Nucleic acid amplification method, detection method, and nucleic acid amplification system

Abstract

Provided is a simple nucleic acid amplification method and a nucleic acid amplification system. This nucleic acid amplification method comprises: a first preparation step of preparing a first liquid by adding at least a nonionic surfactant and an RNA decomposition enzyme inhibitor to a specimen including an analyte; a first incubation step of keeping the first liquid within a first temperature band for the duration of a first time period; a second preparation step of preparing a second liquid by adding an enzyme and a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from the analyte to the first liquid; and a second incubation step of keeping the second liquid within the second temperature band for the duration of a second time period.

Description

Nucleic acid amplification method, detection method, and nucleic acid amplification system

[0001] The present disclosure relates to nucleic acid amplification methods, detection methods and nucleic acid amplification systems.

[0002] When testing whether a subject is infected with a virus or bacteria, a detection method for amplifying nucleic acid is known. Specifically, in the detection method, first, a target nucleic acid derived from a virus or a target nucleic acid derived from a bacterium contained in a sample collected from the subject is amplified by a predetermined amplification method. Then, the amplified target nucleic acid can be detected to test for infection.

[0003] When the target nucleic acid is RNA (ribonucleic acid), for example, the NASBA (Nucleic Acid Sequence-Based Amplification) method is applied as an amplification method (Non-Patent Document 1).

[0004] Compton, J. “Nucleic acid sequence-based amplification.” Nature 350, 91-92, 1991.

[0005] A nucleic acid amplification method according to one embodiment of the present disclosure includes a first preparation step of preparing a first liquid by adding at least an RNase inhibitor to a sample containing a target nucleic acid molecule to be detected; a first incubation step of maintaining the first liquid at a first temperature range for a first time; a second preparation step of preparing a second liquid by adding a primer and an enzyme having a sequence complementary to the sequence of a nucleic acid molecule derived from the target nucleic acid molecule to the first liquid; and a second incubation step of maintaining the second liquid at a second temperature range for a second time.

[0006] A nucleic acid amplification method according to one embodiment of the present disclosure includes a first preparation step of preparing a first liquid containing a nucleic acid molecule derived from a detection target; a first incubation step of holding the first liquid at a first temperature range for a first time; a second preparation step of, after the first incubation step, adding an RNase inhibitor, a primer having a sequence complementary to the sequence of the nucleic acid molecule, and an enzyme to the first liquid to prepare a second liquid; and a second incubation step of holding the second liquid at a second temperature range for a second time.

[0007] A nucleic acid amplification system according to one embodiment of the present disclosure comprises an amplifier and a temperature control device, wherein the amplifier comprises a first preparation unit that prepares a first liquid by adding at least an RNase inhibitor to a sample containing a target to be detected, a first incubation unit in which the first liquid is maintained at a first temperature range for a first period of time, a second preparation unit that prepares a second liquid by adding a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from the target to the first liquid, and an enzyme, and a second incubation unit in which the second liquid is maintained at a second temperature range for a second period of time, and the temperature control device comprises an installation unit in which the amplifier is installed, and a temperature control unit that controls the temperatures of the first incubation unit and the second incubation unit.

[0008] A nucleic acid amplification system according to one embodiment of the present disclosure comprises an amplifier and a temperature control device, wherein the amplifier comprises a first preparation unit that prepares a first liquid containing a nucleic acid molecule derived from a detection target, a first incubation unit in which the first liquid is maintained at a first temperature range for a first period of time, a second preparation unit that prepares a second liquid by adding an RNase inhibitor, a primer having a sequence complementary to the sequence of the nucleic acid molecule, and an enzyme to the first liquid, and a second incubation unit in which the second liquid is maintained at a second temperature range for a second period of time, and the temperature control device comprises an installation unit in which the amplifier is installed and a temperature control unit that controls the temperatures of the first incubation unit and the second incubation unit.

[0009] FIG. 1 is a diagram illustrating an overview of a diagnostic system according to an embodiment of the present disclosure. FIG. 2 is an external view illustrating an example of a cartridge provided in a nucleic acid amplification system. FIG. 3 is a schematic view illustrating an example of a flow channel substrate and a temperature control device according to an embodiment of the present disclosure. FIG. 4 is a block diagram illustrating a configuration of a detection system according to an embodiment of the present disclosure. FIG. 5 is a flowchart illustrating the flow of a nucleic acid amplification method and a detection method. FIG. 6 is a flowchart illustrating another flow of a nucleic acid amplification method and a detection method. FIG. 7 is a diagram illustrating electrophoresis results and fluorescence observation results according to Examples 1 to 3 and Comparative Examples 1 to 3. FIG. 8 is a diagram illustrating electrophoresis results and fluorescence observation results according to Examples 4 to 6 and Comparative Examples 4 to 6. FIG. 9 is a diagram illustrating electrophoresis results and fluorescence observation results according to Comparative Examples 7 to 12. FIG. 10 is a diagram illustrating electrophoresis results and fluorescence observation results according to Examples 7 to 12 and Comparative Examples 13 to 18.

[0010] When extracting a target nucleic acid from a sample, there is a demand for a simple nucleic acid amplification method, a nucleic acid amplification system, etc. According to one aspect of the present disclosure, a simple nucleic acid amplification method and a nucleic acid amplification system can be provided.

[0011] [Embodiment] Hereinafter, one embodiment of the present disclosure will be described in detail.

[0012] <Technical Concept of the Present Disclosure> A nucleic acid amplification method according to one embodiment of the present disclosure includes a first preparation step, a first incubation step, a second preparation step, and a second incubation step. In the nucleic acid amplification method according to one embodiment of the present disclosure, an RNase inhibitor is added before the second incubation step. That is, in the first preparation step, at least the RNase inhibitor is added to a sample containing a target substance to prepare a first liquid. In the first incubation step, the prepared first liquid is maintained in a first temperature range for a first hour. In the second preparation step, a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from the target substance and an enzyme are added to the first liquid after the first incubation step to prepare a second liquid. In the second incubation step, the second liquid is maintained in a second temperature range for a second hour.

[0013] The RNase inhibitor may be added in the second preparation step, not in the first preparation step. In this case, in the first preparation step, a first solution containing a nucleic acid molecule derived from the target nucleic acid is prepared. In the first incubation step, the prepared first solution is maintained at a first temperature range for a first time. In the second preparation step, a second solution is prepared by adding an RNase inhibitor, a primer having a sequence complementary to the sequence of the nucleic acid molecule, and an enzyme to the first solution after the first incubation step. In the second incubation step, the second solution is maintained at a second temperature range for a second time.

[0014] In the nucleic acid amplification method according to one aspect of the present disclosure, an RNase inhibitor may be added in the first preparation step and the second preparation step.

[0015] When an RNase inhibitor is added in the second preparation step, the second preparation step may include a third preparation step in which the RNase inhibitor and a primer having a sequence complementary to the sequence of the nucleic acid molecule are added to the first solution after the first incubation step to prepare a third solution, and a fourth preparation step in which an enzyme is added.

[0016] In the first preparation step, a surfactant may be further added to the first liquid to improve the efficiency of extraction of the target substance contained in the sample. The surfactant may be, for example, a nonionic surfactant.

[0017] When the nucleic acid molecule derived from the target of detection is single-stranded RNA, the RNA degrading enzyme derived from the sample and the RNA degrading enzyme contaminated from the first liquid from the outside may degrade the RNA, resulting in inaccurate detection results. Furthermore, to detect the nucleic acid molecule derived from the target of detection contained in the sample, a reaction that amplifies RNA using the nucleic acid molecule derived from the target of detection as a template may be used. In this case, the RNA degrading enzyme derived from the sample and the RNA degrading enzyme contaminated from the first or second liquid from the outside may degrade the RNA, resulting in inaccurate detection results. For example, when the sample contains cells, the RNA degrading enzyme may be released from within the cells. Alternatively, the RNA degrading enzyme may be contaminated by fingers that touch the sample during collection.

[0018] Therefore, as described above, by adding an RNase inhibitor before the first incubation step and / or before the second incubation step, the unintended influence of RNase can be reduced.By using the nucleic acid amplification method according to one aspect of the present disclosure and labeling the amplified nucleic acid molecule, it is possible to accurately detect nucleic acid molecules derived from the detection target.

[0019] By employing a nucleic acid amplification method and a nucleic acid amplification system according to one embodiment of the present disclosure, nucleic acids contained in a collected sample can be extracted and amplified quickly and easily. Therefore, a detection method and a detection system using the nucleic acid amplification method and the nucleic acid amplification system can quickly, easily, and sensitively detect bacteria, viruses, and the like contained in a sample. Here, the sample may be saliva, urine, sweat, nasal mucus, blood, cells, etc. collected from a subject's body, or soil and water collected from a test object such as the ground, a river, or the sea. Alternatively, the sample may be a deposit collected from the surface of a test object such as a handrail, a door, clothing, shoes, or a toilet. The target of nucleic acid amplification and detection using the nucleic acid amplification method and the nucleic acid amplification system according to one embodiment of the present disclosure is, for example, a virus or bacteria. The type of virus or bacteria contained in a sample is not limited to one type, and may be two or more types. When the detection target is a virus, examples of the virus include influenza virus, coronavirus (e.g., SARS-CoV-2), respiratory syncytial virus, human metapneumovirus, norovirus, HIV virus, herpes virus, and foot-and-mouth disease virus. When the detection target is a bacterium, examples of the bacterium include Streptococcus mutans, Porphyromonas gingivalis (pathogenic oral bacteria), hemolytic streptococcus, mycoplasma, Streptococcus pneumoniae, and Bordetella pertussis (infectious disease pathogens). The subject is not limited to humans, and may be any living organism that can harbor a virus or bacterium, such as mammals, birds, reptiles, and amphibians.

[0020] Hereinafter, a diagnostic system 1 that detects a target substance contained in a specimen by applying a nucleic acid amplification method and a nucleic acid amplification system according to an embodiment of the present disclosure will be described as an example.

