Primer sets, probes, and detection kits

Abstract

To more effectively amplify the target nucleic acid. [Solution] The primer set is represented by SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 10, or SEQ ID NOs: 9 and 12.

Description

[Technical Field]

[0001] This disclosure relates to a primer set, probe, and detection kit for detecting Cnm-positive Streptococcus mutans. [Background technology]

[0002] When testing whether a subject is infected with a virus or bacteria, the target nucleic acid derived from the virus or bacteria contained in the sample taken from the subject is amplified using a predetermined amplification method, and the amplified target nucleic acid is detected. The target nucleic acid is extracted from the sample through specialized pretreatment using instruments and equipment used in molecular biological research, etc.

[0003] For example, when the target nucleic acid is RNA, an amplification method is the NASBA (Nucleic A1cid Sequence-Based Amplification) method (Non-Patent Literature 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Compton, J. Nucleic acid sequence-based amplification. Nature 350, 91-92(1991). [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] There is still room for improvement in methods for more effectively amplifying target nucleic acids. [Means for solving the problem]

[0006] The primer set according to one aspect of the present disclosure is represented by SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 10, or SEQ ID NO: 9 and SEQ ID NO: 12.

[0007] The probe according to one aspect of the present disclosure specifically recognizes a part of the nucleic acid sequence commonly possessed by Cnm-positive Streptococcus mutans bacteria.

Advantages of the Invention

[0008] According to one aspect of the present disclosure, a target nucleic acid can be amplified more effectively.

Brief Description of the Drawings

[0009] [Figure 1] It is a diagram showing an overview of a diagnostic system according to an embodiment of the present disclosure. [Figure 2] It is an external view showing an example of a cartridge included in a nucleic acid amplification system. [Figure 3] It is a general view showing an example of a flow path substrate and a temperature control device according to an embodiment of the present disclosure. [Figure 4] It is a block diagram showing the configuration of a detection system according to an embodiment of the present disclosure. [Figure 5] It is a flowchart showing the flow of a nucleic acid amplification method and a detection method. [Figure 6] It is a diagram showing the gene sequence of the TW295 strain, which is one of the Cnm-positive strains. [Figure 7] It is a diagram showing the electrophoresis results according to Examples 1 to 3. [Figure 8] It is a diagram showing the electrophoresis results according to 4 to 7 and Comparative Examples 1 to 2. [Figure 9] It is a diagram showing the electrophoresis results according to Examples 8 to 10 and Comparative Examples 3 to 5. [Figure 10] It is a diagram showing the electrophoresis results according to Examples 11 to 14 and Comparative Examples 6 to 7. [Figure 11]This figure shows the electrophoresis results for Examples 15-16 and Comparative Example 8. [Figure 12] This figure shows the electrophoresis results and fluorescence observation results for Examples 17-18 and Comparative Examples 9-12. [Figure 13] This figure shows the electrophoresis results and fluorescence observation results for Examples 19-20 and Comparative Examples 13-16. [Figure 14] This figure shows the electrophoresis results for Example 21 and Comparative Examples 17-18. [Modes for carrying out the invention]

[0010] [Embodiment] One embodiment of this disclosure will be described in detail below.

[0011] <Technical Concept of This Disclosure> It has been known that some species of Streptococcus mutans are Cnm-positive. Cnm-positive Streptococcus mutans can be an indicator of the risk of inflammatory bowel disease (ulcerative colitis), stroke, and dementia. Therefore, it is necessary to detect Cnm-positive Streptococcus mutans in the saliva of subjects. Hereafter, Streptococcus mutans will also be referred to as "Streptococcus mutans." Furthermore, Cnm-positive Streptococcus mutans will also be referred to as "Cnm-positive strains."

[0012] The NASBA method is a method for amplifying nucleic acids, and by detecting the amplified nucleic acids, the presence of substances containing those nucleic acids can be detected.

[0013] Here, Cnm-positive strains possess both a nucleic acid sequence common to all Cnm-positive strains and a nucleic acid sequence unique to each species. Hereafter, the nucleic acid sequence common to all Cnm-positive strains will also be referred to as the "common nucleic acid sequence." The inventors conducted extensive research and designed a primer set capable of recognizing and amplifying sequences contained within the common nucleic acid sequence.

[0014] By performing the NASBA method using the primer set described herein, nucleic acid sequences included in the common nucleic acid sequence of Cnm-positive strains can be amplified. This makes it possible to detect the presence of Cnm-positive strains in a sample.

[0015] Furthermore, while Cnm-positive strains include multiple species, using different primer sets for each species to detect each Cnm-positive strain is cumbersome. By realizing a primer set that specifically recognizes a common nucleic acid sequence of Cnm-positive strains, the gene sequences of multiple Cnm-positive strains can be amplified with a single primer set. Therefore, using the primer set according to this disclosure allows for simpler detection of multiple Cnm-positive strains than using multiple primer sets.

[0016] Furthermore, the inventors have designed a probe (molecular beacon) capable of specifically recognizing nucleic acid sequences amplified by the primer set according to this disclosure. The nucleic acid sequences amplified by the primer set according to this disclosure are common nucleic acid sequences of Cnm-positive strains. Therefore, by using this probe, gene amplification products from multiple types of Cnm-positive strains obtained using the primer set according to this disclosure can be detected using only one type of probe.

[0017] The following describes an overview of a nucleic acid amplification method and nucleic acid amplification system using a detection kit including a primer set and probe. The nucleic acid amplification method includes a pretreatment step, a first preparation step, a first incubation step, a second preparation step, and a second incubation step. In the pretreatment step, a bacterial cell treatment solution is prepared by adding a nonionic surfactant to a sample containing the target for detection and heating it. In the first preparation step, a first solution is prepared by adding a NASBA reagent solution containing a NASBA reagent and a primer set having a sequence complementary to the sequence of nucleic acid molecules derived from the target for detection to the bacterial cell treatment solution. This primer set is capable of specifically recognizing a portion of the common nucleic acid sequence commonly found in Cnm-positive Streptococcus mutans bacteria and amplifying this common nucleic acid sequence. In the first incubation step, the prepared first solution is maintained at a first temperature for one hour. In the second preparation step, a second solution is prepared by adding a NASBA enzyme solution containing a probe and enzyme capable of detecting the amplification product from the primer set to the first solution after the first incubation step. In the second incubation step, the second solution is kept at the second temperature for two hours.

[0018] Furthermore, in the first preparation step, a first solution may be prepared by adding a surfactant to improve the extraction efficiency of the target substance contained in the sample. The surfactant may be, for example, a nonionic surfactant.

