Nucleic acid amplification device, nucleic acid amplification method and kit

Abstract

The present invention particularly relates to a nucleic acid amplification device, a nucleic acid amplification method, and a kit. The nucleic acid amplification device includes: a container, the container includes a reagent chamber and a reaction chamber, a first end of the reagent chamber is in fluid communication with a first end of the reaction chamber, a second end of the reagent chamber is an open end and is provided with an outer edge, and a second end of the reaction chamber is a closed end; a seal, one end of the seal is provided with an outer edge for longitudinal sealing; a lid, the lid includes a locking mechanism, and the locking mechanism can cooperate with the outer edge of the reagent chamber so that a main body portion of the seal is locked inside the second end of the reagent chamber. The present invention can store reagents at room temperature, achieve one-step sample loading, avoid reagent preparation before reaction (no manual operation, no automation, no cartridge), have no leakage and no pollution, and achieve low-cost, constant-temperature, multiplex, highly sensitive nucleic acid amplification reaction and target nucleic acid detection.

Description

Technical Field

[0001] The present invention relates to biochemical reactions, and particularly to a nucleic acid amplification device, a nucleic acid amplification method, and a kit. Background Art

[0002] Some nucleic acid amplification devices have been developed, which can amplify and detect target nucleic acids. However, the existing nucleic acid amplification devices have at least the following problems:

[0003] First, at least one essential component in the reagent for nucleic acid amplification reaction is in liquid form, which is not easy to store at room temperature or cannot be stably stored for a long time;

[0004] Second, two or more liquid reagents need to be mixed before being loaded into the reaction tube for nucleic acid amplification reaction;

[0005] Third, the nucleic acid amplification reaction is carried out in a common test tube (EP), which is prone to leakage;

[0006] Fourth, using a thermal cycler for nucleic acid amplification reaction, the instrument cost is relatively high;

[0007] Fifth, using a non-thermal cycling temperature controller for nucleic acid amplification reaction, the sensitivity and specificity are relatively low.

[0008] Therefore, there is an urgent need for a nucleic acid amplification solution that can store reagents at room temperature, achieve one-step sample loading, avoid reagent preparation before reaction (no manual operation, no automation, no cartridge), have no leakage and no pollution, and achieve low-cost, constant-temperature, multiplex, highly sensitive nucleic acid amplification reaction and target nucleic acid detection. Summary of the Invention

[0009] To solve the above problems in the prior art, the present invention provides a nucleic acid amplification device, a nucleic acid amplification method, and a kit.

[0010] The present invention provides a nucleic acid amplification device, which comprises:

[0011] A container, the container includes a reagent chamber and a reaction chamber, the first end of the reagent chamber is in fluid communication with the first end of the reaction chamber, the second end of the reagent chamber is an open end and is provided with an outer edge, and the second end of the reaction chamber is a closed end;

[0012] A seal, one end of the seal is provided with an outer edge for longitudinal sealing;

[0013] A cover, the cover includes a locking mechanism, and the locking mechanism can cooperate with the outer edge of the reagent chamber so that the main body part of the seal is locked inside the second end of the reagent chamber;

[0014] Among them, the reaction chamber is used for performing multiplex nucleic acid amplification reactions under the temperature gradient provided by an asymmetric heater;

[0015] Among them, the reaction chamber is used for receiving excitation light from multiple light sources and emitting corresponding multiplex fluorescence when performing the multiplex nucleic acid amplification reaction.

[0016] In one embodiment of the present invention, the container is transparent to light with wavelengths in the range of 350 nm to 800 nm, and the container does not deform at temperatures below 150 °C.

[0017] In one embodiment of the present invention, the volume of the reagent chamber is 100 μl to 1000 μl, and the inner diameter of the reagent chamber is greater than 3 mm.

[0018] In one embodiment of the present invention, the volume of the reaction chamber is 25 μl to 250 μl, the inner diameter of the reaction chamber is greater than 1 mm, and the length of the reaction chamber is 10 mm to 50 mm.

[0019] In one embodiment of the present invention, the evaporation amount of the aqueous solution in the container is less than 0.15%.

[0020] In one embodiment of the present invention, the seal is a silicone rubber sealing ring.

