A device and method for detecting nucleic acid amplification products using lateral flow test strips

Abstract

This invention discloses an apparatus and method for detecting nucleic acid amplification products using lateral chromatography test strips. The apparatus comprises three chambers: a dilution and sealing chamber for adding and containing the sample solution to be tested, a reaction chamber for the nucleic acid amplification reaction, and a mixing chamber for detection using the lateral chromatography test strip. The reaction chamber contains pre-placed nucleic acid amplification reagents and solutions, and is connected to the bottom of the dilution and sealing chamber via a narrow channel, which is also open to the outside atmosphere. The mixing chamber contains pre-placed nucleic acid lateral chromatography test strips, and its bottom is connected to the reaction chamber via a narrow channel, which is also open to the outside atmosphere. This invention enables lateral chromatography detection of nucleic acid amplification and its products without any manual pipetting steps, and the entire process is conducted in a contamination-free space, offering advantages such as simple operation and high performance.

Description

Technical Field

[0001] This invention relates to a nucleic acid amplification reaction, detection device, and method belonging to the field of nucleic acid analysis, specifically to a device and method for detecting nucleic acid amplification products using lateral chromatography test strips. Background Technology

[0002] Nucleic acid amplification technology, including polymerase chain reaction (PCR) and various isothermal amplification techniques such as loop-mediated isothermal amplification (LAMP) and recombinase amplification (RPA), is a highly sensitive and specific detection method with wide applications in medicine, agriculture, food safety, and environmental monitoring. Currently, the detection of amplified products, also known as amplicones, is mostly achieved using fluorescence detection methods. While fluorescence detection offers high sensitivity and allows for real-time detection of amplified products, it suffers from drawbacks such as high instrument costs and complex operation.

[0003] Lateral chromatography test strips, sometimes called colloidal gold test strips, have been widely used in the field of immunoassay. Colloidal gold pregnancy test strips are widely used in homes. With simple modifications to the labeling and detection substances on the lateral chromatography test strips, they can also be used to detect nucleic acid amplification products. This allows for the visual, on-site detection of amplification products.

[0004] However, there are still some problems with using lateral chromatography test strips to detect nucleic acid amplification products: 1. In most cases, nucleic acid amplification is performed in a separate amplification tube. Then, using various methods, the amplification tube is opened or cut to add the amplified product to the sample pad of the test strip for detection. Since the concentration of amplified products is generally high, opening the amplification tube easily causes aerosol contamination, leading to false positives in subsequent tests. 2. Some integrated systems have been developed that integrate nucleic acid amplification and test strip detection of amplified products into a closed system, or open the amplification tube in a closed system. However, such systems generally require pumps, valves, blades, etc., making them complex and costly. 3. For common lateral chromatography test strips, to achieve detection, for a typical 3-4 mm wide test strip, the solution added to the sample pad needs to be at least 50 μL. Too little solution hinders capillary movement on the chromatography paper, failing to transport sufficient analytes to the T-line for detection, thus affecting the test results. The volume of a nucleic acid amplification reaction system is typically around 25 microliters. Due to the high sensitivity of nucleic acid amplification detection, increasing the reaction volume does not significantly improve sensitivity; instead, it increases the consumption of biological reagents such as amplification enzymes, thus increasing reaction costs. Therefore, in many current nucleic acid amplification product test strips, the amplification solution needs to be diluted to facilitate subsequent chromatographic detection. How to conveniently mix the amplification solution and diluent without introducing aerosol contamination of the amplicon is also a problem that must be solved. To address these issues, this invention designs a novel device and method for detecting nucleic acid amplification products using lateral chromatography test strips, which simplifies the operation steps, reduces the need for related instruments, and effectively improves detection performance. Summary of the Invention

[0005] To address the problems existing in the background art, the present invention proposes an apparatus and method for detecting nucleic acid amplification products using lateral chromatography test strips, which can improve the performance of detecting nucleic acid amplification products using lateral chromatography test strips.

[0006] The technical solution of this invention is:

[0007] I. A device for detecting nucleic acid amplification products using lateral chromatography test strips:

[0008] The device comprises three chambers, namely:

[0009] The dilution chamber is a closed cavity used to add and contain the sample solution to be tested, and the cavity is arranged vertically.

[0010] The reaction chamber is used for nucleic acid amplification reactions. Nucleic acid amplification reagents and solutions are pre-placed inside the chamber. One end of the reaction chamber is connected to the bottom of the dilution and sealing chamber through a narrow channel, and it is also connected to the outside atmosphere through a vertically arranged channel.

