention becomes a microfluidic chip under the injection cooperation of each reagent tube valve (that is, the downward push of the push rod).

[0035] Specifically, the sample tube 15 is used for collecting and lysing the sample to be tested (the sample can be DNA / RNA of a certain concentration, or a pathogenic microorganism sample, including cells, viruses, tissues, bacteria, and fungi); the cleaning solution tube 11 is used to clean the filter paper (disposed in the reaction chamber 24) after capturing nucleic acids, removing certain impurities and residual lysate; the reaction solution tube 12 is used to store liquid reaction reagents, specifically, for example, polymerase chain reaction (PCR), recombinase polymerase reaction (RPA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA) reaction reagents, which are used in combination with the reaction reagents stored in the reconstitution chamber 23, such as lyophilized powder reconstitution or reagents added during nucleic acid amplification (such as magnesium ions required during RPA reaction); the reaction chamber 24 is used for nucleic acid amplification (in-situ amplification), such as polymerase chain reaction (PCR), recombinase polymerase reaction (RPA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), etc. The fluorescence information of the amplified content therein can be obtained through the fluorescence detection component arranged in a matching manner; the waste liquid pool tube 14 is used to store waste liquid.

[0036] It should be particularly noted that the switching valve 13 separates the sample liquid and cleaning liquid pipelines from the reconstitution chamber 23 in the reaction reagent pipeline, avoiding the backflow of liquid into the reconstitution chamber when injecting the sample liquid and cleaning liquid, which may cause the reconstitution reagent to fail. Only when starting to inject the reaction reagent, this switching valve 13 is opened to allow the reagent to flow through, first enter the reconstitution chamber for reconstitution of the lyophilized reagent, and then flow into the reaction chamber for amplification. That is to say, the switching valve 13 of the present invention forms an effective isolation between the injection sample and cleaning pipeline (i.e., the pipeline represented by the flow channel 21) and the reaction reagent pipeline, and only controls the switching valve 13 to open to connect the two pipelines when they need to be connected.

[0037] In some embodiments, a plurality of hollow ejector pins 22 are further provided on the runner plate 2. The plurality of hollow ejector pins 22 are fixed through the pin seat holes. One hollow ejector pin 22 is provided at each position on the runner 21 corresponding to the cleaning liquid pipe 11, the reaction liquid pipe 12, and the waste liquid tank pipe 14 respectively. Two hollow ejector pins 22 are provided at the position on the runner 21 corresponding to the switching valve 13. And the bottoms of the cleaning liquid pipe 11, the reaction liquid pipe 12, the waste liquid tank pipe 14, and the switching valve 13 are all provided with lower sealing rubber plugs 300. The lower sealing rubber plugs 300 have a blocking position for wrapping and sealing the hollow ejector pins 22 and a communicating position where the hollow ejector pins 22 penetrate through so that the corresponding pipe valves communicate with the runner 21 or the chamber. Specifically, an external pushing member (such as a push rod) is used to push the relevant pipe valves to move closer to the hollow ejector pins 22 at the top of the lower sealing rubber plug 300, so as to realize the switching of the lower sealing rubber plug 300 from the blocking position to the communicating position. That is, when the lower sealing rubber plug 300 is in the blocking position, the content in the corresponding pipe valve is stored in the pipe valve and cannot flow out. And when the lower sealing rubber plug 300 is in the communicating position, the content in the corresponding pipe valve is forced out into the runner 21, the reconstitution chamber 23, or the reaction chamber 24, so as to realize the corresponding nucleic acid extraction process, such as extraction, cleaning, reagent reconstitution, amplification reaction, etc. In this technical solution, the outflow and blocking of the content in the chamber valve are realized by the way that the hollow ejector pin 22 pierces the lower sealing rubber plug 300. This process is consistent with the injection action (that is, pushing down each chamber valve) of the corresponding chamber valve. The structure is simple and the control is convenient.