[0021] <Configuration of Diagnostic System> Fig. 1 is a diagram showing an overview of a diagnostic system 1 according to an embodiment of the present disclosure. As shown in Fig. 1, the diagnostic system 1 includes a detection system 1000, a terminal device 40 of a subject, a server 50, and a terminal device 60 of a medical professional. The detection system 1000 includes a cartridge 10 (amplifier) ​​and a detection device 30. The terminal device 40 and the server 50 may be connected to each other so that they can communicate with each other.

[0022] The cartridge 10 functions as a container for extracting nucleic acids from a sample collected from a subject (e.g., saliva, urine, blood, etc.) and amplifying nucleic acids derived from a detection target. The cartridge 10 may be, for example, a disposable cartridge.

[0023] The detection device 30 is a device that detects nucleic acids derived from a target nucleic acid when the nucleic acid is amplified in the cartridge 10. The detection device 30 includes an installation unit 31 for installing the cartridge 10, a temperature control unit 32 for controlling a temperature control device 70 that controls the temperature of the installed cartridge 10, and a detection unit 33 for detecting the target nucleic acid amplified in the installed cartridge 10. The installation unit 31, the temperature control unit 32, and the detection unit 33 will be described later. For example, when detecting a target nucleic acid by fluorescently labeling the target nucleic acid, the detection device 30 functions as a fluorescent microscope capable of measuring fluorescence intensity. In one example, the detection device 30 may output the measured fluorescence intensity as a detection value. The detection device 30 may include a communication unit (not shown) and transmit the detection value to a terminal device 40 of the subject. The detection device 30 may determine whether a certain concentration or more of nucleic acid derived from the target nucleic acid is present. The detection device 30 may determine that the sample is positive when a certain concentration or more of nucleic acid derived from the target nucleic acid is present. Alternatively, the detection device 30 may be a measurement device that measures the concentration of nucleic acid derived from the target nucleic acid.

[0024] Hereinafter, a system consisting of the cartridge 10, the installation unit 31 and temperature control unit 32 provided in the detection device 30, and the temperature control device 70 will be referred to as a nucleic acid amplification system 100. In other words, the nucleic acid amplification system 100 refers to the part of the detection system 1000 excluding the detection unit 33 provided in the detection device 30.

[0025] The terminal device 40 may analyze the detection values ​​acquired by the detection device 30, for example, using an application, and output the analysis results to the server 50. In this case, the server 50 may store the analysis results for each subject. Alternatively, the terminal device 40 may output the detection values ​​acquired by the detection device 30 to the server 50. In this case, the server 50 may analyze the detection values, store the analysis results for each subject, and transmit the analysis results to the terminal device 60 of the medical professional in charge of the subject and the terminal device 40 of the subject.

[0026] In the terminal device 60 that has acquired the analysis results, for example, diagnostic information with comments from a medical professional may be input, and the diagnostic information may be transmitted to the terminal device 40 of the subject.

[0027] The server 50 may be a single server or may include two or more servers. The server 50 may include an analysis server for analyzing test results from test data, or may include a database server for long-term storage of analysis results for each subject.

[0028] <Nucleic Acid Amplification System 100> Fig. 2 is an external view showing an example of the cartridge 10 provided in the nucleic acid amplification system 100. Fig. 3 is a schematic view showing an example of the flow path substrate 4 and temperature control device 70 provided in the cartridge 10. For the sake of simplicity, Fig. 3 does not show the detection device 30 including the temperature control unit 32 that controls the temperature control device 70.

[0029] The nucleic acid amplification system 100 is a system including a cartridge 10, a temperature control device 70, and an installation unit 31 and a temperature control unit 32 provided in a detection device 30. As shown in FIG. 2 , the cartridge 10 may include a main body unit 21 and a bottle unit 22.

[0030] A specimen collected from a user may be contained in the bottle portion 22. The main body portion 21 may include a flow path substrate 4 (see FIG. 3 ) that receives a liquid containing the specimen contained in the bottle portion 22 and, if the specimen contains nucleic acid derived from the detection target, contains a reagent that reacts with the nucleic acid.

[0031] The nucleic acid amplification system 100 is a system including a cartridge 10, which includes a bottle unit 22 having a first preparation unit 11 and a first incubation unit 12, which will be described below, and a main body unit 21 having a microchannel in which a liquid receiving unit 41, a second preparation unit 13, and a second incubation unit 14 are arranged on a single channel. As a result, a sample injected into the liquid receiving unit 41 flows through the microchannel while undergoing various processes, and reaches the second incubation unit 14. In other words, the inside of the microchannel functions as a single reaction system in which multiple processes are performed. The number of channels is not limited to one, as long as multiple processes function as a single reaction system.

[0032] (Cartridge 10) As described above, the cartridge 10 includes the liquid receiving section 41, the first preparation section 11, the first incubation section 12, the second preparation section 13, and the second incubation section .

[0033] The liquid receiving section 41 is one end of the microchannel, and a sample containing the detection target is supplied from the bottle section 22 to the main body section 21 via the liquid receiving section 41.

[0034] (Bottle 22) The first preparation unit 11 included in the bottle 22 is, for example, a container capable of holding a liquid. The first preparation unit 11 may be made of a transparent material, but may also be made of an opaque material. In this case, the liquid held in the first preparation unit 11 flows from the liquid receiving unit 41 to the second preparation unit 13. The first preparation unit 11 may be a flow path through which a sample provided to the liquid receiving unit 41 flows. In this case, the sample provided to the liquid receiving unit 41 flows from the first preparation unit 11 to the second preparation unit 13 along the flow path. The first preparation unit 11 may be a flow path that meanders multiple times. The meandering of the flow path promotes stirring of the sample flowing through the flow path. In the first preparation unit 11, at least an RNase inhibitor may be added to a sample containing a detection target to prepare a first liquid. In addition to the RNase inhibitor, a nonionic surfactant, dNTPs, NTPs, etc. may be added to the sample. The nonionic surfactant may be ethylene glycol (-O-CH ２ -CH ２ -) repeating structure, specifically, Tween-20 (registered trademark) or Triton X-100 (registered trademark).

[0035] For example, when collecting saliva as a specimen, the user inserts the sampler 23 into the user's mouth to attach saliva to the sampler 23. As shown in Fig. 2 , the user attaches saliva to the sampler 23 and then inserts the sampler 23 into the bottle portion 22. When the user inserts the sampler 23 into the bottle portion 22, the saliva attached to the sampler 23 is mixed with a buffer solution containing an RNase inhibitor that is contained in the bottle portion 22.

[0036] In the NASBA method, since the material that has not undergone the specialized pretreatment for extracting and purifying nucleic acid can be used as the sample, the target nucleic acid can be amplified more easily.However, if no pretreatment is performed, the sample used in the NASBA method, for example, saliva, contains substances other than nucleic acid, such as RNase.If the sample contains RNase, the amplified RNA may be decomposed by the RNase.

[0037] In the nucleic acid amplification system 100 according to this embodiment, adding an RNase inhibitor to the first solution reduces the degree to which RNA produced in a subsequent step is degraded by RNase, enabling more effective amplification of the target nucleic acid. As described above, the nucleic acid amplification system 100 does not require specialized pretreatment for extracting and purifying the target nucleic acid from the sample, and can amplify the target nucleic acid more simply and effectively.

[0038] The RNase inhibitor added to the first solution may be any substance that can reduce the degree to which RNA is decomposed by RNase, and a substance that does not inhibit the activity of RNase H may be used.

[0039] In the nucleic acid amplification system 100, the timing at which the RNase inhibitor is added to the sample is not limited to the timing at which the first liquid is prepared in the first preparation unit 11. If the RNase inhibitor is not added to the sample in the first preparation unit 11, the RNase inhibitor may be added at any time up until the step of maintaining the second liquid in the second temperature range in the second incubation unit 14, which will be described later, is started.

[0040] In the first incubator 12, the first liquid is maintained at a first temperature for a first time. The first time for maintaining the first incubator 12 at the first temperature may be, for example, about 10 minutes. The first time may be shorter than a second time for maintaining the second incubator 14 at the second temperature, which will be described later.

[0041] The first incubator 12 may hold the first liquid in the first temperature zone for a first time, and then lower the temperature of the first liquid. The temperature of the first liquid may be lowered at a location other than the first incubator 12. For example, the nucleic acid amplification system 100 may include an additional flow path (not shown) between the first incubator 12 and the second preparation unit 13, and the first incubator 12 may hold the first liquid in the first temperature zone, and then the additional flow path may lower the temperature of the first liquid.

[0042] (Main body 21) As described above, the main body 21 includes a flow path substrate 4. As shown in FIG. 3 , the flow path substrate 4 may include a liquid receiving section 41 for receiving a liquid, a branch flow path 42, a flow path 45, and a reservoir 46 for storing the liquid that has flowed through the flow path substrate 4. The flow path 45 may include a first region 451 in which a reagent containing a primer is disposed, and a second region 452 in which a reagent containing an enzyme that amplifies nucleic acid is disposed at a position different from the first region 451. The flow path 45 in the flow path substrate 4 corresponds to the second preparation section 13 and the second incubation section 14. The housing of the main body 21 may be made of an opaque material. In this case, the main body 21 may include a window 211 so that the interior of the main body 21 can be viewed.

[0043] The branch flow path 42 may be a flow path that connects the liquid receiving section 41 to each of the multiple flow paths included in the flow path 45. In other words, the multiple flow paths included in the flow path 45 may branch off from the branch flow path 42 that is connected to the liquid receiving section 41.

[0044] The plurality of flow paths included in flow path 45 may be flow paths for flowing a liquid. Each of the plurality of flow paths may receive the liquid flowing from liquid receiving portion 41 and flow it to storage portion 46 connected to each of the plurality of flow paths. Each of the plurality of flow paths may be in communication from liquid receiving portion 41 to storage portion 46.

[0045] In the flow path substrate 4 , the branch flow path 42 , the flow path 45 and the reservoir 46 correspond to the second preparation section 13 and the second incubation section 14 .