[0011] The reaction chamber is pre-fixed with all the reagents required for nucleic acid amplification, including amplification enzymes, primers, (dNTPs), and amplification buffer components.

[0012] The mixing chamber used for chromatographic test strip detection has a nucleic acid lateral chromatographic test strip pre-placed inside. The bottom of the mixing chamber and the other end of the reaction chamber are connected by a thin channel, and it is also connected to the outside atmosphere through a vertically arranged channel.

[0013] The term "slender" refers to a length-to-diameter ratio greater than 3.

[0014] A valve is installed in the channel between the reaction chamber and the mixing chamber for opening and closing, and the valve opens or closes the channel as needed.

[0015] The top of the dilution sealing chamber can be fitted with a cap with a breathable structure.

[0016] The volume of the dilution enclosed chamber is larger than the volume of the reaction chamber.

[0017] Filter materials are provided in the channels connecting the reaction chamber and the outside atmosphere, as well as in the channels connecting the mixing chamber and the outside atmosphere. The filter materials may be filter elements, silica particles, or silica gel columns, etc.

[0018] A valve is installed in the channel connecting the mixing chamber to the outside atmosphere for opening and closing. The valve opens or closes the channel as needed, preventing liquid from flowing from the reaction chamber into the mixing chamber through the channel.

[0019] The channels connecting the reaction chamber to the outside atmosphere and the mixing chamber to the outside atmosphere are arranged horizontally, inclined, or vertically.

[0020] A flexible membrane is installed at the top of the channel connecting the reaction chamber to the outside atmosphere, without any filter material.

[0021] A flexible membrane is sealed at the top of the channel connecting the mixing chamber and the outside atmosphere, without any filter material or valve. No valve is installed in the channel between the reaction chamber and the mixing chamber. A rigid material is installed at the top of the channel connecting the mixing chamber and the outside atmosphere, above the flexible membrane.

[0022] II. A detection method applied to the above-mentioned device:

[0023] First, a certain amount of the sample solution to be tested is added to the dilution sealing chamber. Then, the channel between the dilution sealing chamber and the reaction chamber is opened. The liquid level in the dilution sealing chamber is higher than that in the reaction chamber. Under the action of static pressure, the sample solution to be tested enters the reaction chamber through the channel between the dilution sealing chamber and the reaction chamber. At this time, by setting a valve in the channel between the reaction chamber and the mixing chamber and closing it, or by setting a valve in the channel connecting the mixing chamber to the outside atmosphere and closing it, the mixing chamber becomes an airtight chamber, preventing the solution from flowing from the reaction chamber into the mixing chamber.

[0024] After the amplification reaction is completed, a valve is installed and opened in the channel between the reaction chamber and the mixing chamber, thus opening the channel between them. A valve is also installed and opened in the channel connecting the mixing chamber to the outside atmosphere, and the mixing chamber is connected to the outside atmospheric pressure through the channel. The liquid level in the dilution sealing chamber is higher than the liquid levels in the reaction chamber and the mixing chamber. Under the action of the hydrostatic pressure of the solution in the dilution sealing chamber, the solution in the reaction chamber will enter the mixing chamber. Due to the hydrostatic pressure of the solution, the unamplified sample solution in the dilution sealing chamber also passes through the reaction chamber and further enters the mixing chamber until the water pressure in the dilution sealing chamber, the reaction chamber, and the mixing chamber is balanced, that is, the liquid levels in the dilution sealing chamber, the reaction chamber, and the mixing chamber are at the same level.

[0025] After the sample solution enters the reaction chamber, after the pre-placed solution in the reaction chamber dissolves, and before the amplification reaction ends, the temperature of the reaction chamber is controlled by an external or auxiliary temperature control device to carry out the nucleic acid amplification reaction. At this time, the solution in the channel is controlled and adjusted by the geometric dimensions of the channel so that the solution in the channel will not flow under the condition that there is no direct external force and the static pressure at both ends of the channel is in equilibrium. That is, the sample solution in the dilution and sealing chamber no longer enters the reaction chamber through the channel under the action of static pressure. The solute components in the reaction chamber can only enter the channel through diffusion and further enter the dilution and sealing chamber.