[0019] By employing a nucleic acid amplification method and nucleic acid amplification system using a detection kit including a primer set and probe according to one aspect of this disclosure, nucleic acids contained in a collected sample can be easily extracted and amplified in a short time. Therefore, a detection method and detection system using the nucleic acid amplification method and nucleic acid amplification system can easily and sensitively detect bacteria and viruses contained in a sample in a short time. Here, the sample may be saliva, urine, sweat, nasal secretions, blood, cells, etc., collected from the body of a subject, or it may be soil and water collected from an object to be tested, such as the ground, rivers, or the sea. Alternatively, the sample may be a substance attached to the surface of an object to be tested, such as a handrail, door, clothing, shoes, or toilet. The target of nucleic acid amplification and detection using the nucleic acid amplification method and nucleic acid amplification system using a detection kit including a primer set and probe according to one aspect of this disclosure is bacteria, specifically Streptococcus mutans (hereinafter referred to as Streptococcus mutans) that shows Cnm positivity. The type of bacteria contained in the sample is not limited to one type, but may be two or more types. For example, the target of detection may be at least one species of Streptococcus mutans that is Cnm-positive. The subjects are not limited to humans, but may be any organism that can contain the target of detection, such as mammals, birds, reptiles, or amphibians.

[0020] Hereinafter, a diagnostic system 1 for detecting a target substance in a sample will be described as an example, applying a nucleic acid amplification method and nucleic acid amplification system using a detection kit including a primer set and probe according to one embodiment of the present disclosure.

[0021] <Configuration of the diagnostic system> Figure 1 is a diagram showing an overview of a diagnostic system 1 according to one embodiment of the present disclosure. As shown in Figure 1, the diagnostic system 1 comprises a detection system 1000, a patient terminal device 40, a server 50, and a medical personnel terminal device 60. The detection system 1000 comprises a cartridge 10 (amplifier) ​​and a detection device 30. The terminal device 40 and the server 50 may be connected to each other in a manner that allows them to communicate with each other.

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

[0023] The detection device 30 is a device that detects nucleic acids originating from the target of detection when they are 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, temperature control unit 32, and detection unit 33 will be described later. For example, when detecting the target nucleic acid by fluorescently labeling it, the detection device 30 has the function of a fluorescence microscope capable of measuring fluorescence intensity. In one example, the detection device 30 may output the measured fluorescence intensity as the detection value. The detection device 30 may include a communication unit (not shown) and transmit the detection value to the subject's terminal device 40. The detection device 30 may determine whether nucleic acids originating from the target of detection are present at a certain concentration or higher. If nucleic acids originating from the target of detection are present at a certain concentration or higher, the detection device 30 may determine that the sample is positive. Alternatively, the detection device 30 may be a measuring device that measures the concentration of nucleic acids originating from the target of detection.

[0024] Hereinafter, the system consisting of the cartridge 10, the installation unit 31 and temperature control unit 32 of the detection device 30, and the temperature control device 70 will be referred to as the 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 of the detection device 30.

[0025] The terminal device 40 may output the analysis results obtained from the detection device 30 to the server 50, for example, by analyzing the detected values ​​using an application. In this case, the server 50 may store the analysis results for each subject. Alternatively, the terminal device 40 may output the detected values ​​obtained from the detection device 30 to the server 50. In this case, the server 50 may analyze the detected 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 subject's terminal device 40.

[0026] In the terminal device 60 that acquires the analysis results, for example, diagnostic information with findings by medical professionals may be input, and this diagnostic information may be transmitted to the subject's terminal device 40.

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

[0028] <Nucleic Acid Amplification System 100> Figure 2 is an external view showing an example of a cartridge 10 included in the nucleic acid amplification system 100. Figure 3 is an overview view showing an example of a flow channel substrate 4 and a temperature control device 70 included in the cartridge 10. In Figure 3, for the sake of simplicity, the detection device 30, which includes a temperature control unit 32 that controls the temperature control device 70, is not shown.

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

[0030] The bottle section 22 may contain a sample collected from the user. The main body section 21 may receive the liquid containing the sample contained in the bottle section 22 and may include a flow channel substrate 4 (see Figure 3) containing a reagent that reacts with the nucleic acid if the sample contains nucleic acid originating from the target for detection.

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

[0032] (Cartridge 10) As described above, the cartridge 10 comprises a liquid receiving section 41, a first preparation section 11, a first incubation section 12, a second preparation section 13, and a second incubation section 14.

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

[0034] (Bottle section 22) The first preparation section 11 provided in the bottle section 22 is, for example, a container capable of holding liquid. The first preparation section 11 may be made of a transparent material, or it may be made of an opaque material. In this case, the liquid contained in the first preparation section 11 flows from the liquid receiving section 41 to the second preparation section 13. The first preparation section 11 may also be a flow path through which the sample to be supplied to the liquid receiving section 41 flows. In this case, the sample supplied to the liquid receiving section 41 flows along the flow path from the first preparation section 11 to the second preparation section 13. The first preparation section 11 may also be a flow path that meanders multiple times. The meandering of the flow path promotes agitation of the sample flowing through the flow path. In the first preparation section 11, the first solution may be prepared by adding a NASBA reagent solution containing a NASBA reagent and a primer set having a sequence complementary to the sequence of nucleic acid molecules derived from the target to a bacterial cell processing solution containing the target for detection.

[0035] The bacterial cell treatment solution is mixed with the NASBA reagent solution after the temperature has been lowered to room temperature. The bacterial cell treatment solution may be prepared by pre-treating the sample by adding a nonionic surfactant and then heating it at 95°C for 3 minutes. In addition, dNTPs and NTPs may be added to the sample. The nonionic surfactant may be a surfactant having a repeating structure of ethylene glycol (-O-CH2-CH2-), and specifically may be Tween-20® or Triton X-100®.

[0036] For example, when collecting saliva as a sample, the user attaches saliva to the sampler 23 by inserting it into the user's mouth. As shown in Figure 2, after attaching saliva to the sampler 23, the user inserts the sampler 23 into the bottle section 22. When the user inserts the sampler 23 into the bottle section 22, the saliva attached to the sampler 23 is mixed with a buffer solution containing a nonionic surfactant contained in the bottle section 22 to prepare a bacterial cell treatment solution. Furthermore, the bacterial cell treatment solution is pre-treated by heating it at 95°C for 3 minutes in the first incubation section 12.

[0037] Thus, the NASBA method allows the use of samples that have not undergone specialized pretreatment for nucleic acid extraction and purification, making it easier to amplify the target nucleic acid.

[0038] In the first incubation section 12, the bacterial cell treatment solution is maintained at a predetermined temperature range for a predetermined time. The predetermined time for maintaining the first incubation section 12 at the predetermined temperature may be, for example, 3 to 10 minutes. This predetermined time may be shorter than the second time for maintaining the second incubation section 14 at the second temperature, as described later.

[0039] The first incubation unit 12 may maintain the bacterial cell processing solution at a predetermined temperature range for a predetermined time, and then lower the temperature of the bacterial cell processing solution. The temperature reduction of the bacterial cell processing solution may be performed at a location other than the first incubation unit 12. For example, the nucleic acid amplification system 100 may have an additional flow channel (not shown) between the first incubation unit 12 and the second preparation unit 13, and after the first incubation unit 12 maintains the bacterial cell processing solution at a predetermined temperature range, the flow channel may lower the temperature of the bacterial cell processing solution.