[0046] In the second preparation unit 13, a primer having a sequence complementary to the sequence of the nucleic acid molecule derived from the detection target and an enzyme are added to the first solution to prepare a second solution. The primers added to the first solution may include a first primer corresponding to a first region of the nucleic acid molecule and a second primer corresponding to a second region different from the first region. For example, the first primer may be a forward primer that amplifies the nucleic acid molecule derived from the detection target in the sense direction, and the second primer may be a reverse primer that amplifies the nucleic acid molecule in the antisense direction.

[0047] If an RNase inhibitor is not added to the sample in the first preparation unit 11, the RNase inhibitor may be added in the second preparation unit 13. In this case, in the second preparation unit 13, a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from the detection target, an enzyme, and an RNase inhibitor are added to the first solution to prepare a second solution. The primers added to the first solution may include a first primer corresponding to a first region of a predetermined nucleic acid molecule and a second primer corresponding to a second region different from the first region. Primers may be added as needed as long as they are capable of amplifying the target nucleic acid molecule, and may include, for example, one type or three or more types.

[0048] Adding an RNase inhibitor to the first liquid in the second preparation unit 13 also reduces the degree to which RNA produced in a later step will be degraded by RNase, as in the case of adding an RNase inhibitor to the first liquid, thereby enabling more effective amplification of the target nucleic acid. As described above, the nucleic acid amplification system 100 does not require specialized pretreatment for extracting and purifying nucleic acids from a sample, and can amplify the target nucleic acid more simply and effectively.

[0049] The second preparation unit 13 may include a third preparation unit 15 for adding a primer having a sequence complementary to the sequence of the nucleic acid molecule derived from the target nucleic acid, and a fourth preparation unit 16 for adding an enzyme. In this case, in the third preparation unit 15, a primer having a sequence complementary to the sequence of the nucleic acid molecule derived from the target nucleic acid may be added to the first liquid to prepare the third liquid. Alternatively, in the fourth preparation unit 16, an enzyme may be added to the third liquid to prepare the second liquid. Here, when an RNase inhibitor is added to the first liquid in the second preparation unit 13, the RNase inhibitor may be added to the first liquid together with a primer having a sequence complementary to the sequence of the nucleic acid molecule derived from the target nucleic acid, in the third preparation unit 15, to prepare the second liquid. Adding the RNase inhibitor at a stage before the start of the amplification reaction reduces the degree to which the generated RNA is decomposed by RNase in advance, allowing for more effective amplification of the target nucleic acid. As shown in Figure 3, in the flow path substrate 4, the first region 451 in which a reagent containing a primer is arranged corresponds to the third preparation section 15, and the second region 452 in which a reagent containing an enzyme that amplifies nucleic acids is arranged at a position different from the first region 451 corresponds to the fourth preparation section 16.

[0050] When a configuration in which an RNase inhibitor is added to the first liquid in the second preparation unit 13 is adopted, a first liquid containing nucleic acid molecules derived from the detection target is prepared in the first preparation unit 11. For example, the first liquid may be prepared by adding a nonionic surfactant, dNTPs, NTPs, etc. to a sample containing the detection target. Furthermore, the RNase inhibitor added to the first liquid in the second preparation unit 13 may be the same substance as that used when adding the RNase inhibitor when preparing the first liquid.

[0051] The predetermined nucleic acid molecule may be DNA or RNA. The enzymes added in the second preparation section 13 may include reverse transcriptase, DNA polymerase, RNA polymerase, and ribonuclease.

[0052] In the second incubation unit 14, the second liquid is maintained in the second temperature zone for a second time period. The second time period may be any time period that allows the target nucleic acid to be amplified to a detectable extent, and may be, for example, 30 minutes or more and 90 minutes or less.

[0053] The first preparation unit 11 and the first incubation unit 12 are installed in the bottle unit 22 of the cartridge 10, which are connected to each other. The first preparation unit 11 and the first incubation unit 12 may be installed in a container and / or a flow path that are connected to each other. In this manner, in the nucleic acid amplification system 100, the first liquid prepared in the first preparation unit 11 spontaneously reaches the first incubation unit 12 along the microflow path. Therefore, in the nucleic acid amplification system 100, there is no need to transfer the first liquid to an incubation container.

[0054] Furthermore, the first preparation unit 11, the first incubation unit 12, the second preparation unit 13, and the second incubation unit 14 may be installed in the same cartridge 10, which has containers and / or flow paths that are connected to one another. In this way, the nucleic acid amplification system 100 does not need to remove and transfer each liquid after preparing the first liquid, after holding the first liquid in the first temperature range, after preparing the second liquid, and after holding the second liquid in the second temperature range, and therefore each step can be performed within the cartridge 10.

[0055] (1) The first primer, (2) the second primer, (3) the enzyme, and (4) the probe DNA having a predetermined base sequence and capable of binding to the enzymatically amplified nucleic acid molecule may be pre-immobilized in at least one of the second preparation unit 13 and the second incubation unit 14. (1) The first primer, (2) the second primer, (3) the enzyme, and (4) the probe DNA having a predetermined base sequence and capable of binding to the enzymatically amplified nucleic acid molecule may be pre-immobilized in the second preparation unit 13, rather than in the second incubation unit 14. (1) The first primer, (2) the second primer, (3) the enzyme, and (4) the probe DNA having a predetermined base sequence and capable of binding to the enzymatically amplified nucleic acid molecule may be immobilized as a coating. This allows the nucleic acid amplification system 100 to provide each component of the first primer, the second primer, the enzyme, and the probe DNA to the second solution without later adding them. Hereinafter, the probe DNA is also referred to as a molecular beacon.

[0056] (1) The first primer and (2) the second primer may be pre-immobilized in the third preparation unit 15, and (3) the enzyme and (4) the probe DNA having a predetermined base sequence and capable of binding to the nucleic acid molecule amplified by the enzyme may be pre-immobilized in the fourth preparation unit 16.

[0057] A moisture-resistant coating agent, such as a sugar, may be used to immobilize the first primer, the second primer, the enzyme, and the probe DNA. Examples of sugars include trehalose dihydrate, sucrose, lactose, glucose, and fructose. By immobilizing the first primer, the second primer, the enzyme, and the probe DNA in this manner, the nucleic acid amplification system 100 can store each component in an unused cartridge 10 for a long period of time.

[0058] Furthermore, when an RNase inhibitor is added to the first liquid to prepare the second liquid in the second preparation unit 13, the RNase inhibitor may be immobilized in advance in at least one of the second preparation unit 13 and the second incubation unit 14. The RNase inhibitor may be immobilized in advance in the second preparation unit 13. The RNase inhibitor may be immobilized in advance in the third preparation unit 15 of the second preparation unit 13. The RNase inhibitor may be immobilized as a coating. A moisture-resistant coating agent, such as a sugar, may be used to immobilize the RNase inhibitor.

[0059] The temperature control device 70 may also be provided with an installation section 71 for installing the cartridge 10, a temperature control area 72A for controlling the temperature of the first region 451 corresponding to the third preparation section 15, and a temperature control area 72B for controlling the temperature of the second region 452 corresponding to the fourth preparation section 16. In this case, the temperature control area 72A is provided at a position corresponding to the lower part of the third preparation section 15, and the temperature control area 72B is provided at a position corresponding to the lower part of the fourth preparation section 16. The temperature control areas 72A and 72B may control the temperature of regions on the flow path substrate 4 where the second incubation step is performed, and may control the temperature of regions other than the first region 451 and the second region 452. For example, if the second incubation step is performed in the storage section 46, the temperature control area 72B may be provided below the storage section 46 and control the temperature of the storage section 46, as shown in FIG. 3 .

[0060] The temperature control device 70 may control the first temperature zone to a temperature zone higher than the second temperature zone. The first temperature zone may be a temperature for inactivating the detection target, and the second temperature zone may be a temperature for activating the enzyme.

[0061] The first temperature zone may be in the range of 80°C or higher and 95°C or lower, and the second temperature zone may be in the range of 37°C or higher and 41°C or lower.

[0062] The first and second temperature zones may be temperatures set by the temperature control device 70. Alternatively, the first and second temperature zones may be the liquid temperature of the liquid whose temperature is to be controlled by the temperature control device 70. When the first and second temperature zones are liquid temperatures, for example, an average temperature or a central temperature may be used. Here, the average temperature may be, for example, the average liquid temperature over a predetermined period of time. The central temperature may be, for example, the temperature at the center between the highest and lowest liquid temperatures. The central temperature may be calculated as the sum of the highest and lowest temperatures divided by two. The detection device 30 may be equipped with a temperature sensor that detects the liquid temperature of the liquid whose temperature is to be controlled by the first incubator 12 and the second incubator 14. The first incubator 12 and the second incubator 14 may heat the liquid whose temperature is to be detected by the temperature sensor so as to maintain the liquid temperature detected by the temperature sensor.

[0063] <Detection System> Fig. 4 is a block diagram showing the configuration of a detection system 1000 according to an embodiment of the present disclosure. As shown in Fig. 4, the detection system 1000 includes a cartridge 10 and a detection device 30.

[0064] As shown in Fig. 2, the cartridge 10 includes a main body 21 having a flow path substrate 4, and a bottle 22 having a first preparation unit 11 and a first incubation unit 12. As shown in Fig. 3, the flow path substrate 4 includes a second preparation unit 13 and a second incubation unit 14. Here, in the detection system 1000, the cartridge 10 may have a structure (detection preparation unit) in the second incubation unit 14 that adds, to the second liquid, a probe DNA that has a predetermined base sequence and is capable of binding to a nucleic acid molecule amplified by an enzyme. The detection preparation unit may be installed subsequent to the second incubation unit 14.