[0026] A sealed flexible membrane is installed at the top of the channel connecting the mixing chamber and the outside atmosphere. A rigid material is placed on top of the flexible membrane, preventing the mixing chamber from communicating with the outside atmospheric pressure through the channel. After the amplification reaction is completed, the rigid material is torn open, and a valve is installed and opened in the channel between the reaction chamber and the mixing chamber. This allows the solution to enter the mixing chamber from the reaction chamber for chromatography detection. At the same time, the original gas in the mixing chamber is compressed, and the flexible membrane responds by bulging outward to maintain the pressure balance between the gas inside the mixing chamber and the external gas pressure. The solution in the reaction chamber and the dilution chamber can then enter the mixing chamber.

[0027] The beneficial effects of this invention are:

[0028] The device and method of this invention can realize lateral chromatography detection of nucleic acid amplification and its products. The entire detection process requires only two manual steps from the user. First, a certain amount of the sample solution to be tested is added to the dilution chamber; second, after waiting for a certain nucleic acid amplification time, a valve is opened to complete the entire detection process. Through the device design, the volume of the nucleic acid amplification reaction can be controlled between a few microliters and tens of microliters, and such volume control does not require the use of high-precision pipetting instruments such as pipettes. After nucleic acid amplification is complete, only one valve needs to be opened to complete the dilution of the amplification solution and the addition of it to the sample pad of the chromatography test strip, without any manual pipetting steps, and the entire process is carried out in a contamination-free space.

[0029] Meanwhile, under the control of the supporting instruments, the opening of the second valve can also be completed automatically by the instruments.

[0030] Compared with existing methods, the apparatus and method of the present invention have the advantages of simple operation and good performance. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the device of the present invention. 1. Substrate, 2. Dilution sealing chamber 2, 3. Channel, 4. Reaction chamber 4, 5. Valve, 6. Channel, 7. Mixing chamber 7, 8. Filter material, 9. Channel, 10. Valve, 11. Filter material, 12. Channel.

[0032] Figure 2 It is a chromatographic test strip for detecting amplified products.

[0033] Figure 3 These are amplification gel electrophoresis images. The left side shows standard nucleic acid fragments of various lengths, the middle side shows the electrophoresis results of amplification products in a standard reaction tube, and the right side shows the electrophoresis results of amplification products in reaction chamber 4 of the device of this invention.

[0034] Figure 4 It is a ferromagnetic valve in the channel. 51. Ferromagnetic material, 6. Channel.

[0035] Figure 5 It utilizes flexible thin-film materials to achieve airtightness and pressure balance. 12. Channel; 13. Flexible thin film.

[0036] Figure 6 It utilizes flexible thin-film materials and rigid materials to achieve airtightness and valve functions. 9. Channel, 13. Flexible thin film, 14. Rigid material. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0038] Combination Figure 1 This paper introduces the method and supporting apparatus of the present invention. Figure 1 Primarily for the purpose of facilitating the explanation of the relevant content of this invention, and to facilitate the description and highlight structural details, it is not drawn according to the actual scale.

[0039] The device has three independent chambers inside the substrate 1: a dilution and sealing chamber 2, a reaction chamber 4, and a mixing chamber 7. The dilution and sealing chamber 2 and the reaction chamber 4 are connected by a channel 3, and the reaction chamber 4 and the mixing chamber 7 are connected by a channel 6.

[0040] A valve 5 is installed in the channel 6 between the reaction chamber 4 and the mixing chamber 7 to open or close the channel as needed.

[0041] Reaction chamber 4 contains pre-immobilized all reagents required for nucleic acid amplification, including amplification enzymes, primers, dNTPs, and amplification buffer components. Mixing chamber 7 contains pre-placed corresponding nucleic acid lateral chromatography test strips.

[0042] Both reaction chamber 4 and mixing chamber 7 maintain the same and balanced gas pressure with the outside through attached channels 12 and 9. Meanwhile, filters and sealing materials 11 and 8 can be installed in channels 12 and 9, such as filter cartridges, silica particles, or silica gel columns, to ensure that only small molecules such as gases can pass through the channels. This maintains the pressure balance between the inside and outside of the chambers while preventing nucleic acid amplification products from leaking into the external environment and causing amplicon contamination.

[0043] A valve 10 is installed in the capillary channel 9 of the mixing chamber 7 to open or close the connection between the channel and the external environment. That is, when the valve 10 is closed, the mixing chamber 7 can be set to an airtight mode to prevent liquid from entering the mixing chamber 7 from the reaction chamber 4 through the channel 6.