[0021] The present invention also provides a nucleic acid amplification method, which includes:

[0022] Providing the nucleic acid amplification device according to the above, wherein the reagent chamber of the nucleic acid amplification device is filled with a lyophilized reagent, and the lyophilized reagent contains components sufficient for performing multiplex nucleic acid amplification reactions, and the components include polymerase, nucleic acid primers and probes;

[0023] Loading a liquid containing the target nucleic acid to be detected into the reagent chamber to dissolve the lyophilized reagent and form a reaction system in the reaction chamber of the nucleic acid amplification device, and the liquid does not contain any critical amounts of components for multiplex nucleic acid amplification reactions except for the sample preservation solution and the target nucleic acid;

[0024] Locking the nucleic acid amplification device through the cooperation of the locking mechanism of the lid of the nucleic acid amplification device and the outer edge of the reagent chamber;

[0025] Providing a temperature gradient through an asymmetric heater, so that the reaction system generates liquid thermal convection and performs multiplex nucleic acid amplification reactions.

[0026] In one embodiment of the present invention, the asymmetric heater includes a heating ring and a heating resistor. The heating ring is capable of accommodating the high-temperature region of the reaction chamber, and the heating resistor is disposed on one side of the heating ring such that the heat generated by the heating resistor is conducted from one side of the heating ring to the high-temperature region of the reaction chamber.

[0027] In one embodiment of the present invention, after locking the nucleic acid amplification device, the air in the container of the nucleic acid amplification device is compressed to 0.5 times its original volume.

[0028] In one embodiment of the present invention, the method further includes:

[0029] Detecting a plurality of fluorescence signals of different wavelengths from the container;

[0030] Based on the plurality of fluorescence signals, determining the detection result of the target nucleic acid.

[0031] In one embodiment of the present invention, determining the detection result of the target nucleic acid based on the plurality of fluorescence signals includes:

[0032] Based on the plurality of fluorescence signals, forming two-dimensional reaction information;

[0033] Based on the characteristics of the two-dimensional reaction information, determining the detection result of the target nucleic acid.

[0034] The present invention also provides a kit, which includes:

[0035] According to the nucleic acid amplification device described above, the reagent chamber of the nucleic acid amplification device is filled with a lyophilized reagent, and the lyophilized reagent contains components sufficient for a multiplex nucleic acid amplification reaction. The components include a polymerase, nucleic acid primers, and probes;

[0036] A sample tube filled with a sample preservation solution, which is used to collect the target nucleic acid to be detected to form a liquid containing the target nucleic acid. The liquid does not contain any critical amounts of components for a multiplex nucleic acid amplification reaction other than the sample preservation solution and the target nucleic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1A and Figure 1B are respectively a top view and a front view of the structure of a nucleic acid amplification device according to an embodiment of the present invention;

[0038] Figure 2 is a schematic diagram comparing the leakage effects of the container of a nucleic acid amplification device according to an embodiment of the present invention with an existing PCR 8-tube strip;

[0039] Figure 3ASchematic diagram of the cooperation between the container of the nucleic acid amplification device and the asymmetric heater according to an embodiment of the present invention;

[0040] Figure 3B Top view of the structure of the asymmetric heater according to an embodiment of the present invention;

[0041] Figure 4 Schematic diagram of the establishment process of the nucleic acid amplification method according to an embodiment of the present invention;

[0042] Figure 5 Schematic diagram of temperature control and multiplex detection of the nucleic acid amplification method according to an embodiment of the present invention;

[0043] Figure 6 Schematic diagram of the test results of multiplex detection of the nucleic acid amplification method according to an embodiment of the present invention. Detailed implementation manners

[0044] The embodiments of the present invention will be described below with reference to the accompanying drawings.

[0045] Here, the exemplary embodiments will be described in detail, and the examples are shown in the accompanying drawings. When the following description refers to the accompanying drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementation manners described in the following exemplary embodiments do not represent all implementation manners consistent with the present application. On the contrary, they are merely examples of devices and methods consistent with some aspects of the present application as detailed in the appended claims.

[0046] The terms used in the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The singular forms "a", "the" and "said" used in the present application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that "one kind" or "multiple kinds" generally refer to the number of types of different material structures or phenomena or physical characteristics; "one" or "multiple" can refer to the number of individuals of the same material structure or phenomenon or physical characteristic, and can also refer to the number of types of different material structures or phenomena or physical characteristics, and its meaning is determined by the meaning expressed in the context. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more of the associated listed items. The words such as "including" or "comprising" etc. mean that the elements or objects appearing before "including" or "comprising" cover the elements or objects listed after "including" or "comprising" and their equivalents, and do not exclude other elements or objects.

[0047] The first implementation manner

[0048] The first implementation manner of the present invention provides a nucleic acid amplification device.

[0049] Figure 1A and Figure 1B are respectively a top view and a front view of the structure of a nucleic acid amplification device according to an embodiment of the present invention.