[0040] (Main body 21) As described above, the main body 21 has a flow channel substrate 4. As shown in Figure 3, the flow channel substrate 4 may include a liquid receiving section 41 for receiving liquid, a branched flow channel 42, a flow channel 45, and a storage section 46 for storing the liquid that has flowed through the flow channel substrate 4. The flow channel 45 may have a first region 451 where a reagent including a primer set is placed, and a second region 452 where a reagent including an enzyme for nucleic acid amplification is placed at a different position from the first region 451. The flow channel 45 in the flow channel 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 have a window section 211 so that the inside of the main body 21 can be seen.

[0041] The branched channel 42 may be a channel that connects to each of the multiple channels provided by the liquid receiving section 41 and the channel 45. In other words, the multiple channels provided by the channel 45 may branch off from the branched channel 42 connected to the liquid receiving section 41.

[0042] The multiple channels provided in the flow path 45 may be channels for flowing liquid. Each of the multiple channels may be a channel that receives liquid flowing in from the liquid receiving section 41 and flows it to the storage section 46 which is connected to each of the multiple channels. Each of the multiple channels may be in communication from the liquid receiving section 41 to the storage section 46.

[0043] In the flow channel substrate 4, the branched flow channel 42, the flow channel 45, and the storage section 46 correspond to the second preparation section 13 and the second incubation section 14.

[0044] In the second preparation unit 13 and the second incubation unit 14, the preparation of the first liquid, the first incubation step, the preparation of the second liquid, and the second incubation step are performed.

[0045] As shown in Figure 3, the second preparation unit 13 may include a third preparation unit 15 for adding a primer set having a sequence complementary to the sequence of nucleic acid molecules derived from the target for detection, and a fourth preparation unit 16 for adding an enzyme. In this case, in the third preparation unit 15, a NASBA reagent solution containing a primer set having a sequence complementary to the sequence of nucleic acid molecules derived from the target for detection may be added to the bacterial cell treatment solution to prepare the first solution. The prepared first solution may also be heated in the third preparation unit 15. That is, the first preparation step and the first incubation step may be performed in the third preparation unit 15 of the second preparation unit 13. In addition, in the fourth preparation unit 16, a NASBA enzyme solution may be added to the first solution to prepare the second solution. In this case, as shown in Figure 3, in the flow channel substrate 4, the first region 451 where the reagent containing the primer set is located corresponds to the third preparation unit 15, and the second region 452 where the reagent containing the enzyme that amplifies nucleic acid is located at a different position from the first region 451 corresponds to the fourth preparation unit 16.

[0046] The NASBA reagent solution may be prepared by dissolving the NASBA reagent with NASBA dissolving solution and a primer set. Alternatively, when using the NASBA reagent solution as a dried reagent, it may be prepared by further dissolving it with trehalose and freeze-drying it for 3 hours.

[0047] A primer set may include a first primer corresponding to a first region of a 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 target in the sense direction, and the second primer may be a reverse primer that amplifies the nucleic acid molecule in the antisense direction.

[0048] The primer set relating to this disclosure may be any of the following primer sets 1 to 7. In each primer set, the first primer may be a forward primer containing a T7 promoter sequence, and the second primer may be a reverse primer containing an RNA polymerase recognition sequence. Hereinafter, when the primer sets 1 to 7 are not distinguished, they will simply be referred to as "primer set". • First primer set: A set consisting of the first primer represented by Sequence ID No. 1 and the second primer represented by Sequence ID No. 2. • Second primer set: A set consisting of the first primer represented by Sequence ID No. 3 and the second primer represented by Sequence ID No. 4. • Third primer set: A set consisting of the first primer represented by SEQ ID NO: 5 and the second primer represented by SEQ ID NO: 6. • Fourth primer set: A set consisting of the first primer represented by SEQ ID NO: 7 and the second primer represented by SEQ ID NO: 8. • Fifth primer set: A set consisting of the first primer represented by SEQ ID NO: 9 and the second primer represented by SEQ ID NO: 10. • Sixth primer set: A set consisting of the first primer represented by SEQ ID NO: 11 and the second primer represented by SEQ ID NO: 10. • Seventh primer set: A set consisting of the first primer represented by Sequence ID No. 9 and the second primer represented by Sequence ID No. 12.

[0049] In the third preparation section 15 of the second incubation section 14, the first liquid is maintained at a first temperature for a first hour. The first hour may be, for example, 3 to 10 minutes. The first hour may be shorter than the second hour for which the second liquid is maintained at a second temperature in the fourth preparation section 16 of the second incubation section 14, which will be described later.

[0050] The second incubation unit 14 may maintain the first solution in a first temperature range for a first hour, and then lower the temperature of the first solution. The temperature reduction of the first solution may be performed at a location other than the second incubation unit 14. For example, the nucleic acid amplification system 100 may have an additional flow path (not shown) between the third preparation unit 15 and the fourth preparation unit 16, and after the second incubation unit 14 maintains the first solution in a first temperature range, this flow path may lower the temperature of the first solution.

[0051] In the fourth preparation section 16 of the second preparation section 13, the second solution is prepared by adding a NASBA enzyme solution containing a probe and enzyme capable of detecting the amplification product by the primer set to the first solution.

[0052] The NASBA enzyme solution may be prepared by dissolving the NASBA enzyme in sterile water and a probe aqueous solution. Furthermore, when using the enzyme solution as a dry reagent, it may be prepared by further dissolving it with trehalose, then adding the NASBA reagent solution, and freeze-drying for 3 hours.

[0053] The probe may be a probe that specifically recognizes a portion of the nucleic acid sequence commonly present in Cnm-positive strains. Specifically, the probe may be the probe represented by Sequence ID No. 13. Hereafter, unless otherwise specified, the probe relating to this disclosure will be referred to as "probe" or "probe DNA".

[0054] The specified nucleic acid molecule may be DNA or RNA. In addition, the enzymes added in the second preparation unit 13 may include reverse transcriptase, DNA polymerase, RNA polymerase, and ribonuclease.

[0055] In the fourth preparation section 16 of the second incubation section 14, the second solution is maintained in the second temperature zone for two hours. The second hour can be any time required for the target nucleic acid to be amplified to a detectable level, for example, 30 minutes or more and 90 minutes or less.

[0056] The first preparation unit 11 and the first incubation unit 12 are installed in the bottle portion 22 of the cartridge 10, which are in communication with each other. The first preparation unit 11 and the first incubation unit 12 may be installed in a container and / or flow channel that are in communication with each other. In this configuration, the nucleic acid amplification system 100 allows the bacterial cell treatment solution prepared in the first preparation unit 11 to spontaneously reach the first incubation unit 12 along the microflow channel. Therefore, the nucleic acid amplification system 100 does not require the bacterial cell treatment solution to be transferred to an incubation container.