[0044] The following is the implementation process of this invention.

[0045] 1. In practical applications, a certain amount of the sample solution to be tested is added to the closed chamber 2 using a dropper for isotropic dilution.

[0046] Depending on the specific nucleic acid amplification reagents, the test sample solutions can be prepared using a standard nucleic acid extraction process, or they can be prepared through simple heating or dilution. The amount of test sample solution added can be controlled between approximately 50 and 1000 microliters. A smaller amount can be added if subsequent chromatographic testing requirements are met.

[0047] After the sample solution is added, the dilution chamber 2 can be sealed with a cap or similar device with a breathable structure. This prevents large molecules from entering the surrounding environment from the dilution chamber 2, while also maintaining pressure balance between the inside and outside of the chamber. The breathable structure on the cap can be achieved using a filter element, silica particles, or a silica gel column.

[0048] 2. Nucleic acid amplification reaction

[0049] After the sample solution to be tested is added to the dilution sealing chamber 2, since the dilution sealing chamber 2 and the reaction chamber 4 are a connected structure, the liquid level in the dilution sealing chamber 2 is higher than the liquid level in the reaction chamber 4. Therefore, under the action of static pressure, the sample solution to be tested will enter the reaction chamber 4.

[0050] At this time, the reaction chamber 4 and the mixing chamber 7 are controlled by valve 5 or valve 10: when valve 5 is set, valve 5 is closed, and the solution cannot enter the mixing chamber 7; when valve 5 is not set, valve 10 can be used to close the air pressure connection between the channel 9 and the outside, so that the mixing chamber 7 becomes an airtight chamber and prevents the liquid from flowing in.

[0051] Meanwhile, the design of channel 12 allows it to function as a single gas channel, making its volume relatively small compared to the reaction chamber 4. Thus, the volume of liquid flowing into the reaction chamber 4 is primarily determined by the volume of the reaction chamber 4. The design allows the volume of the reaction chamber 4 to be controlled between 3 and 80 microliters. This means that by adding an appropriate sample to the dilution chamber 2, a corresponding volume of solution can be obtained in the reaction chamber 4 for reaction, according to the device's design. A volume of 3 to 80 microliters typically requires a high-precision pipette. For ordinary users, using a pipette is quite complex. However, this invention, through its device design, allows users to achieve this simply by adding a certain amount of sample solution to the dilution chamber 2. This is an advantage of this invention.

[0052] In this invention, according to functional requirements, the volume of the dilution sealing chamber 2 is larger than that of the reaction chamber 4. This allows more solution to be stored in the dilution sealing chamber 2, which is beneficial for the movement of liquid throughout the device under hydrostatic pressure.

[0053] After the sample solution enters reaction chamber 4, it will dissolve the pre-placed reaction solution in reaction chamber 4. The temperature of reaction chamber 4 can be controlled by an external or auxiliary temperature control device to carry out the nucleic acid amplification reaction. Such a reaction can be a PCR reaction or an isothermal amplification reaction, such as LAMP or RPA.

[0054] During the nucleic acid amplification reaction, reaction chamber 4 and dilution sealing chamber 2 are connected by channel 3. In this invention, by controlling the geometry of channel 3, the solution in channel 3 will not flow under conditions where no external force acts directly and the hydrostatic pressure at both ends is balanced. Therefore, the solute components in reaction chamber 4 can only enter channel 3 by diffusion and further enter dilution sealing chamber 2. Solute amplification in solution is a slow process, while nucleic acid amplification reactions generally last within one hour.

[0055] Experiments show that, on this timescale, when the diameter of channel 3 is between 50 micrometers and 1 millimeter, as long as its length is greater than 5 millimeters, the exchange of solute components between reaction chamber 4 and dilution sealing chamber 2 due to diffusion can be ignored.

[0056] 3. The reaction solution and reagents also reach the sample pad of the chromatography strip to carry out the chromatography reaction.

[0057] After the amplification reaction is complete,

[0058] With valve 5 installed, it can be opened, simultaneously connecting the mixing chamber 7 to the external air pressure via channel 9. Under the influence of hydrostatic pressure, the solution in reaction chamber 4 enters mixing chamber 7. Simultaneously, due to hydrostatic pressure, the unamplified sample solution in dilution chamber 2 also passes through reaction chamber 4 and further into mixing chamber 7, until the water pressure in dilution chamber 2, reaction chamber 4, and mixing chamber 7 reaches equilibrium. This valve opening action dilutes the nucleic acid amplification product to a certain volume, meeting the requirements for detection by the chromatography strip.