[0050] As Figure 1B shown, the nucleic acid amplification device includes a container 10, and the container 10 includes a reagent chamber 101 and a reaction chamber 102. The first end of the reagent chamber 101 is in fluid communication with the first end of the reaction chamber 102. The second end of the reagent chamber 101 is an open end, and the second end of the reaction chamber 102 is a closed end. The reagent chamber 101 can accommodate reagents for nucleic acid amplification reactions (for example, the lyophilized reagents to be described below). The reaction chamber 102 is used for multiplex nucleic acid amplification reactions carried out under a temperature gradient provided by an asymmetric heater, and the reaction chamber 102 is also used for receiving excitation light from multiple light sources and emitting corresponding multiplex fluorescence when performing the multiplex nucleic acid amplification reaction. Here, the multiplex nucleic acid amplification reaction refers to a nucleic acid amplification reaction for simultaneously detecting two or more targets.

[0051] The lyophilized reagents can contain components sufficient for multiplex nucleic acid amplification reactions. The components include polymerase, nucleic acid primers, and probes, and can be stored at room temperature. All the liquids required for the multiplex nucleic acid amplification reaction come from the sample. When nucleic acid amplification is to be performed, a sampling device (such as a swab) can be used to collect the sample and mix it with the sample preservation solution into a sample tube to release the target nucleic acid to be detected. Then, the liquid containing the target nucleic acid can be loaded into the reagent chamber 101 to dissolve the lyophilized reagents and form a reaction system in the reaction chamber 102. This liquid does not contain any critical amounts of components for multiplex nucleic acid amplification reactions other than the sample preservation solution and the target nucleic acid, enabling one-step sample loading and eliminating the need for reagent preparation before the reaction, that is, no mixing of two or more liquids is required. In addition, the asymmetric heater is a non-thermal cycling constant temperature heater, which has low cost and high sensitivity.

[0052] In one embodiment, the container 10 is transparent to light in the wavelength range of 350 nm to 800 nm, so that light in this wavelength range can irradiate the reaction system inside the container 10, and light in this wavelength range from the reaction system can be detected externally through the container 10.

[0053] In one embodiment, the container 10 does not deform at temperatures below 150 °C, and the temperature required for nucleic acid amplification reactions generally does not exceed 150 °C, so that it can be ensured that the container 10 does not deform during nucleic acid amplification reactions. Here, the container 10 not deforming means that the container 10 does not have a volume change greater than 10% due to temperature change.

[0054] In one embodiment, as Figure 1A and Figure 1BAs shown, the nucleic acid amplification device is generally cylindrical. Among them, the inner diameter of the reagent chamber 101 can be relatively large to accommodate the lyophilized reagent, and the inner diameter of the reaction chamber 102 can be relatively small to suit liquid heat convection. As an example, the volume of the reagent chamber 101 is 100 μl to 1000 μl, and the inner diameter of the reagent chamber 101 is greater than 3 mm. As another example, the volume of the reaction chamber 102 is 25 μl to 250 μl, the inner diameter of the reaction chamber 102 is greater than 1 mm, and the length of the reaction chamber 102 is 10 mm to 50 mm. It can be understood that the shapes and sizes of the reagent chamber 101 and the reaction chamber 102 can be adjusted according to actual needs and are not limited herein.

[0055] In one embodiment, as Figure 1B shown, an outer edge 1011 is provided at the second end of the reagent chamber 101, and the nucleic acid amplification device further includes a lid 20. The lid 20 includes a locking mechanism 201, and the locking mechanism 201 can cooperate with the outer edge 1011 of the reagent chamber 101 to achieve the locking of the container 10. After locking, the container 10 will not be accidentally opened, and the lid 20 will not be pushed open by the high pressure inside the container 10, so as to ensure that the amplification product will not escape therefrom and cause leakage and contamination.

[0056] In one embodiment, as Figure 1B shown, the nucleic acid amplification device further includes a seal 30. When the locking mechanism 201 cooperates with the outer edge 1011 of the reagent chamber 101 for locking, the main body part of the seal 30 is locked inside the second end of the reagent chamber 101, which can further improve the sealing performance of the container 10. As an example, the seal 30 is a silicone rubber sealing ring, the shape of the seal 30 matches the second end of the reagent chamber 101, and one end of the seal 30 is provided with an outer edge 301 (such as an annular eaves-shaped outer edge) for longitudinal sealing.