[0038] See Figure 6As shown, in some embodiments, the switching valve 13 includes a valve body. An inlet chamber 131 and an outlet chamber 132 are configured in the valve body. The inlet chamber 131 and the outlet chamber 132 are connected through an intermediate hole 133. Two hollow ejector pins 22 corresponding to the switching valve 13 are respectively an inlet ejector pin 221 corresponding to the inlet chamber 131 and an outlet ejector pin 222 corresponding to the outlet chamber 132. Among them, the inlet ejector pin 221 is connected to the reconstitution chamber 23, and the outlet ejector pin 222 is connected to the reaction chamber 24. In this way, the state switching of effective isolation and connection between the reaction reagent pipeline where the reconstitution chamber 23 is located and the injection sample and cleaning pipeline where the reaction chamber 24 is located is realized. It should be particularly noted that when the reaction liquid tube 12 is driven to move close to the hollow ejector pin 22, the hollow ejector pin 22 will finally pierce the lower sealing rubber plug 300. The reaction liquid in the reaction liquid tube 12 enters the reconstitution chamber 23, mixes and dissolves with the freeze-dried reagent therein, and then enters the inlet chamber 131 through the inlet ejector pin 221 (at this time, the inlet ejector pin 221 has been controlled to pierce the lower sealing rubber plug 300 in the switching valve 13). After being fully mixed therein, it further flows into the outlet chamber 132 through the intermediate hole 133, and finally flows out to the reaction chamber 24 through the outlet ejector pin 222 to react with the content in the reaction chamber 24. During this process, the reaction reagents (including the aforementioned reaction liquid and freeze-dried reagent) can be fully dissolved in the inlet chamber 131 and the outlet chamber 132, ensuring the subsequent reaction effect. When the reaction reagent flows through the intermediate hole 133, due to the small diameter of the intermediate hole 133, a speed increasing effect can be formed on the reaction reagent, further ensuring the full dissolution of the two reagents.

[0039] In a preferred embodiment, a switching valve ejector rod 134 is provided at the top of the valve body. Through the setting of the switching valve ejector rod 134, the volume of the switching valve 13 can be designed to be relatively smaller, which is more matched with the reagent usage amount. The switching valve ejector rod 134 is in the top position of the switching valve 13 and can also fix the position of the switching valve 13 to ensure the relative stability of the position of the switching valve 13.

[0040] In some embodiments, upper sealing rubber plugs 301 are also provided in the cleaning liquid pipe 11, the reaction liquid pipe 12, the waste liquid pool pipe 14, and the switching valve 13. The upper sealing rubber plugs 301 are in the upper region of the lower sealing rubber plugs 300 and can be controlled to move closer to the lower sealing rubber plugs 300. Thus, when the corresponding pipe valves keep moving closer to the hollow thimble 22 and the contents inside them completely flow out, the corresponding pipe valves can be blocked to prevent the liquid from flowing back into the pipe valves during other processing steps. In a preferred embodiment, a hard layer is provided on the side of the upper sealing rubber plug 301 facing away from the lower sealing rubber plug 300. For example, hard plastic is provided on the top surface of the upper sealing rubber plug 301, so that the upper sealing rubber plug 301 forms a rubber plug with a combination of hardness and softness. Specifically, at this time, the end of the external push rod can directly apply force to the top surface of the upper sealing rubber plug 301. The setting of the hard layer can effectively prevent the uneven deformation and inclination of the rubber plug during the force application process, ensuring the smooth, reliable and stable downward movement of the corresponding pipe valve.

[0041] Specifically refer to Figure 4 As shown, the figure specifically shows a disassembled structure of a sample tube 15 in an embodiment. It includes a sample tube body 153, which is a cylindrical structure that penetrates up and down. Upper sealing rubber plugs 301 and lower sealing rubber plugs 300 are respectively plugged at its upper and lower ends. There is a gap between the two rubber plugs in the initial state, and this gap constitutes the accommodating space of the sample tube. The outer side of the lower sealing rubber plug 300 further includes an aluminum pressing cover 154, which cooperates with the lower sealing rubber plug 300 to form a reliable seal for the top end of the sample tube 15. A fixed cover 152 and an upper end cap 151 combined with the fixed cover 152 are also sleeved on the outer side of the upper sealing rubber plug 301. The fixed cover 152 has an internal thread, and it is combined with the upper end cap 151 to seal the sample tube 15.