[0065] The detection preparation unit may include, for example, a plurality of reaction fields, and each reaction field may add a probe DNA capable of binding to a nucleic acid molecule derived from a different target substance to the second liquid. The detection preparation unit may be located in the second preparation unit 13 and / or the second incubation unit 14. For example, a probe DNA capable of binding to a nucleic acid molecule of influenza virus and a probe DNA capable of binding to a nucleic acid molecule of coronavirus (SARS-CoV-2) may be added to the second liquid in each reaction field. This allows detection of multiple targets by collecting a sample once.

[0066] As described above, the detection device 30 includes the installation unit 31, the temperature control unit 32, and the detection unit 33. The temperature control unit 32 can individually control the temperatures of the temperature control areas 72A and 72B of the temperature control device 70 in FIG.

[0067] The detection unit 33 may have a fluorescence intensity detection function that detects nucleic acid molecules amplified by the enzyme based on the intensity of fluorescence emitted from the probe DNA. The detection unit 33 may have, for example, the function of a fluorescence microscope. The detection unit 33 may have, for example, an excitation light source for exciting the probe DNA to be measured, and a light receiving unit that receives fluorescence emitted by the probe DNA upon receiving the excitation light. The detection unit 33 may also be a camera that can capture images of the fluorescent emission.

[0068] Each molecule of the probe DNA is modified with a fluorescent molecule and a quencher molecule. The quencher molecule is configured, for example, to absorb light in a wavelength band corresponding to the fluorescence wavelength of the fluorescent molecule. The distance between the fluorescent molecule and the quencher molecule in the probe DNA not bound to the enzymatically amplified nucleic acid molecule is shorter than the distance between the fluorescent molecule and the quencher molecule in the probe DNA bound to the enzymatically amplified nucleic acid molecule. Examples of fluorescent molecules include 6-carboxyfluorescein (FAM), Texas Red (TR), and cyanine (CY)-based materials. When the fluorescent substance is 6-FAM, it emits fluorescence with a peak wavelength of 517 nm when irradiated with excitation light with a peak wavelength of 494 nm. When the fluorescent substance is Texas Red, it emits fluorescence with a peak wavelength of 615 nm when irradiated with excitation light with a peak wavelength of 596 nm. When the fluorescent substance is Cy3-carboxylic acid, it emits fluorescence with a peak wavelength of 570 nm when irradiated with excitation light with a peak wavelength of 555 nm. The base sequence of the probe DNA may be appropriately designed based on the base sequence of the nucleic acid molecule to be detected.

[0069] (1) The first primer, (2) the second primer, (3) the enzyme, and (4) the probe DNA having a predetermined base sequence and capable of binding to the enzymatically amplified nucleic acid molecule may be pre-immobilized in at least one of the second preparation unit 13 and the second incubation unit 14. (1) The first primer, (2) the second primer, (3) the enzyme, and (4) the probe DNA having a predetermined base sequence and capable of binding to the enzymatically amplified nucleic acid molecule may not be immobilized in the second incubation unit 14 but may be pre-immobilized in the second preparation unit 13. (1) The first primer, (2) the second primer, (3) the enzyme, and (4) the probe DNA having a predetermined base sequence and capable of binding to the enzymatically amplified nucleic acid molecule may be immobilized as a coating. This allows the detection system 1000 to provide the probe DNA to the second liquid without later adding the probe DNA.

[0070] (1) The first primer and (2) the second primer may be pre-immobilized in the third preparation unit 15, and (3) the enzyme and (4) the probe DNA having a predetermined base sequence and capable of binding to the nucleic acid molecule amplified by the enzyme may be pre-immobilized in the fourth preparation unit 16.

[0071] A moisture-resistant coating agent, such as a sugar, may be used to immobilize the probe DNA. Examples of sugars include trehalose dihydrate, sucrose, lactose, glucose, and fructose. The immobilization of the probe DNA allows the detection unit 33 to detect fluorescence at the immobilization position.

[0072] <Nucleic Acid Amplification Method and Detection Method> A nucleic acid amplification method and a detection method will be described with reference to Figure 5. Figure 5 is a flowchart showing the flow of the nucleic acid amplification method and the detection method. In the nucleic acid amplification method and detection method described using Figure 5, an RNase inhibitor is added when the first solution is prepared in the first preparation step. Although a nucleic acid amplification method and a detection method using a nucleic acid amplification system 100 are described below, nucleic acid amplification and detection may also be performed using conventional laboratory equipment including pipettes, microtubes, test tubes, etc., without using the nucleic acid amplification system 100.

[0073] In S1, the first preparation unit 11 prepares a first liquid by adding at least an RNase inhibitor to a sample containing a detection target (first preparation step). In addition to the nonionic surfactant and the RNase inhibitor, dNTPs and NTPs may be added to the sample. The nonionic surfactant is ethylene glycol (-O-CH ２ -CH ２ The surfactant may be a surfactant having a repeating structure of (-), specifically, Tween-20 (registered trademark) or Triton X-100 (registered trademark). Here, the RNase inhibitor used is one that inhibits the activity of RNases that have the activity of degrading single-stranded RNA. In other words, an RNase inhibitor that does not inhibit the activity of RNase H is added.

[0074] Next, in S2, the first incubator 12 holds the first liquid in a first temperature range for a first time (first incubation step). The first temperature range may be higher than the second temperature range in S4 described below. The first temperature range may be in the range of 80°C or higher and 95°C or lower. The first time may be shorter than the second time in S4 described below.

[0075] Next, in S3, the first incubation unit 12 or the flow path between the first incubation unit 12 and the second preparation unit 13 lowers the temperature of the first liquid. In S3, the nucleic acid amplification system 100 may actively lower the temperature of the first liquid present in the first incubation unit 12 or the first liquid moving from the first incubation unit 12, or may utilize the natural decrease in temperature of the first liquid as it flows through the flow path. In other words, S3 is not an essential step.

[0076] The first preparation step S1 and the first incubation step S2 may be performed in the same reaction system. The step of lowering the temperature of the first liquid S3 may also be performed in the same reaction system as S1 and S2. This configuration eliminates the need for steps such as extracting nucleic acid molecules from the solution in the system and using the extracted nucleic acid molecules in another system, and each step is performed consecutively. This allows nucleic acids to be amplified quickly and easily.

[0077] In S4, after the first incubation step, the second preparation unit 13 adds a primer having a sequence complementary to the sequence of the nucleic acid molecule derived from the target to the first liquid and an enzyme to prepare a second liquid (second preparation step). The nucleic acid molecule may be RNA, and the enzyme may include reverse transcriptase, RNA polymerase, and ribonuclease. The primer and enzyme may be added together with sugars. The primer may include, for example, a first primer corresponding to a first region and a second primer corresponding to a second region different from the first region. In S4, the third preparation unit 15 may add a primer having a sequence complementary to the sequence of the nucleic acid molecule derived from the target to the first liquid (third preparation step). In this case, in S4, the fourth preparation unit 16 may add an enzyme to the third liquid to prepare a second liquid (fourth preparation step).

[0078] The temperature of the solution during the second preparation step may be 37°C or higher and 95°C or lower. The temperature of the solution during the third preparation step and the temperature of the solution during the fourth preparation step may be different; for example, the temperature of the solution during the third preparation step may be higher than the temperature of the solution during the fourth preparation step. For example, the temperature of the solution during the third preparation step may be 80°C or higher and 95°C or lower. The temperature of the solution during the fourth preparation step may be 37°C or higher and 41°C or lower. In S4, the third preparation unit 15 and the flow path between the third preparation unit 15 and the fourth preparation unit 16 may lower the temperature of the solutions flowing through the flow paths containing the first liquid and the third liquid.

[0079] In S5, the second preparation unit 13 or the second incubation unit 14 adds a probe DNA having a predetermined base sequence and capable of binding to the enzymatically amplified nucleic acid molecule to the second liquid (probe DNA addition step). The probe DNA may be added together with sugars. Each molecule of the probe DNA is modified with a fluorescent molecule and a quencher molecule. The distance between the fluorescent molecule and the quencher molecule in the probe DNA that is not bound to the enzymatically amplified nucleic acid molecule is closer than the distance between the fluorescent molecule and the quencher molecule in the probe DNA that is bound to the enzymatically amplified nucleic acid molecule. S5 (probe DNA addition step) may be performed simultaneously with S4 (second preparation step). When S5 is performed simultaneously with S4, step S5 may be performed in the fourth preparation step of S4.

[0080] In S6, the second incubator 14 holds the second liquid in a second temperature range for a second time (second incubation step). The second temperature range may be in the range of 37°C or higher and 41°C or lower.

[0081] The first preparation step S1, the first incubation step S2, the second preparation step S4, and the second incubation step S6 may be performed in the same reaction system. Furthermore, the step of lowering the temperature of the first liquid S3 may also be performed in the same reaction system as steps S1-S2 and S4-S6. The same reaction system may be, for example, within the same flow channel or the same specific region of the flow channel. According to the above configuration, steps such as extracting nucleic acid molecules from the solution in the system and using the extracted nucleic acid molecules in another system are not required, and each step is performed consecutively. This allows nucleic acids to be amplified quickly and easily.

[0082] Nucleic acid derived from the detection target is amplified by steps S1 to S6.

[0083] Next, in S7, the nucleic acid molecules amplified by the enzyme in S1 to S6 are detected based on the intensity of the fluorescence emitted from the fluorescent molecules (detection step).

[0084] In the nucleic acid amplification method described above, a first liquid is prepared by adding at least an RNase inhibitor to a sample containing a target nucleic acid molecule, and the solution is maintained at a first temperature for a first hour. Subsequently, a second liquid containing a primer having a sequence complementary to the sequence of the target nucleic acid molecule and an enzyme is added to the first liquid maintained at the first temperature for the first hour, and the resulting solution is maintained at a second temperature, thereby amplifying the target nucleic acid molecule. Furthermore, in the detection method described above, the nucleic acid molecules amplified by the nucleic acid amplification methods S1 to S6 are detected based on the intensity of fluorescence emitted from the fluorescent molecules. In the nucleic acid amplification and detection methods described above, the RNase inhibitor is added in S1, whereby the RNase inhibitor inhibits the activity of the RNase. This reduces the degree to which the nucleic acid molecules amplified in S6 are degraded by the RNase inhibitor, allowing for more effective amplification of the target nucleic acid.