[0057] Figure 2 is a schematic diagram of the leakage effect comparison between the container of the nucleic acid amplification device according to an embodiment of the present invention and the existing PCR 8-tube strip.

[0058] 50 μl of aqueous solution is filled in each test tube of the existing PCR 8-tube strip, and a standard PCR thermal cycle is performed (a total of 30 cycles, in each cycle, 95 °C is maintained for 30 seconds, 55 °C is maintained for 30 seconds, and 72 °C is maintained for 30 seconds). 50 μl of aqueous solution is also filled in multiple containers 10 of the present application. The temperature at the bottom of the container 10 is set to 103 °C, the temperature at the top of the container 10 is set to 55 °C, and this temperature is maintained for 30 minutes. The mass changes of the PCR 8-tube strip and the container 10 are measured before and after the reaction respectively, and the measurement results are as Figure 2 shown.

[0059] Figure 2The left half shows the mass changes of the PCR 8-well strip and the container 10 respectively. Among them, the mass change of each test tube in the PCR 8-well strip is within the range of 1.5% to 3.5%, while the mass changes of multiple containers 10, as shown by the dashed box, are all significantly less than 0.5%. Figure 2 The right half magnifies the dashed box, and it can be seen that the mass changes of multiple containers 10 are all less than 0.15%.

[0060] In other words, through the cooperation and locking of the locking mechanism 201 with the outer edge 1011 of the reagent chamber 101, plus the sealing of the seal 30, the evaporation amount of the aqueous solution in the container 10 can be less than 1.5%, further less than 0.5%, and further less than 0.15%.

[0061] Figure 1A and Figure 1B Not shown in [description], the nucleic acid amplification device further includes an asymmetric heater 40. The asymmetric heater 40 can be independent of the nucleic acid amplification device or can be a part of the nucleic acid amplification device, and the number of the asymmetric heaters 40 can be one or more, so as to provide one or more constant temperature heating zones.

[0062] Figure 3A is a schematic diagram of the cooperation between the container and the asymmetric heater of the nucleic acid amplification device according to an embodiment of the present invention. Figure 3B is a top view of the structure of the asymmetric heater according to an embodiment of the present invention.

[0063] As Figure 3A and Figure 3B shown, the asymmetric heater 40 includes a heating ring 401 and heating resistors 402a, 402b. The heating ring 401 can accommodate the high-temperature area 1021 of the reaction chamber 102, and the heating resistors 402a, 402b are arranged on one side of the heating ring 401 (for example, Figure 3B the left side of the dashed axis in [figure reference]), so that the heat generated by the heating resistors 402a, 402b is conducted from one side of the heating ring 401 to the high-temperature area 1021 of the reaction chamber 102, realizing asymmetric heating. Asymmetric heating can make the reaction system in the reaction chamber 102 more likely to form regular thermal convection. It can be understood that although Figure 3B shows two heating resistors 402a, 402b, the number of heating resistors can be adjusted according to actual needs and is not limited herein.

[0064] In one embodiment, as Figure 3B shown, the asymmetric heater 40 further includes a temperature sensor 403, and the temperature sensor 403 can detect the temperature of the heating area.

[0065] In one embodiment, as Figure 3BAs shown, the asymmetric heater 40 further includes a circuit connection socket 404. The circuit connection socket 404 can be respectively connected to the control circuit of the heating device, the heating resistors 402a, 402b, and the temperature sensor 403 described below, so as to obtain the temperature feedback information from the temperature sensor 403, and control the power on and off of the heating resistors 402a, 402b according to the temperature control signal from the control circuit, thereby realizing the constant temperature of the heating zone.

[0066] In one embodiment, as Figure 3B shown, the asymmetric heater 40 further includes a heating plate 405. The heating ring 401, the heating resistors 402a, 402b, the temperature sensor 403, and the circuit connection socket 404 are all arranged on the heating plate 405, and the heating zone (such as the heating ring 401, the heating resistors 402a, 402b, and the temperature sensor 403) is thermally insulated from the non-heating zone (such as the circuit connection socket 404) through a heat insulation air gap 406.

[0067] It can be understood that although Figure 3A the heating ring 401 of the asymmetric heater 40 is shown to accommodate the high-temperature zone 1021 of the reaction chamber 102, the heating ring 401 can also accommodate the low-temperature zone (not shown) of the reaction chamber 102, which is not limited herein. For example, two asymmetric heaters are provided as the upper heater and the lower heater respectively to accommodate the low-temperature zone and the high-temperature zone of the reaction chamber respectively.