[0042] Specifically refer to Figure 5 As shown, the figure shows a disassembled structure of a waste liquid pool pipe 14 in an embodiment. It specifically includes a waste liquid pool pipe body 142 and a waste liquid pool cover 141 sealed and connected to its top. The lower sealing rubber plug 300 and the upper sealing rubber plug 301 are arranged at intervals up and down, and the interval between them forms the liquid inlet channel of the waste liquid pool pipe body 142. This gap will be reduced during the downward movement of the waste liquid pool pipe 14 until the upper sealing rubber plug 301 completely wraps and blocks the hollow thimble 22 below it, realizing the sealing of the waste liquid pool pipe 14 and separating the waste liquid pool pipe 14 from the reaction chamber 24 to avoid the leakage of amplification products and causing nucleic acid contamination.

[0043] The nucleic acid capture filter paper 242 is specifically a chitosan-modified capture filter paper, which is hydrophilic. In some embodiments, a pressure ring 241 is also provided in the reaction chamber 24, and the pressure ring 241 is used to fix the nucleic acid capture filter paper 242, and the pressure ring 241 is a C-ring structure. This C-ring structure allows the reagent to flow through the nucleic acid capture filter paper 242, first filling the internal space of the pressure ring 241, and then flowing to the side C-ring outlet, thereby ensuring that there are no bubbles in the reaction chamber 24, and will not interfere with subsequent reactions. It should be noted that the flow channel 21 in the present invention is divided into two sections by the nucleic acid capture filter paper 242 at the reaction chamber 24, one of which has an outlet in the upper area of ​​the filter paper, and the other has an inlet in the lower area of ​​the filter paper and is connected to the waste liquid pool pipe 14.

[0044] In some embodiments, a tube rack 101 is placed in the outer shell 1, and the cleaning liquid tube 11, the reaction liquid tube 12, the switch valve 13, and the waste liquid pool tube 14 are placed in the tube rack 101, that is, the tube rack 101 arranged in the outer shell 1 is located between the inner wall of the outer shell 1 and the outer wall of each tube valve, and can effectively and reliably fix each tube valve. At this time, the outer shell 1 has the effect of enhancing the aesthetic appearance of the entire chip. The top opening of the outer shell 1 is covered and connected with a cover plate 102, which can prevent the various tube valves in the outer shell 1 from falling out from the top opening, prevent the loss of the internal tube valves, and ensure that the position of the internal tube valves is reliable and stable.

[0045] The present invention also provides a nucleic acid detector, including a push rod, which is used in conjunction with the above-mentioned integrated pathogen nucleic acid detection chip. When the integrated pathogen nucleic acid detection chip includes an upper sealing plug 301 and a hollow ejector pin 22, the push rod can apply force to the upper sealing plug 301 to make the contents in the corresponding tube valve flow out or make the upper sealing plug 301 wrap and seal the corresponding hollow ejector pin 22.

[0046] The technical solution of the present invention is further described below in conjunction with a specific embodiment.

[0047] Taking the detection of novel coronavirus in air bioaerosol by recombinase polymerase amplification reaction (RPA) as an example, the specific implementation method of the chip is as follows:

[0048] Storage Reagents: Each independent reagent tube, together with an upper rubber stopper (i.e., the aforementioned upper sealing rubber stopper 301, the same hereinafter) and a lower rubber stopper (i.e., the aforementioned lower sealing rubber stopper 300, the same hereinafter), can be used for pre-storing reagents for subsequent experiments. The reagent stored in the sample tube (i.e., the aforementioned sample tube 15, the same hereinafter) can be 1 mL of virus lysis solution. The reagent in the cleaning solution chamber (i.e., the aforementioned cleaning solution tube 11, the same hereinafter) can be 1 mL of DEPC water (DNase, RNase free) (Product number: R0022, Manufacturer: Beyotime). The reagent stored in the reaction reagent sleeve (i.e., the aforementioned reaction solution tube 12, the same hereinafter) can be the RNA isothermal rapid amplification reagent corresponding to the recombinase polymerase amplification reaction. On the surface of the reconstitution chamber 23, there is 2.5 μL of 280 nM magnesium acetate in a solid state after drying.

[0049] Sample Collection: Use the sample tube to collect samples and release nucleic acids in the sample tube. The sample can be collected by using a bioaerosol sampler (Specification: Portable Bioaerosol Sampler, Manufacturer: Beijing Craftsman Biotechnology Co., Ltd.) for pathogens in the environment, or by rinsing nasal / pharyngeal swabs, or other liquid or solid reagents to be detected. In this experiment, a novel coronavirus (2019-nCoV) pseudovirus ribonucleic acid reference material is selected for substitution (Product number: NIM-RM5203, Manufacturer: National Institute of Metrology, China).