[0057] 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 having containers and / or channels that communicate with each other. With this arrangement, the nucleic acid amplification system 100 does not need to take out and transfer each liquid after preparing the bacterial cell treatment solution, after preparing the first solution, after holding the first solution in the first temperature zone, after preparing the second solution, and after holding the second solution in the second temperature zone, so that each step can be performed within the cartridge 10.

[0058] (1) The first primer, (2) the second primer, (3) the enzyme, and (4) the probe DNA may be pre-immobilized in at least one of the second preparation section 13 and the second incubation section 14. (1) The first primer, (2) the second primer, (3) the enzyme, and (4) the probe DNA may not be immobilized in the second incubation section 14, but may be pre-immobilized in the second preparation section 13. (1) The first primer, (2) the second primer, (3) the enzyme, and (4) the probe DNA may be immobilized as a coating. According to this, the nucleic acid amplification system 100 can supply each component to the first or second solution without later adding the first primer, the second primer, the enzyme, and the probe DNA. Hereafter, the probe DNA will also be referred to as a molecular beacon.

[0059] (1) The first primer and (2) the second primer may be pre-immobilized in the third preparation section 15, and (3) the enzyme and (4) the probe DNA may be pre-immobilized in the fourth preparation section 16.

[0060] A moisture-resistant coating agent may be used to immobilize the first primer, second primer, enzyme, and probe DNA, for example, a sugar may be used. Examples of sugars include trehalose dihydrate, sucrose, lactose, glucose, and fructose. By immobilizing the first primer, second primer, enzyme, and probe DNA in this way, the nucleic acid amplification system 100 allows each component to be stored for a long period of time in an unused cartridge 10.

[0061] Furthermore, the temperature control device 70 may 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.

[0062] The temperature control device 70 may control the first temperature range to a higher temperature range than the second temperature range. The first temperature range may be a temperature for inactivating the target to be detected, and the second temperature range may be a temperature for activating the enzyme.

[0063] The first temperature zone may be in the range of 80°C to 95°C, and the second temperature zone may be in the range of 37°C to 41°C.

[0064] 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 temperatures of the liquid whose temperature is controlled by the temperature control device 70. When the first and second temperature zones are liquid temperatures, for example, average temperature or core temperature may be used. Here, the average temperature may be, for example, the average of the liquid temperatures over a predetermined period of time. The core temperature may be, for example, the temperature midway between the highest and lowest values ​​of the liquid temperature. The core temperature can be calculated as the sum of the highest and lowest temperatures divided by 2. The detection device 30 may include a temperature sensor that detects the liquid temperature of the liquid whose temperature is controlled by the first incubation unit 12 and the second incubation unit 14. The first incubation unit 12 and the second incubation unit 14 may heat the liquid whose temperature is detected by the temperature sensor so as to maintain the liquid temperature detected by the temperature sensor.

[0065] <Detection System> Figure 4 is a block diagram showing the configuration of a detection system 1000 according to one embodiment of the present disclosure. As shown in Figure 4, the detection system 1000 comprises a cartridge 10 and a detection device 30.

[0066] As shown in Figure 2, the cartridge 10 comprises a main body 21 having a flow channel substrate 4, and a bottle 22 having a first preparation section 11 and a first incubation section 12. As shown in Figure 3, the flow channel substrate 4 comprises a second preparation section 13 and a second incubation section 14. In the detection system 1000, the cartridge 10 may have a structure (detection preparation section) in the second incubation section 14 for adding probe DNA having a predetermined base sequence and capable of binding to nucleic acid molecules amplified by the enzyme to the second solution. The detection preparation section may be installed downstream of the second incubation section 14. As described above, the DNA may be contained in the NASBA enzyme solution. In this case, the step of adding probe DNA to the second solution in the detection preparation section is unnecessary.

[0067] The detection preparation unit may, for example, include multiple reaction fields, and each reaction field may have probe DNA capable of binding to nucleic acid molecules derived from different targets added to the second solution. The detection preparation unit may be located in the second preparation unit 13 and / or the second incubation unit 14. For example, probe DNA capable of binding to the nucleic acid molecule of influenza virus and probe DNA capable of binding to the nucleic acid molecule of coronavirus (SARS-CoV-2) may be added to the second solution in their respective reaction fields. This makes it possible to detect multiple targets with a single sample collection.

[0068] As described above, the detection device 30 comprises an installation unit 31, a temperature control unit 32, and a detection unit 33. The temperature control unit 32 can individually control the temperatures of the temperature control area 72A and temperature control area 72B of the temperature control device 70 shown in Figure 3.

[0069] The detection unit 33 may include 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 also include, for example, a fluorescence microscope function. The detection unit 33 may include, for example, an excitation light source for exciting the probe DNA to be measured, and a light receiving unit that receives the fluorescence emitted by the probe DNA in response to the excitation light. The detection unit 33 may also be a camera capable of acquiring images of fluorescence emission.

[0070] Each molecule in the probe DNA is modified with a fluorescent molecule and a quenching molecule. The quenching molecule is configured, for example, to absorb light in the wavelength band corresponding to the fluorescence wavelength of the fluorescent molecule. In probe DNA not bound to an enzyme-amplified nucleic acid molecule, the distance between the fluorescent molecule and the quenching molecule is closer than in probe DNA bound to an enzyme-amplified nucleic acid molecule. Examples of fluorescent molecules include 6-caroxyfluorescein (FAM), Texas Red (TR), and cyanine (CY)-based materials. When the fluorescent substance is 6-FAM, irradiation with excitation light at a peak wavelength of 494 nm produces fluorescence at a peak wavelength of 517 nm. When the fluorescent substance is Texas Red, irradiation with excitation light at a peak wavelength of 596 nm produces fluorescence at a peak wavelength of 615 nm. When the fluorescent substance is Cy3-carboxylic acid, irradiation with excitation light at a peak wavelength of 555 nm produces fluorescence at a peak wavelength of 570 nm. The base sequence of the probe DNA can be appropriately designed based on the base sequence of the nucleic acid molecule to be detected.

[0071] (1) The first primer, (2) the second primer, (3) the enzyme, and (4) the probe DNA may be pre-immobilized in at least one of the second preparation section 13 and the second incubation section 14. (1) The first primer, (2) the second primer, (3) the enzyme, and (4) the probe DNA may not be immobilized in the second incubation section 14, but may be pre-immobilized in the second preparation section 13. (1) The first primer, (2) the second primer, (3) the enzyme, and (4) the probe DNA may be immobilized as a coating. According to this, the detection system 1000 can supply probe DNA to the second solution without adding the probe DNA later.

[0072] (1) The first primer and (2) the second primer may be pre-immobilized in the third preparation section 15, and (3) the enzyme and (4) the probe DNA may be pre-immobilized in the fourth preparation section 16.

[0073] A moisture-resistant coating agent may be used to immobilize the probe DNA, for example, a sugar may be used. Examples of sugars include trehalose dihydrate, sucrose, lactose, glucose, and fructose. Because the probe DNA is immobilized, the detection unit 33 can detect fluorescence at the immobilization site.