[0068] Second Embodiment

[0069] The second embodiment of the present invention provides a nucleic acid amplification method, which is applicable to the nucleic acid amplification device according to the first embodiment of the present invention. The relevant technical details mentioned in the first embodiment are still valid in this embodiment. To reduce repetition, they will not be elaborated here. Correspondingly, the relevant technical details mentioned in this embodiment can also be applied in the first embodiment.

[0070] Figure 4 is a schematic diagram of the establishment process of a nucleic acid amplification method according to an embodiment of the present invention.

[0071] As Figure 4 shown, the nucleic acid amplification method includes:

[0072] Step 1: Provide a nucleic acid amplification device. The reagent chamber of the nucleic acid amplification device is filled with a lyophilized reagent, and the lyophilized reagent contains components sufficient to perform a multiplex nucleic acid amplification reaction. The components include a polymerase, nucleic acid primers, and probes. It can be understood that according to different detection types, the nucleic acid primers and probes can be different, and there can be multiple nucleic acid primers and probes, so as to realize a multiplex nucleic acid amplification reaction.

[0073] Step 2: Load a liquid containing the target nucleic acid to be detected into the reagent chamber to dissolve the lyophilized reagent and form a reaction system in the reaction chamber of the nucleic acid amplification device. This liquid does not contain any critical amounts of components for multiplex nucleic acid amplification reactions other than the sample preservation solution and the target nucleic acid. As an example, the sample preservation solution contains water, 0.1% Tween-20, 1 mM EDTA, and 10 mM Tris-HCl with a pH of 8.8. Here, the critical amount refers to the concentration or amount of a given component that neither significantly promotes nor significantly inhibits a specific multiplex nucleic acid amplification reaction.

[0074] Step 3: Lock the nucleic acid amplification device by the cooperation of the locking mechanism of the lid of the nucleic acid amplification device with the outer edge of the reagent chamber. In one embodiment, after locking the nucleic acid amplification device, the air in the container of the nucleic acid amplification device is compressed to 0.5 times its original volume.

[0075] Step 4: Provide a temperature gradient through an asymmetric heater so that the reaction system generates liquid thermal convection and undergoes multiplex nucleic acid amplification reactions. In one embodiment, a heating device with multiple asymmetric heaters can be provided. The heating device includes a control circuit to achieve temperature control of the multiple asymmetric heaters, and the multiple asymmetric heaters can simultaneously perform batch multiplex nucleic acid amplification reactions on multiple nucleic acid amplification devices.

[0076] In one embodiment, the nucleic acid amplification method further includes:

[0077] Step 5: Detect multiple fluorescence signals of different wavelengths from the container. In one embodiment, multiple LEDs emit light of different wavelengths to irradiate the reaction system in the container, and the reaction system generates multiple fluorescence signals of different wavelengths and is received by multiple photodetectors or photosensors (PDs).

[0078] Step 6: Determine the detection result of the target nucleic acid based on the multiple fluorescence signals. In one embodiment, determining the detection result of the target nucleic acid based on the multiple fluorescence signals includes: forming two-dimensional reaction information (such as a reaction signal curve) based on the multiple fluorescence signals; determining the detection result of the target nucleic acid based on the characteristics (such as shape characteristics) of the two-dimensional reaction information. In one embodiment, the two-dimensional reaction information includes time information and corresponding fluorescence information. The two-dimensional reaction information can be visual and non-visual. The two-dimensional reaction information can be used to extract amplification reaction characteristics and for result judgment. As an example, the control circuit of the heating device can automatically collect multiple fluorescence signals at regular intervals, form a reaction signal curve, and display the shape characteristics of the reaction signal curve on the display screen of the heating device, or light up the corresponding color lamp of the heating device based on the shape characteristics for the user to determine the detection result of the target nucleic acid.

[0079] Figure 5Schematic diagram of temperature control and multiplex detection of a nucleic acid amplification method according to an embodiment of the present invention.

[0080] As Figure 5 shown, a temperature gradient is provided for a single nucleic acid amplification device by a heating device (not shown). Among them, the reaction chamber has a high-temperature area (greater than 95 °C, such as 100 °C or 103 °C or 105 °C) at the bottom and a low-temperature area (such as 55 °C) at the top. The high-temperature area and the low-temperature area are heated by asymmetric heaters serving as the upper heater and the lower heater respectively. A liquid thermal convection is generated in the reaction system in the reaction chamber between the high-temperature area and the low-temperature area. Among them, the reaction system denatures in the high-temperature area, anneals in the low-temperature area, and extends in the middle of the two areas, thereby performing a multiplex nucleic acid amplification reaction.