[0085] Another embodiment of the nucleic acid amplification method and detection method will be described with reference to Fig. 6. Fig. 6 is a flowchart showing the flow of a nucleic acid amplification method and detection method different from the nucleic acid amplification method and detection method described with reference to Fig. 5. In the nucleic acid amplification method and detection method described with reference to Fig. 6, an RNase inhibitor is added at the timing when the second liquid is prepared in the second preparation step.

[0086] In S11, the first preparation unit 11 prepares a first liquid containing nucleic acid molecules derived from the detection target (first preparation step). In this step, the first liquid may be prepared by adding a nonionic surfactant, dNTPs, and NTPs. The nonionic surfactant is ethylene glycol (—O—CH ２ -CH ２ -) repeating structure, specifically, Tween-20 (registered trademark) or Triton X-100 (registered trademark).

[0087] Steps S12 and S13 (the first incubation step and the step of lowering the temperature of the first liquid) are the same as steps S2 and S3 described with reference to FIG. 5, and therefore will not be described further.

[0088] In S14, after the first incubation step, the second preparation unit 13 prepares a second liquid by adding a primer having a sequence complementary to the sequence of the nucleic acid molecule derived from the detection target, an enzyme, and an RNase inhibitor to the first liquid (second preparation step). The nucleic acid molecule may be RNA, and the enzyme may include reverse transcriptase, RNA polymerase, and ribonuclease. The primer may include a first primer corresponding to a first region and a second primer corresponding to a second region different from the first region. The first primer, second primer, enzyme, and RNase inhibitor may also be added together with sugars.

[0089] The second preparation step of S14 may include a third preparation step of adding a primer having a sequence complementary to the sequence of the nucleic acid molecule derived from the target nucleic acid, and a fourth preparation step of adding an enzyme. In this case, in S14, the third preparation unit 15 may prepare the third liquid by adding a primer having a sequence complementary to the sequence of the nucleic acid molecule derived from the target nucleic acid to the first liquid after the first incubation step (third preparation step). Alternatively, in S4, the fourth preparation unit 16 may prepare the second liquid by adding an enzyme to the third liquid (fourth preparation step). Here, when an RNase inhibitor is added to the first liquid in the second preparation step, the third preparation unit 15 may prepare the second liquid by adding the RNase inhibitor to the first liquid together with a primer having a sequence complementary to the sequence of the nucleic acid molecule derived from the target nucleic acid in the third preparation step. Adding the RNase inhibitor before the start of the amplification reaction reduces the degree to which the generated RNA is degraded by RNase in advance, allowing for more effective amplification of the target nucleic acid.

[0090] The temperature of the solution during the second preparation step may be 37°C or higher and 95°C or lower. The temperature of the solution during the third preparation step and the temperature of the solution during the fourth preparation step may be different; for example, the temperature of the solution during the third preparation step may be higher than the temperature of the solution during the fourth preparation step. For example, the temperature of the solution during the third preparation step may be 80°C or higher and 95°C or lower. The temperature of the solution during the fourth preparation step may be 37°C or higher and 41°C or lower. In S4, the third preparation unit 15 and the flow path between the third preparation unit 15 and the fourth preparation unit 16 may lower the temperature of the solutions flowing through the flow paths containing the first liquid and the third liquid.

[0091] Steps S15 to S17 (probe DNA addition step, second incubation step, and detection step) are the same as steps S5 to S7 described with reference to FIG. 5, and therefore will not be described here.

[0092] In the nucleic acid amplification method described with reference to FIG. 6 , a first liquid containing a nucleic acid molecule derived from a target nucleic acid is maintained at a first temperature for a first time. After the first time, a second liquid containing a primer having a sequence complementary to the nucleic acid molecule, an enzyme, and an RNase inhibitor is added to the first liquid, and the second liquid is maintained at a second temperature, thereby amplifying the nucleic acid molecule derived from the target nucleic acid. The RNase inhibitor is added in S14, whereby the RNase inhibitor inhibits the activity of the RNase. This reduces the degree to which the amplified nucleic acid molecule is decomposed by the RNase inhibitor in S15, allowing for more effective amplification of the target nucleic acid. Furthermore, in the above detection method, the nucleic acid molecule amplified by the nucleic acid amplification method in S1 to S6 is detected based on the intensity of fluorescence emitted from the fluorescent molecule.

[0093] By using the nucleic acid amplification methods S1 to S6 and S11 to S16 and the detection methods S1 to S7 and S11 to S17, it is possible to amplify the nucleic acid molecules derived from the target substance and detect the target substance in a short time and easily, since extraction and purification of the target substance derived from the target substance from the first liquid is not required.

[0094] The invention according to the present disclosure has been described above based on the drawings and examples. However, the invention according to the present disclosure is not limited to the above-described embodiments. In other words, the invention according to the present disclosure can be modified in various ways within the scope of the present disclosure, and embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the invention according to the present disclosure. In other words, it should be noted that a person skilled in the art can easily make various modifications or corrections based on the present disclosure. It should also be noted that these modifications or corrections are included in the scope of the present disclosure.

[0095] [Summary] As described above, the nucleic acid amplification method according to aspect 1 of the present disclosure includes a first preparation step of preparing a first liquid by adding at least an RNase inhibitor to a sample containing a target to be detected; a first incubation step of holding the first liquid at a first temperature range for a first time; a second preparation step of preparing a second liquid by adding a primer and an enzyme having a sequence complementary to the sequence of a nucleic acid molecule derived from the target to the first liquid; and a second incubation step of holding the second liquid at a second temperature range for a second time.

[0096] A nucleic acid amplification method according to aspect 2 of the present disclosure includes a first preparation step of preparing a first liquid containing a nucleic acid molecule derived from a detection target; a first incubation step of holding the first liquid at a first temperature range for a first time; a second preparation step of, after the first incubation step, adding an RNase inhibitor, a primer having a sequence complementary to the sequence of the nucleic acid molecule, and an enzyme to the first liquid to prepare a second liquid; and a second incubation step of holding the second liquid at a second temperature range for a second time.

[0097] In the nucleic acid amplification method according to aspect 3 of the present disclosure, in the above-described aspect 1, the second preparation step may include a third preparation step of adding a primer having a sequence complementary to the sequence of the nucleic acid molecule to the first liquid to prepare a third liquid, and a fourth preparation step of adding an enzyme to the third liquid to prepare a second liquid.

[0098] In the nucleic acid amplification method according to aspect 4 of the present disclosure, in the method according to aspect 2, the second preparation step may include a third preparation step of adding an RNase inhibitor and a primer having a sequence complementary to the sequence of the nucleic acid molecule to the first solution to prepare a third solution, and a fourth preparation step of adding an enzyme to the third solution to prepare a second solution.

[0099] The nucleic acid amplification method according to Aspect 5 of the present disclosure, in any one of Aspects 1 to 4, may further include a step of lowering the temperature of the first liquid after the first incubation step.

[0100] In the nucleic acid amplification method according to Aspect 6 of the present disclosure, in any one of Aspects 1 to 5, the first temperature zone may be in the range of 80°C or higher and 95°C or lower.

[0101] In the nucleic acid amplification method according to Aspect 7 of the present disclosure, in any one of Aspects 1 to 6, the second temperature zone may be in the range of 37°C or higher and 41°C or lower.

[0102] In the nucleic acid amplification method according to Aspect 8 of the present disclosure, in any one of Aspects 1 to 7, the first preparation step and the first incubation step may be performed in the same reaction system.

[0103] A nucleic acid amplification method according to aspect 9 of the present disclosure is the same as that of aspect 8, wherein the first preparation step, the first incubation step, the second preparation step, and the second incubation step may be performed in the same reaction system.

[0104] A tenth aspect of the present disclosure provides a nucleic acid amplification method according to any one of the first to ninth aspects, wherein the detection target is a virus or a bacterium, and the enzymes may include a reverse transcriptase, an RNA polymerase, and a ribonuclease.

[0105] A nucleic acid amplification method according to an eleventh aspect of the present disclosure is the method according to any one of the first to tenth aspects, wherein the RNase inhibitor does not inhibit the activity of RNase H.

[0106] In a nucleic acid amplification method according to aspect 12 of the present disclosure, in any one of aspects 1 to 11, the primers may include a first primer corresponding to a first region of the nucleic acid molecule and a second primer corresponding to a second region of the nucleic acid molecule that is different from the first region.

[0107] A nucleic acid amplification method according to aspect 13 of the present disclosure, in any one of aspects 1 to 12, further includes a probe DNA addition step of adding probe DNA having a predetermined base sequence and capable of binding to the nucleic acid molecule amplified by the enzyme to the second liquid, wherein each molecule of the probe DNA is modified with a fluorescent molecule and a quencher molecule, and the distance between the fluorescent molecule and the quencher molecule in the probe DNA that is not bound to the nucleic acid molecule amplified by the enzyme may be closer than the distance between the fluorescent molecule and the quencher molecule in the probe DNA that is bound to the nucleic acid molecule amplified by the enzyme.

[0108] A nucleic acid amplification method according to Aspect 14 of the present disclosure is the method according to Aspect 13, wherein the RNase inhibitor, the primer, the enzyme, and the probe DNA may be added together with sugars.

[0109] A detection method according to aspect 15 of the present disclosure may further comprise, in the nucleic acid amplification method according to aspect 13 or 14, a detection step of detecting the nucleic acid molecules amplified by the enzyme based on the intensity of fluorescence emitted from the fluorescent molecules.

[0110] A nucleic acid amplification system according to aspect 16 of the present disclosure comprises an amplifier and a temperature control device, wherein the amplifier comprises a first preparation unit that prepares a first liquid by adding at least an RNase inhibitor to a sample containing the target of detection, a first incubation unit in which the first liquid is maintained at a first temperature range for a first period of time, a second preparation unit that prepares a second liquid by adding a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from the target of detection and an enzyme to the first liquid, and a second incubation unit in which the second liquid is maintained at a second temperature range for a second period of time, and the temperature control device comprises an installation unit in which the amplifier is installed, and a temperature control unit that controls the temperatures of the first incubation unit and the second incubation unit.