[0050] Nucleic Acid Release: The storage solution in the sample tube is lysis solution. Once the sample enters, the pathogens can be lysed to release nucleic acids.

[0051] Nucleic Acid Capture: Cooperate with the instrument push rod to drive the combined soft and hard rubber stopper (i.e., the aforementioned upper sealing rubber stopper 301) in the sample tube by plunger pressure. During injection, the reagent tube (corresponding to the sample tube) will first move downward by a certain distance, so that the hollow thimble (i.e., the hollow thimble 22, the same hereinafter) fixed on the lower part of the pipeline layer (i.e., the aforementioned flow channel layer 2, the same hereinafter) pierces the lower rubber stopper of the reagent tube (i.e., the aforementioned lower sealing rubber stopper 300, the same hereinafter). Then, the fluid can flow through the sample liquid outlet, enter the lower part of the pipeline layer, and then flow through the filter paper for nucleic acid capture.

[0052] Nucleic Acid Cleaning: Cooperate with the instrument push rod to drive the combined soft and hard rubber stopper in the cleaning solution tube 11 by plunger pressure. During injection, the reagent tube (corresponding to the cleaning solution tube 11) will first move downward by a certain distance, so that the thimble fixed on the lower part of the pipeline layer pierces the lower rubber stopper of the reagent tube. Then, the cleaning solution can flow through the cleaning solution outlet, enter the lower part of the pipeline layer, and then flow through the filter paper for cleaning to wash away the impurities on the filter paper.

[0053] Reagent reconstitution: The combined instrument push rod is used to drive the soft-hard combined rubber stopper in the reagent tube (corresponding to the reaction liquid tube 12 mentioned above) by plunger pressure. During injection, the reagent tube will first move downward by a certain distance, so that the thimble fixed on the lower pipe layer pierces the lower rubber stopper of the reagent tube, and then the liquid reagent can flow through the liquid reagent outlet and flow into the reconstitution chamber for reconstitution of the solid reagent.

[0054] Open the switching valve (i.e., the switching valve 13 mentioned above, the same below): The switching valve is initially in a closed state, that is, the thimble pierces the lower rubber stopper of the switching valve, and the pinhole is blocked, preventing fluid flow. When the switching valve is pressed downward by a certain stroke in cooperation with the instrument, the thimble pierces through the lower rubber stopper and enters the switching valve chamber, allowing fluid to flow and the switching valve to open.

[0055] Nucleic acid amplification: The combined instrument push rod continues to drive the soft-hard combined rubber stopper in the reagent tube by plunger pressure, injecting the fluid in the reconstitution chamber into the amplification chamber (i.e., the reaction chamber 24 mentioned above), and then controlling the temperature in cooperation with the instrument to perform nucleic acid amplification.

[0056] Output the test result: Cooperate with the fluorescence signal reading module of the fully integrated instrument to perform real-time fluorescence detection, give the fluorescence curve, and finally interpret the positive and negative results.

[0057] It has been verified that the present invention can adopt the isothermal amplification method to perform ultra-fast nucleic acid amplification, and can give the test result within half an hour, which is especially suitable for on-site detection in unique scenarios to achieve ultra-fast detection. The detection sensitivity of the traditional detection method can only reach 200 copies / mL. This detection chip, combined with the fully integrated instrument, can further improve the detection sensitivity to 20 copies / mL. The highly sensitive nucleic acid detection is especially suitable for some samples with low pathogen loads.

[0058] It is easy for those skilled in the art to understand that, on the premise of no conflict, the above-mentioned advantageous methods can be freely combined and superimposed.

[0059] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. The above is only the preferred implementation manner of the present invention. It should be noted that for those of ordinary skill in the art, several improvements and variations can be made without departing from the technical principle of the present invention, and these improvements and variations should also be regarded as the protection scope of the present invention.