[0081] A plurality of LEDs are arranged at the bottom of the reaction chamber, and emit lights of different wavelengths through a filter and a light guide medium to irradiate the reaction system. A plurality of PDs are arranged at the top of the reaction chamber, and receive a plurality of fluorescence signals of different wavelengths generated by the reaction system through a filter and a light guide medium, thereby realizing multiplex nucleic acid detection.

[0082] Figure 6 Schematic diagram of the multiplex detection test result of a nucleic acid amplification method according to an embodiment of the present invention.

[0083] As described above, there can be various nucleic acid primers and probes, so as to realize a multiplex nucleic acid amplification reaction. As an example, the reaction system contains dNTP with a concentration of 0.3 mM, MgSO4 with a concentration of 3 mM, K2SO4 with a concentration of 20 mM, NaCl with a concentration of 40 mM, TrisHCl with a concentration of 25 mM and a pH of 8.8, Tween-20 with a concentration of 0.1%, polymerase with a concentration of 0.08 U / µl, and a pair of primers with a concentration of 0.3 µM for detecting the heme oxygenase gene (pbsA), a probe corresponding to the heme oxygenase gene (pbsA) with a concentration of 0.2 µM, and in this probe, the four fluorescence labels (FAM, Hex, ROX, Cy5) each account for 25%.

[0084] As Figure 6 shown, 4-fold fluorescence signals are detected from the container of a single nucleic acid amplification device. Each fluorescence signal corresponds to the corresponding fluorescence label and is excited by the corresponding LED. Among them, the abscissa represents time, the ordinate represents intensity, the 1st fluorescence signal corresponds to the Hex fluorescence label, the 2nd fluorescence signal corresponds to the Cy5 fluorescence label, the 3rd fluorescence signal corresponds to the FAM fluorescence label, and the 4th fluorescence signal corresponds to the ROX fluorescence label.

[0085] The third embodiment

[0086] The third embodiment of the present invention provides a kit, which includes a nucleic acid amplification device according to the first embodiment of the present invention and is applicable to the nucleic acid amplification method according to the second embodiment of the present invention. The relevant technical details mentioned in the first and second embodiments are still valid in this embodiment. To avoid repetition, they will not be elaborated here. Correspondingly, the relevant technical details mentioned in this embodiment can also be applied to the first and second embodiments.

[0087] The kit includes a nucleic acid amplification device. In the reagent chamber of the nucleic acid amplification device, there is a freeze-dried reagent, which contains components sufficient for a multiplex nucleic acid amplification reaction. The components include a polymerase, nucleic acid primers, and probes.

[0088] In one embodiment, the kit further includes a sample tube containing a sample preservation solution. The sample tube is used to collect the target nucleic acid to be detected to form a liquid containing the target nucleic acid. The liquid does not contain any critical amount of components for the multiplex nucleic acid amplification reaction other than the sample preservation solution and the target nucleic acid.

[0089] In one embodiment, the nucleic acid amplification device containing the freeze-dried reagent is stored in a moisture-proof sealed bag, and the kit further includes a swab, a pipette, and an instruction manual.

[0090] Example 1

[0091] The user collects a sample from the patient's oral cavity using a swab according to the instruction manual. The swab after sampling is placed into a sample tube containing 500 μl of the sample preservation solution. The sample preservation solution contains water, 0.1% Tween-20, 1 mM EDTA, and 10 mM Tris-HCl with a pH of 8.8. After sealing the sample tube with a cap, the sample solution is heated to 98 °C and maintained for 3 minutes to inactivate the virus and release the viral nucleic acid.

[0092] After the sample solution is cooled to below 40 °C, use a pipette to aspirate the supernatant of the sample, and then load 55 μl of the sample solution into the reagent chamber of the nucleic acid amplification device. The reagent chamber is pre-loaded with a lyophilized reagent. Except for the sample preservation solution and the target nucleic acid, the lyophilized reagent contains components sufficient to perform a multiplex nucleic acid amplification reaction in a 55 μl reaction volume. After dissolving the lyophilized reagent in a 55 μl reaction volume, the components of the reaction system include dNTP at a concentration of 0.3 mM, MgSO4 at a concentration of 3 mM, K2SO4 at a concentration of 20 mM, NaCl at a concentration of 40 mM, TrisHCl at a concentration of 25 mM and pH 8.8, Tween-20 at a concentration of 0.1%, EDTA at a concentration of 1 mM, DTT at a concentration of 3 mM, polymerase at a concentration of 0.08 U / µl, reverse transcriptase at a concentration of 0.4 U / µl, a pair of primers for detecting the nucleic acid of the novel coronavirus at a concentration of 0.3 µM, a pair of primers for detecting an internal reference gene (ribonuclease P protein or hRPP) at a concentration of 0.3 µM, a probe corresponding to the nucleic acid of the novel coronavirus at a concentration of 0.2 µM (FAM fluorescence-labeled), and a probe corresponding to the internal reference gene at a concentration of 0.2 µM (Hex fluorescence-labeled).