[0111] A nucleic acid amplification system according to aspect 17 of the present disclosure comprises an amplifier and a temperature control device, wherein the amplifier comprises a first preparation unit that prepares a first liquid containing a nucleic acid molecule derived from a detection target, a first incubation unit in which the first liquid is maintained at a first temperature range for a first time, a second preparation unit that prepares a second liquid by adding an RNase inhibitor, a primer having a sequence complementary to the sequence of the nucleic acid molecule, and an enzyme to the first liquid, and a second incubation unit in which the second liquid is maintained at a second temperature range for a second time, and the temperature control device comprises an installation unit in which the amplifier is installed and a temperature control unit that controls the temperatures of the first incubation unit and the second incubation unit.

[0112] In the nucleic acid amplification system of aspect 18 of the present disclosure, in aspects 16 or 17, the first preparation section and the first incubation section may be installed in mutually communicating containers and / or flow paths arranged within the same amplifier.

[0113] In the nucleic acid amplification system of aspect 19 of the present disclosure, in aspect 18, the first preparation unit, the first incubation unit, the second preparation unit, and the second incubation unit may be installed in mutually communicating containers and / or flow paths arranged within the same amplifier.

[0114] In a nucleic acid amplification system according to aspect 20 of the present disclosure, in any one of aspects 16 to 19, the RNase inhibitor, the primer, the enzyme, and a probe DNA having a predetermined base sequence and capable of binding to a nucleic acid molecule amplified by the enzyme may be pre-immobilized in at least one of the second preparation section and the second incubation section.

[0115] A nucleic acid amplification system according to Aspect 21 of the present disclosure is the nucleic acid amplification system of Aspect 20, wherein a sugar is used for the immobilization.

[0116] An embodiment of the present disclosure is described below.

[0117] <Reagents Used> In this example, reagents, enzymes, etc. used in the conventional NASBA (Nucleic Acid Sequence-Based Amplification) method were used.

[0118] (Detection target) Streptococcus mutans bacteria (hereinafter referred to as "mutans bacteria") contained in saliva collected from the subject (Kit) NASBA amplification kit (Kainos Co., Ltd.) (Sugars) Trehalose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (Surfactant) Tween-20 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) RNase inhibitor (Other) Primer (synthesized on request by Fasmac) Molecular beacon (synthesized on request by Fasmac).

[0119] The primer pair used was primer_F17, a forward primer containing a T7 promoter sequence, and primer_R12, a reverse primer containing an RNA polymerase recognition sequence.

[0120] The molecular beacon used for S. mutans was MB3, which contains a fluorescent molecule (carboxyfluorescein, FAM, λex = 492 nm, λem = 518 nm) at the 5' end and a quencher molecule (black hole quencher 1, BHQ1) corresponding to the fluorescent molecule at the 3' end.

[0121] (NASBA reagents) Ribonucleoside triphosphates (NTPs), deoxyribonucleoside triphosphates (dNTPs) Lysis buffer (contains Tris buffer, DMSO, and metal ions) (Enzymes) AMV-RT RNase H T7RNAP.

[0122] Example 1 [Amplification Method] (Preparation of First Liquid: First Adjustment Step and First Incubation Step) 28.5 μL of aqueous saliva solution and 1.5 μL of 1 vol% Tween-20 aqueous solution were added to a 2 mL Eppendorf tube and mixed at room temperature to prepare a first liquid containing nucleic acid molecules derived from the detection target. The Tween-20 concentration in the first liquid after mixing was 0.05 vol%, and the saliva concentration after mixing was 50%. The obtained first liquid was pretreated by heating at 95°C for 10 minutes. Thereafter, the temperature of the first liquid was lowered to room temperature, and 10 mutans streptococci (Cnm positive strains) were added to the first liquid after the temperature drop. ４ 1 / mL was added.

[0123] (Preparation of Solution 3) Subsequently, primers (primer_F17 and primer_R12) and 40 units of an RNase inhibitor were added to Solution 1 to prepare Solution 3. The concentration of each primer after addition was 0.34 μM.

[0124] The prepared third solution was incubated for 3 minutes at 95° C. After incubation, the third solution was used in the next step of preparing second solution without being subjected to further steps such as purification.

[0125] (Preparation of Solution 2: Second Adjustment Step and Second Incubation Step) A molecular beacon (MB) was mixed with a 1 / 6 kit of NASBA kit containing NASBA reagents and enzymes to a final concentration of 1.6 μM, and the mixture was freeze-dried for 3 hours. 5 μL of Solution 3 was added to a 0.2 mL PCR tube, and the temperature of Solution 3 was then lowered to 41°C. The freeze-dried kit was then added to Solution 3, and the mixture was mixed at 41°C to obtain 15 μL of NASBA amplification solution (Solution 2). Solution 2 was then incubated at 41°C for 90 minutes.

[0126] Example 2 A first liquid according to Example 2 was obtained in the same manner as in Example 1, except that the saliva concentration after mixing the saliva solution with the Tween-20 aqueous solution was set to 33%. Thereafter, a third liquid and a second liquid were prepared, and the second liquid was incubated in the same manner as in Example 1.

[0127] Example 3 A first liquid according to Example 2 was obtained in the same manner as in Example 1, except that the saliva concentration after mixing the saliva solution with the Tween-20 aqueous solution was set to 25%. Thereafter, a third liquid and a second liquid were prepared, and the second liquid was incubated in the same manner as in Example 1.

[0128] Example 4 Liquid 1 of Example 2 was obtained in the same manner as in Example 1, except that the saliva concentration after mixing the saliva solution with the Tween-20 aqueous solution was 20%. Then, liquids 3 and 2 were prepared, and liquid 2 was incubated in the same manner as in Example 1.

[0129] Example 5 Liquid 1 of Example 2 was obtained in the same manner as in Example 1, except that the saliva concentration after mixing the saliva solution with the Tween-20 aqueous solution was 10%. Then, liquids 3 and 2 were prepared, and liquid 2 was incubated in the same manner as in Example 1.

[0130] Example 6 Liquid 1 of Example 2 was obtained in the same manner as in Example 1, except that the saliva concentration after mixing the saliva solution with the Tween-20 aqueous solution was 5%. Then, liquids 3 and 2 were prepared, and liquid 2 was incubated in the same manner as in Example 1.

[0131] Comparative Example 1 A third solution according to Comparative Example 1 was obtained in the same manner as in Example 1, except that no primer was added when preparing the third solution. Thereafter, a second solution was prepared and incubated in the same manner as in Example 1.

[0132] Comparative Example 2 A third solution according to Comparative Example 2 was obtained in the same manner as in Example 2, except that no primer was added when preparing the third solution. Thereafter, a second solution was prepared and incubated in the same manner as in Example 1.

[0133] Comparative Example 3 A third solution according to Comparative Example 2 was obtained in the same manner as in Example 3, except that no primer was added when preparing the third solution. Thereafter, a second solution was prepared and incubated in the same manner as in Example 1.

[0134] Comparative Example 4 A third solution according to Comparative Example 4 was obtained in the same manner as in Example 4, except that no primer was added when preparing the third solution. Thereafter, a second solution was prepared and incubated in the same manner as in Example 1.

[0135] Comparative Example 5 A third solution according to Comparative Example 5 was obtained in the same manner as in Example 5, except that no primer was added when preparing the third solution. Thereafter, a second solution was prepared and incubated in the same manner as in Example 1.

[0136] Comparative Example 6 A third solution according to Comparative Example 6 was obtained in the same manner as in Example 6, except that no primer was added when preparing the third solution. Thereafter, a second solution was prepared and incubated in the same manner as in Example 1.

[0137] Comparative Example 7 Liquid 3 of Comparative Example 7 was obtained in the same manner as in Example 1, except that the saliva concentration after mixing the saliva solution with the Tween-20 aqueous solution was 20% and that no RNase inhibitor was added when preparing Liquid 3. Liquid 2 was then prepared and incubated in the same manner as in Example 1.

[0138] Comparative Example 8 A third solution according to Comparative Example 8 was obtained in the same manner as in Comparative Example 7, except that no primer was added when preparing the third solution. Thereafter, a second solution was prepared and incubated in the same manner as in Example 1.

[0139] Comparative Example 9: Liquid 3 of Comparative Example 9 was obtained in the same manner as in Example 1, except that the saliva concentration after mixing the saliva solution with the Tween-20 aqueous solution was 10% and no RNase inhibitor was added when preparing Liquid 3. Liquid 2 was then prepared and incubated in the same manner as in Example 1.

[0140] Comparative Example 10: A third liquid according to Comparative Example 10 was obtained in the same manner as in Comparative Example 9, except that no primer was added when preparing the third liquid. Thereafter, a second liquid was prepared and incubated in the same manner as in Example 1.

[0141] Comparative Example 11: A third solution according to Comparative Example 11 was obtained in the same manner as in Example 1, except that the saliva concentration after mixing the saliva solution with the Tween-20 aqueous solution was 5% and that no RNase inhibitor was added when preparing the third solution. Thereafter, a second solution was prepared and incubated in the same manner as in Example 1.

[0142] Comparative Example 12 A third solution according to Comparative Example 12 was obtained in the same manner as in Comparative Example 11, except that no primer was added when preparing the third solution. Thereafter, a second solution was prepared and incubated in the same manner as in Example 1.

[0143] [Detection and Observation of Amplified Nucleic Acid] The second solution after incubation in each of Examples 1 to 6 and Comparative Examples 1 to 12 was analyzed by electrophoresis using a 3% agarose gel. The gel after electrophoresis was observed using a fluorescence microscope (IX83, manufactured by Olympus) equipped with an LED light emitter (X-Cite XYLIS, manufactured by Excelitas Technologies) and optical filter sets (IX3-FGFPXL; λex: 460-480 nm, λem: 495-540 nm, U-RFP; λex: 535-555 nm, λem: 570-625 nm, manufactured by Olympus).