[0059] Alternatively, valve 5 can be omitted, and valve 10 can be installed in channel 9 instead. In this case, when valve 10 is closed, mixing chamber 7 is an airtight space. When the solution enters through the narrow channel 6, it compresses the gas in mixing chamber 7, causing a pressure increase that prevents the solution from entering. When valve 10 is open, the solution in reaction chamber 4 and dilution sealing chamber can enter mixing chamber 7 for chromatographic detection.

[0060] In actual device design, the hydrostatic pressure relationship between the various chambers can be controlled by adjusting the relative positions of the dilution sealing chamber, reaction chamber 4, and mixing chamber 7, so as to improve the detection effect.

[0061] Specific implementation examples:

[0062] 1. Nucleic acid amplification and test strip detection of Vibrio parahaemolyticus

[0063] Device structure as follows Figure 1 As shown, the material used is polymethyl methacrylate (PMMA) sheet. The sheet is machined to create a pattern on one side. Figure 1 The diagram shown (please note) Figure 1 For ease of explanation, this drawing is not based on actual dimensions. The dimensions of each structure in the device are as described below.

[0064] The depth mentioned here refers to the direction perpendicular to the paper surface, the height refers to the vertical direction of the paper surface, and the width refers to the horizontal direction of the paper surface.

[0065] The depth of the dilution sealing chamber 2, reaction chamber 4, and mixing chamber 7 is 2 mm. The width of the dilution sealing chamber 2 is 3 mm, and the height is 60 mm. The width of the reaction chamber 4 is 4 mm, and the height is 3 mm. The width of the mixing chamber 7 is 4 mm, and the height is 60 mm.

[0066] Channel 3 has a depth of 1 mm, a height of 1 mm, and a length of 5 mm. Channel 6 has a depth of 1 mm, a height of 1 mm, and a length of 5 mm. Channel 9 has a depth of 0.1 mm, a width of 0.1 mm, and a height of 10 mm. Channel 12 has a depth of 0.1 mm, a width of 0.1 mm, and a height of 59 mm. Silica gel particles are placed in both channels 9 and 12 to maintain unobstructed airflow while preventing the diffusion of large molecules such as nucleic acid amplification products to the outside. Capillary valves can be installed in channels 9 and 12 as needed to prevent liquid flow within the channels. For specific capillary valve settings, refer to the relevant literature Sensors and Actuators A 130–131(2006)601–608.

[0067] After the above structure is fabricated, it can be bonded to another PMMA sheet using pressure-sensitive adhesive to construct a complete chamber. An airtight adhesive tape is bonded to the top of channel 9, serving as valve 10.

[0068] This embodiment uses Vibrio parahaemolyticus as an example, and uses the thermolabilehemolysin (tlh) gene in Vibrio parahaemolyticus as a target to perform LAMP amplification reaction.

[0069] The test strips used in the device are common nucleic acid detection chromatography test strips. Streptavidin is fixed on the T line, and FAM antibody is attached to the colloidal gold. For more information on nucleic acid chromatography test strips, please refer to the literature PLoS ONE8(7):e69355.doi:10.1371 / journal.pone.0069355.

[0070] For details on the relevant reaction system and its curing in the reaction chamber 4 of the device, please refer to Sensors & Actuators: B. Chemical 305 (2020) 127440 and Talanta 214 (2020) 120818.

[0071] The concentration of Vibrio parahaemolyticus used in the sample was approximately 250 CFU / g. 120 μL of Vibrio parahaemolyticus nucleic acid extraction solution was added to dilution chamber 2. The reaction chamber 4 in the apparatus was heated to 65 degrees Celsius for 40 minutes using an external temperature-controlled heating device. After heating, the airtight tape attached to the top of channel 9 was removed, allowing the solutions from reaction chamber 4 and dilution chamber 2 to enter mixing chamber 7. These solutions contacted the sample pad of the chromatography strip and underwent chromatographic detection under capillary force. After approximately 5 minutes, the T line appeared. Figure 2 As shown in the figure. This result demonstrates that the apparatus and method of the present invention can effectively perform nucleic acid amplification and detection.