[0074] <Methods for nucleic acid amplification and detection> Based on Figure 5, the nucleic acid amplification method and detection method will be described. Figure 5 is a flowchart showing the flow of the nucleic acid amplification method and detection method. The nucleic acid amplification method and detection method using the nucleic acid amplification system 100 are shown below, but nucleic acid amplification and detection may also be performed using conventionally known experimental equipment, including pipettes, microcentrifuge tubes, test tubes, etc., without using the nucleic acid amplification system 100.

[0075] In S1, the first preparation unit 11 adds a nonionic surfactant to the sample containing the target for detection and mixes it, and then the first incubation unit 12 pre-treats the mixed solution by heating it at 95°C for 3 minutes to prepare the bacterial cell treatment solution (pre-treatment step). The bacterial cell treatment solution is then used in the next step after its temperature has been lowered to room temperature.

[0076] In addition to the nonionic surfactant, dNTPs and NTPs may be added to the sample. The nonionic surfactant may be a surfactant having a repeating structure of ethylene glycol (-O-CH2-CH2-), and specifically, it may be Tween-20® or Triton X-100®.

[0077] Subsequently, the flow path between the first incubation section 12 and the second preparation section 13 lowers the temperature of the bacterial cell processing solution. In S4, the nucleic acid amplification system 100 may actively lower the temperature of the bacterial cell processing solution present in the first incubation section 12 or the bacterial cell processing solution moving from the first incubation section 12, or it may take advantage of the fact that the temperature of the bacterial cell processing solution naturally decreases as it flows through the flow path.

[0078] All pretreatment steps in S1 may be carried out in the same reaction system. Similarly, the step of lowering the temperature of the bacterial cell treatment solution may also be carried out in the same reaction system as S1. With this configuration, steps such as removing nucleic acid molecules from the solution within the system and using the removed nucleic acid molecules in another system are unnecessary, and each step is performed sequentially. This makes it possible to amplify nucleic acids quickly and easily.

[0079] In S2, the third preparation unit 15 of the second preparation unit 13 prepares the first solution by adding a NASBA reagent solution containing the NASBA reagent and a primer set having a sequence complementary to the sequence of the nucleic acid molecule derived from the target to the bacterial cell treatment solution after the pretreatment step (first preparation step). The primer set 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.

[0080] Next, in S3, the second incubation unit 14 maintains the first liquid in a first temperature range for a first hour (first incubation step). The first temperature range may be higher than the second temperature range in S6 described later. The first temperature range may be in the range of 80°C to 95°C. Also, the first hour may be shorter than the second hour in S6 described later.

[0081] Next, in S4, the flow path between the third preparation section 15 and the fourth preparation section 16 of the second preparation section 13 lowers the temperature of the first solution. In S4, the nucleic acid amplification system 100 may actively lower the temperature of the first solution present in the third preparation section 15, or the first solution moving from the third preparation section 15, but it may also utilize the fact that the temperature of the first solution naturally decreases as it flows through the flow path. In other words, S4 is not an essential step.

[0082] In S5, the fourth preparation section 16 of the second preparation section 13 prepares the second solution by adding a NASBA enzyme solution containing a probe and enzymes capable of detecting the amplification product by the primer set after the first incubation step (second preparation step). The nucleic acid molecule may be RNA, and the enzymes may include reverse transcriptase, RNA polymerase, and ribonuclease. The primer set and enzymes may be added together with sugars.

[0083] 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 first preparation step and the temperature of the solution during the second preparation step may be different. For example, the temperature of the solution during the first preparation step may be higher than the temperature of the solution during the second preparation step. For example, the temperature of the solution during the first preparation step may be 80°C or higher and 95°C or lower. The temperature of the solution during the second preparation step may be 37°C or higher and 41°C or lower. After S5, 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 solution flowing through the flow path containing the first liquid.

[0084] Each molecule in the probe DNA is modified with a fluorescent molecule and a quenching molecule. In probe DNA that is not bound to an enzyme-amplified nucleic acid molecule, the distance between the fluorescent molecule and the quenching molecule is closer than in probe DNA that is bound to an enzyme-amplified nucleic acid molecule.

[0085] In step S6, the second incubation unit 14 maintains the second liquid in the second temperature zone for two hours (second incubation step). The second temperature zone may be in the range of 37°C to 41°C.

[0086] The pretreatment step S1, the first preparation step S2, the first incubation step S3, the second preparation step S5, and the second incubation step S6 may be carried out in the same reaction system. Similarly, the step S4, which lowers the temperature of the first solution, may also be carried out in the same reaction system as S1-S3 and S5-S6. The same reaction system may be, for example, the same channel or a specific same region within the channel. With the above configuration, steps such as removing nucleic acid molecules from the solution in the system and using the removed nucleic acid molecules in another system are unnecessary, and each step is carried out continuously. This makes it possible to amplify nucleic acids quickly and easily.

[0087] Nucleic acid amplification from the target of detection is performed by S1-S6.

[0088] Next, in step S7, nucleic acid molecules amplified by the enzyme in steps S1 to S6 are detected based on the intensity of fluorescence emitted from the fluorescent molecule (detection step).

[0089] In the nucleic acid amplification method described above, a bacterial cell treatment solution is prepared by adding a nonionic surfactant to a sample containing the target to be detected and heating it. Then, a first solution is prepared by adding a NASBA reagent solution containing the primer set according to this disclosure, and this solution is maintained at a first temperature for one hour. Subsequently, a second solution is prepared by adding a NASBA enzyme solution containing the probe according to this disclosure to the first solution, which has been maintained at the first temperature for one hour, and maintaining this solution at a second temperature to amplify nucleic acid molecules derived from the target to be detected. Furthermore, in the detection method described above, nucleic acid molecules amplified by the nucleic acid amplification methods S1 to S6 are detected based on the intensity of fluorescence emitted from a fluorescent molecule. In the nucleic acid amplification method and detection method described above, by using a detection kit containing the primer set and probe according to this disclosure, nucleic acid sequences commonly possessed by Cnm-positive strains can be specifically amplified, and the presence of Cnm-positive strains in the sample can be detected.

[0090] Figure 6 shows the gene sequence of strain TW295, one of the Cnm-positive strains. In Figure 6, the region indicated by reference numeral 601 shows the common nucleic acid sequence of Cnm-positive strains, and the region indicated by reference numeral 602 shows the nucleic acid sequence unique to strain TW295. The primer set according to this disclosure specifically recognizes the region indicated by reference numeral 601 in the sequence shown in Figure 6, that is, the common nucleic acid sequence which is a nucleic acid sequence commonly found in Cnm-positive strains. Therefore, by using any of the primer sets according to this disclosure, i.e., primer sets 1 to 7, it is possible to amplify the nucleic acid sequence found in Cnm-positive strains regardless of the type of Cnm-positive strain.