[0093] After sealing and locking the container of the nucleic acid amplification device with a lid, place the container in a heating device for nucleic acid amplification reaction. Both temperature control and signal acquisition are automatically completed by the control circuit of the heating device. Specifically, first set the high-temperature zone to 45 °C and the low-temperature zone to 37 °C so that the reverse transcription (RT) reaction proceeds for 5 minutes, and then set the high-temperature zone to 100 °C and the low-temperature zone to 50 °C so that the nucleic acid amplification reaction proceeds for 25 minutes.

[0094] During the nucleic acid amplification reaction, the control circuit of the heating device automatically collects fluorescence signals at regular intervals to form two-dimensional reaction information (such as a reaction signal curve), and displays the characteristics of the two-dimensional reaction information (such as shape characteristics) on the display screen of the heating device for the user to determine the test result of the nucleic acid of the novel coronavirus. After the detection is completed, the container containing the amplification product can be treated harmlessly and without pollution.

[0095] Example 2

[0096] According to the instruction manual, the user uses a swab to wipe the solid surface or immerses it in a grinding material to collect a sample. Place the sampled swab into a sample tube containing 500 μl of sample preservation solution. Among them, the sample preservation solution contains water, Tween-20 at a concentration of 0.1%, EDTA at a concentration of 1 mM, and Tris-HCl at a concentration of 10 mM and pH 8.8. After sealing the sample tube with a lid, heat the sample solution to 98 °C and keep it for 3 minutes to inactivate the virus and release the viral nucleic acid.

[0097] After the sample solution is cooled to below 40°C, use a pipette to aspirate the supernatant of the sample, and then load 55 μl of the sample solution into the reagent chamber of the nucleic acid amplification device. The reagent chamber is pre-loaded with a lyophilized reagent. Except for the sample preservation solution and the target nucleic acid, the lyophilized reagent contains components sufficient to perform a multiplex nucleic acid amplification reaction in a 55 μl reaction volume. After dissolving the lyophilized reagent in a 55 μl reaction volume, the components of the reaction system include dNTP with a concentration of 0.3 mM, MgSO4 with a concentration of 3 mM, K2SO4 with a concentration of 20 mM, NaCl with a concentration of 40 mM, TrisHCl with a concentration of 25 mM and a pH of 8.8, Tween-20 with a concentration of 0.1%, EDTA with a concentration of 1 mM, polymerase with a concentration of 0.08 U / μl, a pair of primers with a concentration of 0.3 μM for detecting African swine fever virus (ASFV) nucleic acid, a pair of primers with a concentration of 0.3 μM for detecting an exogenous reference gene (Bordetella bronchiseptica), a probe with a concentration of 0.2 μM corresponding to ASFV nucleic acid (FAM fluorescence-labeled), and a probe with a concentration of 0.2 μM corresponding to the internal reference gene (Hex fluorescence-labeled).

[0098] After sealing and locking the container of the nucleic acid amplification device with a lid, place the container in a heating device to perform a nucleic acid amplification reaction. Both temperature control and signal acquisition are automatically completed by the control circuit of the heating device. Specifically, set the high-temperature zone to 100°C and the low-temperature zone to 50°C, so that the nucleic acid amplification reaction proceeds for 25 minutes.

[0099] During the nucleic acid amplification reaction, the control circuit of the heating device automatically collects fluorescence signals at regular intervals to form two-dimensional reaction information (such as a reaction signal curve), and displays the characteristics of the two-dimensional reaction information (such as shape characteristics) on the display screen of the heating device for the user to determine the detection result of ASFV nucleic acid. After the detection is completed, the container containing the amplification product can be treated harmlessly and without pollution.

[0100] Although the present invention has been illustrated and described with reference to certain preferred embodiments, those of ordinary skill in the art should understand that various changes can be made in form and detail without departing from the spirit and scope of the present invention.