[0144] [Results] Fig. 7 shows the results of electrophoresis and fluorescence observation for Examples 1 to 3 and Comparative Examples 1 to 3. Fig. 8 shows the results of electrophoresis and fluorescence observation for Examples 4 to 6 and Comparative Examples 4 to 6. Fig. 9 shows the results of electrophoresis and fluorescence observation for Comparative Examples 7 to 12.

[0145] The saliva concentration in the reaction solution was 20% in Example 4 and Comparative Example 7, 10% in Example 5 and Comparative Example 9, and 5% in Example 6 and Comparative Example 11.

[0146] In both Example 4 and Comparative Example 7, where the saliva concentration was 20%, Mutans streptococci in saliva could be detected with equivalent intensity. On the other hand, when Example 5 and Comparative Example 9, where the saliva concentration was 10%, were compared, Example 5, where an RNase inhibitor was added, was able to detect Mutans streptococci in saliva better than Comparative Example 9, where no RNase inhibitor was added. Even in Example 6 and Comparative Example 11, where the saliva concentration was 5%, Example 6, where an RNase inhibitor was added, was able to detect Mutans streptococci in saliva with higher accuracy than Comparative Example 11, where no RNase inhibitor was added.

[0147] These results demonstrate that the nucleic acid amplification method of the present disclosure can amplify and detect target nucleic acids more effectively than conventional nucleic acid amplification methods that do not add RNase inhibitors. Furthermore, as described above, the nucleic acid amplification method of the present disclosure is simple and does not require specialized pretreatment for extracting target nucleic acids from samples. Therefore, it was found that the nucleic acid amplification method of the present disclosure can amplify target nucleic acids simply and effectively.

[0148] Next, another embodiment of the present disclosure will be described below.

[0149] <Reagents Used> In this example, the protocol of the conventional NASBA (Nucleic Acid Sequence-Based Amplification) method was modified to correspond to the nucleic acid amplification method performed by the nucleic acid amplification system according to the present disclosure.

[0150] (Detection target) Influenza virus (Kit) NASBA amplification kit (Kainos Corporation) (Sugars) Trehalose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (Surfactant) Tween-20 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (NASBA enzyme) AMV (Avian Myeloblastosis Virus) - reverse transcriptase RNase H T7 RNA polymerase (Others) RNAse inhibitor DMSO Primer (synthesized by Fasmac) Molecular beacon (synthesized by Fasmac).

[0151] The primer pair used was primer_F10, a forward primer containing a T7 promoter sequence, and primer_R10, a reverse primer containing an RNA polymerase recognition sequence.

[0152] The molecular beacon used for influenza virus detection was MB1, which contains a fluorescent molecule (carboxyfluorescein, FAM, λex = 492 nm, λem = 518 nm) at the 5' end and a corresponding quencher molecule (black hole quencher 1, BHQ1) at the 3' end.

[0153] (NASBA lysis solution) Ribonucleoside triphosphates (NTPs), deoxyribonucleoside triphosphates (dNTPs) Lysis buffer (containing Tris buffer, DMSO, and metal ions).

[0154] (NASBA enzymes) AMV (Avian Myeloblastosis Virus) - reverse transcriptase RNase H T7 RNA polymerase.

[0155] In this example, an experiment was conducted to verify whether the presence of an RNase inhibitor improves the ability to detect influenza viruses in saliva by amplifying the nucleic acid sequence of the influenza virus to be detected using a sample to which an RNase inhibitor had been added. The details of the experiment are described below.

[0156] [Amplification of Nucleic Acid] Example 7 (Preparation of Sample) 28.5 μL of saliva and 1.5 μL of 1.0 vol% Tween-20 aqueous solution were added to a 1.5 mL Eppendorf tube and mixed at room temperature. The Tween-20 concentration in the container after mixing was 0.05 vol%. The saliva concentration in the obtained solution was set to 100 vol%. The obtained solution was pretreated by heating at 95°C for 10 minutes. The temperature was then lowered to room temperature. Inactivated influenza virus and sterilized water were added to the cooled solution to obtain a sample. The influenza virus and sterilized water were adjusted so that the concentration of saliva contained in the sample was 50 vol% and the concentration of influenza virus contained in the sample was 10 ４ The amount of PFU / mL was determined.

[0157] (Preparation of Lyophilized Reagent A) 108 μL of NASBA solution containing dNTPs and NTPs, 6 μL of 10 μM first primer, 6 μL of 10 μM second primer, and 40 μL of trehalose were mixed to obtain 160 μL of solution.

[0158] The resulting solution was dispensed into 12 0.2 mL PCR tubes, each containing 13.4 μL, and frozen at −80°C for 40 minutes. The tubes were then freeze-dried for an additional 3 hours to obtain 12 sets of freeze-dried reagent A. One set of freeze-dried reagent B contained 5.0 × 10 －３ The lyophilized reagent A contained 5 nmol (5 pmol) of each of the first primer and the second primer. The lyophilized reagent A was stored frozen at -20°C until use.

[0159] (Preparation of Lyophilized Reagent B) 30 μL of a solution containing the NASBA enzyme, 30 μL of a 10 μM probe, and 20 μL of trehalose were mixed to obtain 80 μL of a solution.

[0160] The resulting solution was dispensed into 12 0.2 mL PCR tubes at 6.7 μL each and frozen at −80°C for 40 minutes. After that, the tubes were freeze-dried for an additional 3 hours to obtain 12 sets of freeze-dried reagent B. Each set of freeze-dried reagent B contained 2.5 × 10 －２ The lyophilized reagent B contained 25 nmol (25 pmol) of probe. The lyophilized reagent B was stored frozen at -20°C until use.

[0161] (Detection Method) (Preparation of First Solution: First Preparation Step) To a 1.5 mL Eppendorf tube were added 1.0 μL of 1.0 vol% Tween-20 aqueous solution, 15 vol% DMSO, 1.0 μL of 40 units / μL RNase inhibitor, 5.0 μL of sample, and 5.0 μL of sterile water to obtain 20 μL of First Solution.

[0162] (First incubation step) The first liquid was heated and stirred, and kept at 95° C. for 3 minutes, and then cooled to room temperature.

[0163] (Preparation of Second Liquid: Second Preparation Step) While the temperature of the First Liquid was decreasing, the First Liquid was added to one set of freeze-dried reagent A when the temperature of the First Liquid reached 80°C. As a result, the concentrations of the first primer and the second primer contained in the solution after addition were each 0.25 mM. Thereafter, when the mixed solution of the First Liquid and the freeze-dried reagent A reached 41°C, the mixed solution was added to one set of freeze-dried reagent B to obtain the Second Liquid. As a result, the concentration of the probe contained in the Second Liquid became 1.25 mM.

[0164] (Second Incubation Step) Thereafter, the second solution was kept at 41° C. for 30 minutes to carry out the NASBA reaction.

[0165] Example 8 A sample according to Example 8 was prepared in the same manner as in Example 7, except that the sample was adjusted so that the concentration of saliva contained in the sample was 40 vol %. Thereafter, the preparation of the first liquid, the first incubation step, the preparation of the second liquid, and the second incubation step were carried out in the same manner as in Example 7.

[0166] Example 9 A sample according to Example 9 was prepared in the same manner as in Example 7, except that the sample was adjusted so that the concentration of saliva contained in the sample was 30 vol %. Thereafter, the preparation of the first liquid, the first incubation step, the preparation of the second liquid, and the second incubation step were carried out in the same manner as in Example 7.

[0167] Example 10 A sample according to Example 10 was prepared in the same manner as in Example 7, except that the sample was adjusted so that the concentration of saliva contained in the sample was 20 vol %. Thereafter, the preparation of the first liquid, the first incubation step, the preparation of the second liquid, and the second incubation step were carried out in the same manner as in Example 7.

[0168] Example 11 A sample according to Example 11 was prepared in the same manner as in Example 7, except that the sample was adjusted so that the concentration of saliva contained in the sample was 10 vol %. Thereafter, the preparation of the first liquid, the first incubation step, the preparation of the second liquid, and the second incubation step were carried out in the same manner as in Example 7.

[0169] Example 12 A sample according to Example 12 was prepared in the same manner as in Example 7, except that the sample was adjusted so that the concentration of saliva contained in the sample was 0 vol %. Thereafter, the preparation of the first liquid, the first incubation step, the preparation of the second liquid, and the second incubation step were carried out in the same manner as in Example 7.

[0170] <Comparative Example 13> A sample according to Comparative Example 13 was prepared in the same manner as in Example 7, except that 1.0 μL of sterile water was used instead of 1.0 μL of 40 units / μL RNase inhibitor. Thereafter, the preparation of the first liquid, the first incubation step, the preparation of the second liquid, and the second incubation step were carried out in the same manner as in Example 7.

[0171] <Comparative Example 14> A sample according to Comparative Example 14 was prepared in the same manner as in Example 8, except that 1.0 μL of sterile water was used instead of 1.0 μL of 40 units / μL RNase inhibitor. Thereafter, the preparation of the first liquid, the first incubation step, the preparation of the second liquid, and the second incubation step were carried out in the same manner as in Example 7.

[0172] Comparative Example 15 A sample according to Comparative Example 15 was prepared in the same manner as in Example 9, except that 1.0 μL of sterile water was used instead of 1.0 μL of 40 units / μL RNase inhibitor. Thereafter, the preparation of the first liquid, the first incubation step, the preparation of the second liquid, and the second incubation step were carried out in the same manner as in Example 7.

[0173] <Comparative Example 16> A sample according to Comparative Example 16 was prepared in the same manner as in Example 10, except that 1.0 μL of sterile water was used instead of 1.0 μL of 40 units / μL RNase inhibitor. Thereafter, the preparation of the first liquid, the first incubation step, the preparation of the second liquid, and the second incubation step were carried out in the same manner as in Example 7.