[0091] The inventions described in this disclosure have been explained above based on the drawings and embodiments. However, the inventions described in this disclosure are not limited to the embodiments described above. That is, the inventions described in this disclosure can be modified in various ways within the scope shown in this disclosure, and embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the inventions described in this disclosure. In other words, it should be noted that it is easy for those skilled in the art to make various modifications or alterations based on this disclosure. Furthermore, it should be noted that these modifications or alterations are included in the scope of this disclosure.

[0092] 〔summary〕 As described above, the primer set according to Embodiment 1 of this disclosure is represented by SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 10, or SEQ ID NOs: 9 and 12.

[0093] A primer set according to aspect 2 of the present disclosure may be represented in aspect 1 by SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 10, or SEQ ID NOs: 9 and 12.

[0094] The primer set according to aspect 3 of this disclosure may be represented in aspect 1 by SEQ ID NOs. 1 and SEQ ID NOs. 2, SEQ ID NOs. 3 and SEQ ID NOs. 4, or SEQ ID NOs. 9 and SEQ ID NOs. 12.

[0095] The primer set according to aspect 4 of this disclosure may be represented in aspect 1 by SEQ ID NOs: 3 and SEQ ID NOs: 4, or SEQ ID NOs: 9 and SEQ ID NOs: 12.

[0096] The primer set relating to aspect 5 of this disclosure may be represented by SEQ ID NO: 9 and SEQ ID NO: 12 in aspect 1 above.

[0097] The probe according to aspect 6 of this disclosure specifically recognizes a portion of the nucleic acid sequence commonly found in Cnm-positive Streptococcus mutans.

[0098] A probe relating to aspect 7 of this disclosure may be represented by sequence number 13 in aspect 6.

[0099] A detection kit according to aspect 7 of this disclosure includes a primer set according to any of aspects 1 to 5 and a probe according to aspect 6 or 7. [Examples]

[0100] One embodiment of this disclosure is described below.

[0101] <Reagents used> In this example, reagents and enzymes used in the conventional NASBA (Nucleic Acid Sequence-Based Amplification) method were used.

[0102] (Detection target) • Streptococcus mutans showing Cnm positivity (hereinafter referred to as "Streptococcus mutans"). (kit) • NASBA Amplification Kit (Kainos Co., Ltd.) (Sugars) • Trehalose (manufactured by Fujifilm Wako Pure Chemical Corporation) (Surfactants) • Tween-20 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (others) • Primer set (synthesized by Fasmac) • Molecular beacon (synthesized by Fasmac).

[0103] One of the following primer sets, from Primer Set 1 to Primer Set 7, was used as the primer set. In each primer set, Primer 1 is a forward primer containing the T7 promoter sequence, and Primer 2 is a reverse primer containing the RNA polymerase recognition sequence. • First primer set: A set consisting of the first primer represented by Sequence ID No. 1 and the second primer represented by Sequence ID No. 2. • Second primer set: A set consisting of the first primer represented by Sequence ID No. 3 and the second primer represented by Sequence ID No. 4. • Third primer set: A set consisting of the first primer represented by SEQ ID NO: 5 and the second primer represented by SEQ ID NO: 6. • Fourth primer set: A set consisting of the first primer represented by SEQ ID NO: 7 and the second primer represented by SEQ ID NO: 8. • Fifth primer set: A set consisting of the first primer represented by SEQ ID NO: 9 and the second primer represented by SEQ ID NO: 10. • Sixth primer set: A set consisting of the first primer represented by SEQ ID NO: 11 and the second primer represented by SEQ ID NO: 10. • Seventh primer set: A set consisting of the first primer represented by Sequence ID No. 9 and the second primer represented by Sequence ID No. 12.

[0104] For the *Streptococcus mutans* probe (molecular beacon), we used the probe represented by Sequence ID No. 13. This probe contains a fluorescent molecule (carboxyfluorescein, FAM, λex=492nm, λem=518nm) at its 5' end and a quenching molecule corresponding to the fluorescent molecule (black hole quencher 1, BHQ1) at its 3' end.

[0105] (NASBA reagent) Ribonucleoside triphosphates (NTPs), deoxyribonucleoside triphosphates (dNTPs) • Dissolution buffer (Tris buffer, DMSO, containing metal ions) (enzyme) AMV (Avian Myeloblastosis Virus) - Reverse Transcriptase ·RNaseH • T7 RNA polymerase

[0106] In this example, experiments were conducted to amplify the nucleic acid sequence of the target bacterium, Streptococcus mutans, using primer sets 1 through 7 and probes, in order to confirm whether the presence of Streptococcus mutans could be detected. The details of the experiment are described below.

[0107] [Amplification of nucleic acids] <Example 1> (Preparation of bacterial cell treatment solution) In a 1.5 mL Eppendorf tube, add 10 units of TW295 strain, a type of Cnm-positive Streptococcus mutans (Cnm-positive strain). 9 28.5 μL of an aqueous solution containing 28.5 μL of tween-20 at 1 vol% and 1.5 μL of a 1 vol% tween-20 aqueous solution were added and mixed at room temperature. The concentration of tween-20 in the first solution after mixing was 0.05 vol%. The resulting first solution was pre-treated by heating at 95°C for 3 minutes. The temperature was then lowered to room temperature to obtain a bacterial cell treatment solution.

[0108] (Preparation of NASBA reagent solution) A NASBA reagent solution was prepared by dissolving 1 / 6 of the NASBA reagent with 9 μL of NASBA dissolving solution and 0.5 μL × 10 μM of the first primer set (the first primer represented by SEQ ID NO: 1 and the second primer represented by SEQ ID NO: 2).

[0109] (Preparation of NASBA enzyme solution) NASBA enzyme (1 / 6 volume) was dissolved in sterile water and 15 μL × 10 μM of probe solution.

[0110] (Detection method) 10 μL of NASBA reagent solution and 5 μL of bacterial cell treatment solution were added to a 0.2 mL PCR tube to obtain solution 1. Solution 1 was then heated at 95°C for 3 minutes. After that, the temperature was lowered to 41°C, 5 μL of NASBA enzyme solution was added, and solution 2 was obtained by pipetting 5 times. Solution 2 was then reacted for 90 minutes.

[0111] <Example 2> The amount to add TW295 strain is 10 7 A bacterial cell treatment solution according to Example 2 was obtained in the same manner as in Example 1, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 1.

[0112] <Example 3> The amount to add TW295 strain is 10 5A bacterial cell treatment solution according to Example 3 was obtained in the same manner as in Example 1, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 1.

[0113] <Example 4> The amount to add TW295 strain is 10 5 A bacterial cell treatment solution according to Example 4 was obtained in the same manner as in Example 1, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 1, except that the third primer set was used instead of the first primer set.

[0114] <Example 5> The amount to add TW295 strain is 10 4 A bacterial cell treatment solution according to Example 5 was obtained in the same manner as in Example 4, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 4.

[0115] <Comparative Example 1> The amount to add TW295 strain is 10 3 A bacterial cell treatment solution according to Comparative Example 1 was obtained in the same manner as in Example 4, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 4.