Claims

1. A nucleic acid amplification device, characterized in that: The device comprises: A container, the container comprising a reagent chamber and a reaction chamber, the first end of the reagent chamber being in fluid communication with the first end of the reaction chamber, the second end of the reagent chamber being an open end and provided with an outer edge, and the second end of the reaction chamber being a closed end; A sealing member, one end of which is provided with an outer edge for longitudinal sealing; a cover including a locking mechanism capable of cooperating with the outer edge of the reagent chamber so that the main body of the seal is locked within the second end of the reagent chamber and so that air within the container is compressed after locking; an asymmetric heater, wherein the asymmetric heater is capable of heating the reaction chamber from one side of the reaction chamber, wherein the asymmetric heater comprises a heating ring and a heating resistor, the heating ring is capable of accommodating a high-heat zone of the reaction chamber, and the heating resistor is arranged on one side of the heating ring so that heat generated by the heating resistor is conducted from one side of the heating ring to the high-heat zone of the reaction chamber; Wherein, the reagent chamber is capable of accommodating a lyophilized reagent, wherein the lyophilized reagent contains components sufficient for performing a multiple nucleic acid amplification reaction, the components including a polymerase, a nucleic acid primer, and a probe; Wherein, the reaction chamber is used for multiple nucleic acid amplification reactions performed under a temperature gradient provided by an asymmetric heater; In which, the reaction chamber is used to receive excitation light from multiple light sources and emit multiple fluorescence signals of different wavelengths when performing the multiple nucleic acid amplification reaction, wherein two-dimensional reaction information including time information and corresponding fluorescence information is formed based on a fluorescence signal of the same wavelength among the multiple fluorescence signals of different wavelengths, and the detection result of the target nucleic acid is determined based on the characteristics of the two-dimensional reaction information.

2. The device according to claim 1, characterized in that The container is transparent to light with a wavelength in the range of 350 nm to 800 nm, and the container does not deform at a temperature less than 150°C.

3. The device according to claim 2, characterized in that The volume of the reagent chamber is 100 μl to 1000 μl, and the inner diameter of the reagent chamber is greater than 3 mm.

4. The device according to claim 2, characterized in that The volume of the reaction chamber is 25 μl to 250 μl, the inner diameter of the reaction chamber is greater than 1 mm, and the length of the reaction chamber is 10 mm to 50 mm.

5. The device according to claim 1, characterized in that The evaporation amount of the aqueous solution in the container is less than 0.15%.

6. The device according to claim 5, characterized in that The sealing member is a silicone sealing ring.

7. A method for nucleic acid amplification, characterized in that: The method comprises: A nucleic acid amplification device according to any one of claims 1 to 6 is provided, wherein a lyophilized reagent is contained in a reagent chamber of the nucleic acid amplification device, wherein the lyophilized reagent contains components sufficient for performing a multiple nucleic acid amplification reaction, wherein the components include a polymerase, a nucleic acid primer, and a probe; The reagent chamber is charged with a liquid containing a target nucleic acid to be detected to dissolve the lyophilized reagent and form a reaction system in the reaction chamber of the nucleic acid amplification device, wherein the liquid does not contain any critical amount of components for multiple nucleic acid amplification reaction except the sample preservation solution and the target nucleic acid; Locking the nucleic acid amplification device by cooperating the locking mechanism of the cover of the nucleic acid amplification device with the outer edge of the reagent chamber, so that the air in the container of the nucleic acid amplification device is compressed after locking; Providing a temperature gradient by an asymmetric heater so that the reaction system generates liquid thermal convection and performs multiple nucleic acid amplification reactions; detecting a plurality of fluorescent signals at different wavelengths from the container; Based on the fluorescent signal of the same wavelength among the multiple fluorescent signals of different wavelengths, forming two-dimensional reaction information including time information and corresponding fluorescent information; Based on the characteristics of the two-dimensional reaction information, the detection result of the target nucleic acid is determined.

8. The method according to claim 7, characterized in that After the nucleic acid amplification device is locked, the air in the container of the nucleic acid amplification device is compressed to 0.5 times of the original volume.

9. A kit, characterized in that: The kit comprises: The nucleic acid amplification device according to any one of claims 1 to 6, wherein the reagent chamber of the nucleic acid amplification device contains a lyophilized reagent, the lyophilized reagent contains components sufficient for performing a multiple nucleic acid amplification reaction, the components including a polymerase, a nucleic acid primer, and a probe; A sample tube filled with a sample preservative solution, the sample tube is used to collect a target nucleic acid to be detected to form a liquid containing the target nucleic acid, and the liquid does not contain any critical amount of components for multiple nucleic acid amplification reactions except the sample preservative solution and the target nucleic acid.

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