[0072] Meanwhile, to further examine the amplification performance of the device and ensure that connecting reaction chamber 4 and dilution sealing chamber 2 via channel 3 does not significantly affect the overall amplification performance, the device can be opened and the amplification solution removed after amplification is complete for gel electrophoresis detection. Alternatively, the same sample and amplification reagents can be used to perform LAMP amplification in standard PCR tubes followed by gel electrophoresis detection. The results of both methods are as follows: Figure 3 As shown, this demonstrates that effective nucleic acid amplification can also be performed in the device.

[0073] The CRISPR system is a highly sensitive detection method capable of detecting target nucleic acids at around 100 pM. Detailed information about CRISPR can be found in relevant literature (Anal. Chem. 2019, 91, 11362-11366). In this experiment, a gRNA sequence was designed, and the CRISPR / Cas12a system was used to detect amplification products that might be present in dilution chamber 2. Since dilution chamber 2 and reaction chamber 4 are connected by channel 3, a large amount of amplification products are generated in reaction chamber 4 during amplification. These products may potentially move to dilution chamber 2 through channel 3. However, according to the experimental results, even with an extract of a 250 cfu / g positive Vibrio parahaemolyticus sample amplified in reaction chamber 4, amplification products could not be detected in dilution chamber 2 using CRISPR technology after amplification. This result indicates that, under the current device conditions, although there is a channel connection between reaction chamber 4 and dilution chamber 2, effective solute exchange cannot occur due to the limitations of the channel's geometry; in other words, the channel configuration does not significantly affect nucleic acid amplification.

[0074] Since the amplification product cannot effectively enter the dilution sealing chamber 2, as long as there is sufficient liquid in the dilution sealing chamber 2, it effectively functions as a seal for the reaction chamber 4. This seal helps prevent the solution in the reaction chamber 4 from evaporating and entering the external environment, causing contamination. This is why the chamber is named dilution sealing chamber 2.

[0075] For the same reaction system, when other parameters of the apparatus remain unchanged, but the geometry of channel 3 is changed to the following parameters: 1) Depth of channel 3 is 0.1 mm, height is 0.1 mm, and length is 5 mm. 2) Depth of channel 3 is 0.1 mm, height is 0.5 mm, and length is 5 mm. 3) Depth of channel 3 is 0.1 mm, height is 0.1 mm, and length is 2 mm. Similar results were obtained in all cases, indicating that the apparatus can function normally when the geometry of channel 3 is within this range.

[0076] Theoretically, the longer the channel 3, the better it is at preventing solute exchange between the reaction chamber 4 and the dilution sealing chamber 2. However, in practice, an excessively long channel can also cause inconvenience; its length is generally set within 10 centimeters.

[0077] In practice, channel 3 does not necessarily have to be set horizontally; it can also be set at an angle or vertically. In short, a long and narrow structure is preferred for its channel.

[0078] When using LAMP primers F3 and B3, PCR amplification of Vibrio parahaemolyticus can also be achieved in reaction chamber 4. In this case, the 5' end of F3 is modified with FAM, and the 5' end of F3 is modified with biotin. A temperature control device is required to cycle the temperature of reaction chamber 4 between 95°C and 63°C. The remaining operations are similar to the LAMP reaction described above, and similar experimental results can be obtained.

[0079] 2. A device with a ferroaluminum valve is used for the detection of Vibrio parahaemolyticus.

[0080] The target bacteria tested in this embodiment is Vibrio parahaemolyticus. The test reagents used are the same as those in Example 1, but the device structure is different.

[0081] like Figure 1 As shown, in this embodiment, valve 10 is provided so that the mixing chamber 7 maintains pressure balance with the outside environment through channel 9. A valve 10 is provided in channel 6 as shown in the diagram. Figure 4 The ferromagnetic material 51 shown forms an iron wax valve.

[0082] During the nucleic acid amplification reaction in reaction chamber 4, the iron-wax valve is in the closed state. After the amplification reaction is completed, the iron-wax valve opens under laser irradiation, allowing the reaction amplification solution and sample solution to enter mixing chamber 7 and contact the sample pad of the chromatography test strip for sample staining detection.

[0083] Experimental results show that using this structure, similar nucleic acid detection results as in Example 1 can be obtained.

[0084] 3. Detection of Vibrio parahaemolyticus using a flexible membrane-sealed device.

[0085] The target bacteria detected in this embodiment is Vibrio parahaemolyticus, and the detection reagents used are the same as in Example 1. However, the device structure is different.