[0116] <Example 6> The amount to add TW295 strain is 10 5 A bacterial cell treatment solution according to Example 6 was obtained in the same manner as in Example 1, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 1, except that the fourth primer set was used instead of the first primer set.

[0117] <Example 7> The amount to add TW295 strain is 10 4 A bacterial cell treatment solution according to Example 7 was obtained in the same manner as in Example 6, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 6.

[0118] <Comparative Example 2> A cell treatment solution according to Comparative Example 2 was obtained in the same manner as in Example 6, except that the amount of TW295 strain added was 10 3 cells / mL. Thereafter, the first solution and the second solution were prepared and incubated in the same manner as in Example 6.

[0119] <Example 8> A cell treatment solution according to Example 8 was obtained in the same manner as in Example 6, except that a mixture of 10 types of Cnm-positive strains (hereinafter referred to as a mixed strain) was added instead of the TW295 strain. Thereafter, the first solution and the second solution were prepared and incubated in the same manner as in Example 6.

[0120] <Comparative Example 3> A cell treatment solution according to Comparative Example 3 was obtained in the same manner as in Example 8, except that the amount of the mixed strain added was 10 4 cells / mL. Thereafter, the first solution and the second solution were prepared and incubated in the same manner as in Example 6.

[0121] <Example 9> A cell treatment solution according to Example 9 was obtained in the same manner as in Example 6, except that the mixed strain was added instead of the TW295 strain. Thereafter, the first solution and the second solution were prepared and incubated in the same manner as in Example 1, except that the fifth primer set was used instead of the first primer set.

[0122] <Comparative Example 4> A cell treatment solution according to Comparative Example 4 was obtained in the same manner as in Example 9, except that the amount of the mixed strain added was 10 4 cells / mL. Thereafter, the first solution and the second solution were prepared and incubated in the same manner as in Example 9.

[0123] <Example 10> A cell treatment solution according to Example 10 was obtained in the same manner as in Example 6, except that the mixed strain was added instead of the TW295 strain. Thereafter, the first solution and the second solution were prepared and incubated in the same manner as in Example 1, except that the sixth primer set was used instead of the first primer set.

[0124] <Comparative Example 5> The amount to add the mixed strain is 10 4 A bacterial cell treatment solution according to Comparative Example 5 was obtained in the same manner as in Example 10, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 10.

[0125] <Example 11>

[0126] (Preparation of NASBA enzyme-dried reagent) A mixture of NASBA reagent solution and NASBA enzyme solution, prepared in the same manner as in Example 1, was dissolved by adding 20 μL × 0.1 μM of trehalose, and then freeze-dried for 3 hours to obtain a dried NASBA enzyme reagent.

[0127] (Detection method) The amount to add TW295 strain is 10 5 A bacterial cell treatment solution according to Example 11 was obtained in the same manner as in Example 1, except that the concentration was changed to cells / mL. Then, 10 μL of NASBA reagent solution and 5 μL of bacterial cell treatment solution were placed in a 0.2 mL PCR tube and heated at 95°C for 3 minutes to obtain the first solution. The temperature of the first solution was then lowered to 41°C, 15 μL of the first solution was added to the NASBA enzyme dry reagent, and the mixture was pipetted 10 times to obtain the second solution. The second solution was then reacted for 90 minutes.

[0128] <Example 12> The amount to add TW295 strain is 10 4 A bacterial cell treatment solution according to Example 12 was obtained in the same manner as in Example 11, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 11.

[0129] <Comparative Example 6> The first and second solutions were prepared and incubated in the same manner as in Example 11, except that water was added instead of the bacterial cell treatment solution.

[0130] <Example 13> A bacterial cell treatment solution according to Example 13 was obtained in the same manner as in Example 11. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 11, except that the second primer set was used instead of the first primer set.

[0131] <Example 14> The amount to add TW295 strain is 10 4 A bacterial cell treatment solution according to Example 14 was obtained in the same manner as in Example 13, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 13.

[0132] <Comparative Example 7> The first and second solutions were prepared and incubated in the same manner as in Example 13, except that water was added instead of the bacterial cell treatment solution.

[0133] <Example 15> A bacterial cell treatment solution according to Example 15 was obtained in the same manner as in Example 11. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 11, except that the seventh primer set was used instead of the first primer set.

[0134] <Example 16> The amount to add TW295 strain is 10 4 A bacterial cell treatment solution according to Example 16 was obtained in the same manner as in Example 15, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 15.

[0135] <Comparative Example 8> The amount to add TW295 strain is 10 3 A bacterial cell treatment solution according to Comparative Example 8 was obtained in the same manner as in Example 15, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 15.

[0136] <Example 17> The bacterial cell treatment solution for Example 17 was obtained in the same manner as in Example 15, except that a mixed strain was added instead of strain TW295. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 15.

[0137] <Comparative Example 9> The first and second solutions were prepared and incubated in the same manner as in Example 15, except that water was added instead of the bacterial cell treatment solution.

[0138] <Example 18> The amount to add the mixed strain is 10 4 A bacterial cell treatment solution according to Example 18 was obtained in the same manner as in Example 17, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 15.

[0139] <Comparative Example 10> The first and second solutions were prepared and incubated in the same manner as in Example 15, except that water was added instead of the bacterial cell treatment solution.

[0140] <Comparative Example 11> The amount to add the mixed strain is 10 3 A bacterial cell treatment solution according to Comparative Example 11 was obtained in the same manner as in Example 17, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 15.

[0141] <Comparative Example 12> The first and second solutions were prepared and incubated in the same manner as in Example 15, except that water was added instead of the bacterial cell treatment solution.

[0142] <Example 19> (Mixed solution of bacterial cell treatment solution + artificial saliva) TW295 shares 10 9 Instead of cells / mL, use a mixed strain of 10 4A bacterial cell treatment solution according to Example 19 was obtained in the same manner as in Example 1, except that cells / mL were added. 0.5 μL of this bacterial cell treatment solution was added to 4.5 μL of artificial saliva to prepare a mixed solution of the bacterial cell treatment solution and the artificial saliva solution.

[0143] (Detection method) 10 μL of NASBA reagent solution and 5 μL of a mixed solution of bacterial cell treatment solution and artificial saliva solution were placed in a 0.2 mL PCR tube and heated at 95°C for 3 minutes to obtain Solution 1. Then, Solution 1 was cooled to 41°C, and 15 μL of Solution 1 was added to the NASBA enzyme dry reagent to obtain Solution 2. Then, Solution 2 was pipetted 10 times and reacted for 90 minutes.

[0144] <Comparative Example 13> The first and second solutions were prepared and incubated in the same manner as in Example 19, except that water was added instead of the bacterial cell treatment solution.

[0145] <Comparative Example 14> A NASBA reagent solution was prepared in the same manner as in Example 1, except that a primer set was not added. A NASBA enzyme dry reagent was prepared in the same manner as in Example 19, except that the NASBA reagent solution was used. A mixed solution of the bacterial cell treatment solution and the artificial saliva solution was obtained in the same manner as in Example 19. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 19, except that a NASBA reagent solution and a NASBA enzyme dry reagent without a primer set were used.