Claims

1. An integrated pathogen nucleic acid detection chip, characterized in that, Comprising: A housing (1) which is internally provided with a separately sealed cleaning liquid pipe (11), a reaction liquid pipe (12), a switching valve (13), and a waste liquid tank pipe (14), and a sample tube (15) containing pre-set nucleic acid lysis solution is provided on the outer side of the housing (1); A flow channel plate (2) connected to the bottom of the housing (1), with a flow channel (21) constructed therein, and a reconstitution chamber (23) and a reaction chamber (24) are also constructed on the flow channel plate (2), and a nucleic acid capture filter paper (242) is provided in the reaction chamber (24); The content in the sample tube (15) can be controlled to enter the reaction chamber (24) via the flow channel (21), and nucleic acid capture is achieved at the nucleic acid capture filter paper (242); The content in the cleaning liquid pipe (11) can be controlled to enter the reaction chamber (24) via the flow channel (21) to clean the captured nucleic acid; The content in the reaction liquid pipe (12) can be controlled to enter the reconstitution chamber (23) and enter the reaction chamber (24) via the flow channel (21) under the control of the switching valve (13); The content in the reaction chamber (24) can be controlled to be stored in the waste liquid tank pipe (14); A plurality of hollow ejector pins (22) are also provided on the flow channel plate (2), and one hollow ejector pin (22) is provided at each position corresponding to the cleaning liquid pipe (11), the reaction liquid pipe (12), and the waste liquid tank pipe (14) of the flow channel (21), and two hollow ejector pins (22) are provided at the position corresponding to the switching valve (13) of the flow channel (21); The switching valve (13) includes a valve body, an inlet chamber (131) and an outlet chamber (132) are constructed in the valve body, the inlet chamber (131) and the outlet chamber (132) are communicated through an intermediate hole (133), and the two hollow ejector pins (22) correspondingly arranged with the switching valve (13) are respectively an inlet ejector pin (221) corresponding to the inlet chamber (131) and an outlet ejector pin (222) corresponding to the outlet chamber (132).

2. The integrated pathogen nucleic acid detection chip according to claim 1, wherein The bottoms of the cleaning liquid pipe (11), the reaction liquid pipe (12), the waste liquid tank pipe (14), and the switching valve (13) all have a lower sealing rubber plug (300), and the lower sealing rubber plug (300) has a blocking position for wrapping and sealing the hollow ejector pin (22) and a communicating position where the hollow ejector pin (22) penetrates out to enable the corresponding pipe valve to communicate with the flow channel (21) or the chamber.

3. The integrated pathogen nucleic acid detection chip according to claim 2, wherein, A switching valve ejector rod (134) is provided at the top of the valve body.

4. The integrated pathogen nucleic acid detection chip according to claim 2, wherein, Upper sealing rubber plugs (301) are also provided in the cleaning liquid pipe (11), the reaction liquid pipe (12), the waste liquid tank pipe (14), and the switching valve (13), the upper sealing rubber plugs (301) are in the upper region of the lower sealing rubber plug (300), and can be controlled to move close to the lower sealing rubber plug (300).

5. The integrated pathogen nucleic acid detection chip according to claim 4, wherein, A hard layer is provided on the side of the upper sealing rubber plug (301) facing away from the lower sealing rubber plug (300).

6. The integrated pathogen nucleic acid detection chip according to claim 1, characterized in that, A retainer ring (241) is further disposed in the reaction chamber (24). The retainer ring (241) is used to fix the nucleic acid capture filter paper (242), and the retainer ring (241) is a C-ring structure.

7. The integrated pathogen nucleic acid detection chip according to claim 1, wherein A pipe rack (101) is disposed in the housing (1). The cleaning liquid pipe (11), the reaction liquid pipe (12), the switching valve (13), and the waste liquid tank pipe (14) are disposed in the pipe rack (101).

8. The integrated pathogen nucleic acid detection chip according to claim 7, wherein, The top opening of the housing (1) is covered with a cover plate (102).

9. A nucleic acid detector, characterized in that, It includes a push rod, which is used in cooperation with the integrated pathogen nucleic acid detection chip according to any one of claims 1 to 8. When the integrated pathogen nucleic acid detection chip includes an upper sealing rubber plug (301) and a hollow thimble (22), the push rod can apply force to the upper sealing rubber plug (301) to cause the content in the corresponding pipe valve to flow out or cause the upper sealing rubber plug (301) to wrap and block the corresponding hollow thimble (22).

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