[0174] <Comparative Example 17> A sample according to Comparative Example 17 was prepared in the same manner as in Example 11, except that 1.0 μL of sterile water was used instead of 1.0 μL of 40 units / μL RNase inhibitor. Thereafter, the preparation of the first liquid, the first incubation step, the preparation of the second liquid, and the second incubation step were carried out in the same manner as in Example 7.

[0175] <Comparative Example 18> A sample according to Comparative Example 18 was prepared in the same manner as in Example 12, except that 1.0 μL of sterile water was used instead of 1.0 μL of 40 units / μL RNase inhibitor. Thereafter, the preparation of the first liquid, the first incubation step, the preparation of the second liquid, and the second incubation step were carried out in the same manner as in Example 7.

[0176] [Detection and Observation of Amplified Nucleic Acids] 6x loading buffer was added to the second solution after incubation in each of Examples 7 to 12 and Comparative Examples 13 to 18, in an amount equal to 1 / 6 the volume of the second solution, and bands were confirmed by electrophoresis. The gel after electrophoresis was observed using a fluorescence microscope (IX83, manufactured by Olympus) equipped with an LED light emitter (X-Cite XYLIS, manufactured by Excelitas Technologies) and an optical filter set (IX3-FGFPXL; λex: 460-480 nm, λem: 495-540 nm, U-RFP; λex: 535-555 nm, λem: 570-625 nm, manufactured by Olympus). Furthermore, the second solution after incubation was directly observed for fluorescence.

[0177] [Results] FIG. 10 shows the results of electrophoresis and fluorescence observation in Examples 7 to 12 and Comparative Examples 13 to 18.

[0178] Regarding the detection results, 10 ４ If an amplification product is confirmed as a result of performing each treatment using a sample containing PFU / mL of the target substance, it can be said that the nucleic acid sequence of the target substance has been sufficiently amplified and the target substance can be detected.

[0179] In Examples 7 to 12, an RNase inhibitor was added to a specimen containing influenza virus to prepare a first solution, and the first incubation step, second preparation step, and second incubation step were carried out. As a result, as shown in Figure 10, clear bands were observed in Examples 7 to 12. Therefore, it was confirmed that the nucleic acid sequence of influenza virus can be amplified by the methods of Examples 7 to 12.

[0180] On the other hand, in Comparative Examples 13 to 18, the first solution was prepared without adding an RNase inhibitor to the specimen, and the other steps were the same as those of Examples 7 to 12. As a result, as shown in FIG. 10 , no clear band corresponding to influenza virus nucleic acid was observed in Comparative Examples 13 to 18. The results of Examples 7 to 12 and Comparative Examples 13 to 18 demonstrated that adding an RNase inhibitor to the specimen can effectively amplify the influenza virus nucleic acid sequence. Furthermore, since adding an RNase inhibitor to the specimen can effectively amplify the influenza virus nucleic acid sequence, it is believed that the RNase inhibitor inhibits the activity of RNase contained in saliva, thereby reducing the adverse effects on amplification of the target influenza virus nucleic acid sequence, which is caused by RNase degradation of the nucleic acid sequence by the RNase.

[0181] 10, as a result of fluorescence observation of the second liquid, clear fluorescence was observed in Examples 7 to 12, but no clear fluorescence was observed in Comparative Examples 13 to 18. Thus, the results of fluorescence observation also confirmed that the nucleic acid sequence of influenza virus can be amplified by the methods of Examples 7 to 12.

[0182] As described above, it has been found that the nucleic acid sequence of influenza virus can be effectively amplified and detected by using the detection method according to the present disclosure.

[0183] REFERENCE SIGNS LIST 10 Cartridge (amplifier) ​​11 First preparation section 12 First incubation section 13 Second preparation section 14 Second incubation section 15 Third preparation section 16 Fourth preparation section 31, 71 Installation section 22A, 22B Temperature control area 30 Detection device 32 Temperature control section 33 Detection section 70 Temperature control device 100 Nucleic acid amplification system 1000 Detection system S1 First preparation step S2 First incubation step S3 Step of lowering the temperature of the first liquid S4 Second preparation step S5 Second incubation step S6 Probe DNA addition step S7 Detection step

Claims

1. A nucleic acid amplification method comprising: a first preparation step of preparing a first liquid by adding at least an RNase inhibitor to a specimen containing a detection target; a first incubation step of holding the first liquid at a first temperature range for a first hour; a second preparation step of preparing a second liquid by adding a primer having a sequence complementary to a sequence of a nucleic acid molecule derived from the detection target and an enzyme to the first liquid; and a second incubation step of holding the second liquid at a second temperature range for a second hour.

2. A method for amplifying nucleic acid comprising: a first preparation step of preparing a first liquid containing a nucleic acid molecule derived from a detection target; a first incubation step of holding the first liquid at a first temperature range for a first time; a second preparation step of, after the first incubation step, adding an RNase inhibitor, a primer having a sequence complementary to the sequence of the nucleic acid molecule, and an enzyme to the first liquid to prepare a second liquid; and a second incubation step of holding the second liquid at a second temperature range for a second time.

3. The nucleic acid amplification method according to claim 1, wherein the second preparation step includes: a third preparation step of adding a primer having a sequence complementary to the sequence of the nucleic acid molecule to the first liquid to prepare a third liquid; and a fourth preparation step of adding an enzyme to the third liquid to prepare a second liquid.

4. The nucleic acid amplification method according to claim 2, wherein the second preparation step includes: a third preparation step of adding an RNase inhibitor and a primer having a sequence complementary to the sequence of the nucleic acid molecule to the first liquid to prepare a third liquid; and a fourth preparation step of adding an enzyme to the third liquid to prepare a second liquid.

5. The method for amplifying nucleic acid according to any one of claims 1 to 4, further comprising a step of lowering the temperature of the first liquid after the first incubation step.

6. A nucleic acid amplification method according to any one of claims 1 to 5, wherein the first temperature zone is in the range of 80°C or higher and 95°C or lower.

7. A nucleic acid amplification method according to any one of claims 1 to 6, wherein the second temperature zone is in the range of 37°C or higher and 41°C or lower.

8. A nucleic acid amplification method according to any one of claims 1 to 7, wherein the first preparation step and the first incubation step are carried out in the same reaction system.

9. The nucleic acid amplification method according to claim 8, wherein the first preparation step, the first incubation step, the second preparation step, and the second incubation step are carried out in the same reaction system.

10. The nucleic acid amplification method according to any one of claims 1 to 9, wherein the detection target is a virus or a bacterium, and the enzyme includes a reverse transcriptase, an RNA polymerase, and a ribonuclease.

11. The nucleic acid amplification method according to any one of claims 1 to 10, wherein the RNAse inhibitor does not inhibit the activity of RNase H.

12. A nucleic acid amplification method according to any one of claims 1 to 11, wherein the primers include a first primer corresponding to a first region of the nucleic acid molecule and a second primer corresponding to a second region of the nucleic acid molecule different from the first region.

13. A nucleic acid amplification method according to any one of claims 1 to 12, further comprising a probe DNA addition step of adding to the second liquid a probe DNA having a predetermined base sequence and capable of binding to the nucleic acid molecule amplified by the enzyme, each molecule of the probe DNA being modified with a fluorescent molecule and a quenching molecule, and the distance between the fluorescent molecule and the quenching molecule in the probe DNA not bound to the nucleic acid molecule amplified by the enzyme is closer than the distance between the fluorescent molecule and the quenching molecule in the probe DNA bound to the nucleic acid molecule amplified by the enzyme.

14. The method for amplifying nucleic acid according to claim 13, wherein the RNase inhibitor, the primer, the enzyme, and the probe DNA are added together with sugars.

15. A method for amplifying nucleic acid according to claim 13 or 14, further comprising a detection step of detecting the nucleic acid molecule amplified by the enzyme based on the intensity of the fluorescence emitted from the fluorescent molecule.

16. A nucleic acid amplification system comprising an amplifier and a temperature control device, wherein the amplifier comprises: a first preparation unit that prepares a first liquid by adding at least an RNase inhibitor to a sample containing the detection target; a first incubation unit that holds the first liquid at a first temperature zone for a first time; a second preparation unit that prepares a second liquid by adding a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from the detection target and an enzyme to the first liquid; and a second incubation unit that holds the second liquid at a second temperature zone for a second time; and the temperature control device comprises: an installation unit that installs the amplifier; and a temperature control unit that controls the temperatures of the first incubation unit and the second incubation unit.

17. A nucleic acid amplification system comprising an amplifier and a temperature control device, wherein the amplifier comprises: a first preparation unit that prepares a first liquid containing a nucleic acid molecule derived from a detection target; a first incubation unit in which the first liquid is maintained at a first temperature zone for a first time; a second preparation unit that prepares a second liquid by adding an RNase inhibitor, a primer having a sequence complementary to the sequence of the nucleic acid molecule, and an enzyme to the first liquid; and a second incubation unit in which the second liquid is maintained at a second temperature zone for a second time, and the temperature control device comprises: an installation unit that installs the amplifier; and a temperature control unit that controls the temperatures of the first incubation unit and the second incubation unit.

18. The nucleic acid amplification system according to claim 16 or 17, wherein the first preparation section and the first incubation section are installed in mutually communicating containers and / or flow paths arranged within the same amplifier.

19. The nucleic acid amplification system described in claim 18, wherein the first preparation section, the first incubation section, the second preparation section, and the second incubation section are installed in mutually communicating containers and / or flow paths arranged within the same amplifier.

20. A nucleic acid amplification system described in any one of claims 16 to 19, wherein the RNase inhibitor, the primer, the enzyme, and a probe DNA having a predetermined base sequence and capable of binding to a nucleic acid molecule amplified by the enzyme are immobilized in advance to at least one of the second preparation section and the second incubation section.

21. The nucleic acid amplification system according to claim 20, wherein the immobilization is performed using a sugar.

Images