[0086] like Figure 1 As shown, in this embodiment, filter material 11 is not provided in channel 12. Instead, a filter is provided at the top of channel 12, such as... Figure 5 The flexible film 13 shown.

[0087] Valve 5 is installed in channel 6. In channel 9, filter material 8 and valve 10 are not installed; instead, a valve is installed at the top of channel 9. Figure 6 The flexible film 13 shown.

[0088] The flexible film 13 can, on the one hand, cut off the flow of gas in the channel from the outside, thereby achieving an airtight effect and preventing pollution that may be caused by amplicon contamination, which is to say, it plays the role of the original filter material 8.

[0089] Meanwhile, since this is a flexible film, the film can be designed and manufactured to have a large area and flexibility. When the valve 5 in the channel 6 is opened and the solution enters the mixing chamber 7 from the reaction chamber 4, causing the original gas in the mixing chamber 7 to be compressed, this flexible film will respond quickly and bulge outward, thereby maintaining the balance between the gas pressure in the mixing chamber 7 and the external gas pressure, so that the solution in the reaction chamber 4 and the dilution chamber can enter the mixing chamber 7.

[0090] Experimental results show that using this structure, similar nucleic acid detection results as in Example 1 can be obtained.

[0091] Furthermore, when a flexible membrane 13 is used in channel 9, valve 5 can be omitted from channel 6, and instead... Figure 6 As shown, a layer of rigid material 14 is attached to the top of channel 9. Due to the presence of this rigid material 14, mixing chamber 7 is an airtight chamber, preventing the solution from entering. After the nucleic acid amplification reaction is complete, this rigid material 14 is removed, allowing the solution from reaction chamber 4 and dilution sealing chamber 2 to enter mixing chamber 7 for chromatographic detection.

[0092] Experimental results show that using this structure, similar nucleic acid detection results as in Example 1 can be obtained.

Claims

1. A device for detecting nucleic acid amplification product by lateral chromatographic test strip, characterized in that: the device comprises three chambers, which are: a dilution closed chamber (2) for adding and containing sample solution to be detected; a reaction chamber (4) for nucleic acid amplification reaction, in which nucleic acid amplification reagents and solution are pre-placed, and the reaction chamber (4) is connected with the bottom of the dilution closed chamber (2) through an elongated channel (3) and connected with the outside atmosphere through a vertically arranged channel (12); and a mixing chamber (7) for chromatographic test strip detection, in which nucleic acid lateral chromatographic test strip is pre-placed, and the mixing chamber (7) is connected with the other end of the reaction chamber (4) through an elongated channel (6) and connected with the outside atmosphere through a vertically arranged channel (9); the channel (6) connecting the reaction chamber (4) with the outside atmosphere, the channel (9) connecting the mixing chamber (7) with the outside atmosphere are vertically arranged; a flexible film (13) is arranged at the top of the channel (9) connecting the mixing chamber (7) with the outside atmosphere, no valve (5) is arranged in the channel (6) between the reaction chamber (4) and the mixing chamber (7), and a hard material is arranged on the flexible film (13) at the top of the channel (9) connecting the mixing chamber (7) with the outside atmosphere; the flexible film (13) is arranged in the channel (9), and no valve (5) is arranged in the channel (6); a layer of hard material (14) is further pasted at the top of the channel (9), the mixing chamber (7) is an airtight chamber, and solution cannot enter; after the nucleic acid amplification reaction is completed, the layer of hard material (14) is torn, and then the solution in the reaction chamber (4) and the dilution closed chamber (2) can enter the mixing chamber (7) for chromatographic detection. The volume of the dilution closed chamber (2) is greater than the volume of the reaction chamber (4). Filter materials (11, 8) are arranged in the channel (12) connecting the reaction chamber (4) with the outside atmosphere and the channel (9) connecting the mixing chamber (7) with the outside atmosphere, and the filter materials (11, 8) are filter cartridges. The filter materials (11, 8) are silica particles or silica gel columns. ​ ​ ​ ​ 2. The device for detecting nucleic acid amplification product by using lateral chromatographic test strip according to claim 1, characterized in that: ​ 3. The device for detecting nucleic acid amplification product by using lateral chromatographic test strip according to claim 1, characterized in that: ​ 4. The device for detecting nucleic acid amplification product by using lateral chromatographic test strip according to claim 3, characterized in that: ​

Images