[0146] <Example 20> A mixed solution of bacterial cell treatment solution and artificial saliva solution was obtained in the same manner as in Example 19, except that strain TW295 was added instead of the mixed strain. Then, the first and second solutions were prepared and incubated in the same manner as in Example 19.

[0147] <Comparative Example 15> The preparation and incubation of the first and second solutions were carried out in the same manner as in Example 20, except that water was added instead of the mixed solution of bacterial cell treatment solution and artificial saliva solution.

[0148] <Comparative Example 16> A mixed solution of bacterial cell treatment solution and artificial saliva solution was obtained in the same manner as in Example 20, except that a NASBA reagent solution and NASBA enzyme drying reagent without a primer set were used. Then, the first and second solutions were prepared and incubated in the same manner as in Example 19.

[0149] <Example 21> A mixed solution of the bacterial cell treatment solution and the artificial saliva solution was obtained in the same manner as in Example 20. Then, the first and second solutions were prepared and incubated in the same manner as in Example 20, except that the second primer set was used instead of the seventh primer set.

[0150] <Comparative Example 17> The amount to add TW295 strain is 10 4 A bacterial cell treatment solution according to Comparative Example 17 was obtained in the same manner as in Example 20, except that the concentration was changed to cells / mL. Subsequently, the first and second solutions were prepared and incubated in the same manner as in Example 21.

[0151] <Comparative Example 18> The preparation and incubation of the first and second solutions were carried out in the same manner as in Example 21, except that water was added instead of the mixed solution of bacterial cell treatment solution and artificial saliva solution.

[0152] [Detection and observation of amplified nucleic acids] For each of Examples 1-21 and Comparative Examples 1-18, 1 / 6 the volume of 6× Loading Buffer was added to the incubated second solution, and the bands were confirmed by electrophoresis. The gels after electrophoresis were observed using a fluorescence microscope (IX83, Olympus Corporation) equipped with an LED emitter (X-Cite XYLIS, Excelitas Technologies) and an optical filter set (IX3-FGFPXL; λex: 460-480nm, λem: 495-540nm; U-RFP; λex: 535-555nm, λem: 570-625nm, Olympus Corporation).

[0153] Furthermore, for Examples 17-20 and Comparative Examples 9-16, the second solution after incubation was directly observed using fluorescence.

[0154] 〔result〕 Figure 7 shows the electrophoresis results for Examples 1-3. Figure 8 shows the electrophoresis results for Examples 4-7 and Comparative Examples 1-2. Figure 9 shows the electrophoresis results for Examples 8-10 and Comparative Examples 3-5. Figure 10 shows the electrophoresis results for Examples 11-14 and Comparative Examples 6-7. Figure 11 shows the electrophoresis results for Examples 15-16 and Comparative Example 8. Figure 12 shows the electrophoresis results and fluorescence observation results for Examples 17-18 and Comparative Examples 9-12. Figure 13 shows the electrophoresis results and fluorescence observation results for Examples 19-20 and Comparative Examples 13-16. Figure 14 shows the electrophoresis results for Example 21 and Comparative Examples 17-18.

[0155] Regarding the detection results, 10 5 If amplification products are confirmed after performing each treatment on a sample containing detectable cells / mL or less, it can be said that the nucleic acid sequence of the detectable cell has been sufficiently amplified, making it possible to detect the detectable cell.

[0156] In Examples 1-3 (Figure 7) and 11-12 (Figure 10), nucleic acid amplification was performed using a sample containing *Streptococcus mutans* and the first primer set, and a clear band was observed. On the other hand, in Comparative Example 6 (Figure 10), nucleic acid amplification was performed using a sample without *Streptococcus mutans* and the first primer set, and no clear band was observed. Therefore, it was found that the nucleic acid sequence of *Streptococcus mutans* Cnm-positive strains can be amplified using the first primer set, and that Cnm-positive strains can be detected.

[0157] In Examples 13-14 (Figure 10) and Example 21 (Figure 14), nucleic acid amplification was performed using samples containing Streptococcus mutans and the second primer set, and clear bands were observed. On the other hand, in Comparative Example 7 (Figure 10) and Comparative Example 18 (Figure 14), nucleic acid amplification was performed using samples without Streptococcus mutans and the second primer set, and no clear bands were observed. Therefore, it was found that the nucleic acid sequence of Cnm-positive strains of Streptococcus mutans can be amplified using the second primer set, and that Cnm-positive strains can be detected.

[0158] In Example 21 (Figure 14), 10 5 A mixed solution containing a bacterial cell treatment solution with Streptococcus mutans cells / mL and artificial saliva was used, and a clear band was observed. On the other hand, in Comparative Example 17 (Figure 14), 10 4 Nucleic acid amplification was performed using a mixed solution of bacterial cell treatment solution containing Streptococcus mutans cells / mL and artificial saliva, along with a second primer set. As a result, no clear band was observed in the areas where amplification products by the second primer set could be observed. Therefore, if the sample is 10 5 It was found that a mixed solution of a bacterial cell treatment solution containing Streptococcus mutans at a concentration of 1 / mL or higher and artificial saliva could amplify the nucleic acid sequence of Cnm-positive strains of Streptococcus mutans using a second primer set, making it possible to detect Cnm-positive strains.

[0159] In Examples 4-5 (Figure 8), nucleic acid amplification was performed using a sample containing *Streptococcus mutans* and the third primer set, and a clear band was observed. On the other hand, in Comparative Example 1 (Figure 8), nucleic acid amplification was performed using a sample without *Streptococcus mutans* and the third primer set, and no clear band was observed. Therefore, it was found that the nucleic acid sequence of *Streptococcus mutans* Cnm-positive strains can be amplified using the third primer set, and that Cnm-positive strains can be detected.

[0160] In Examples 6-7 (Figure 8) and Example 8 (Figure 9), 10 5Nucleic acid amplification was performed using a sample containing *Streptococcus mutans* at a concentration of 1 / mL or higher and a fourth primer set, and a clear band was observed. On the other hand, in Comparative Example 2 (Figure 8) and Comparative Example 3 (Figure 9), 10 3 pieces / mL or 10 4 When nucleic acid amplification was performed using a sample containing *Streptococcus mutans* at a concentration of 1 / mL and a fourth primer set, no clear band was observed. Therefore, 10 5 It was found that by using a sample containing *Streptococcus mutans* at a concentration of 1 / mL or higher and a fourth primer set, the nucleic acid sequence of *Streptococcus mutans* Cnm-positive strains could be amplified, and these Cnm-positive strains could be detected.

[0161] In Example 9 (Figure 9), 10 5 Nucleic acid amplification was performed using a sample containing Streptococcus mutans at a concentration of 10 cells / mL or higher and the fifth primer set, and a relatively clear band was observed. On the other hand, in Comparative Example 4 (Figure 9), 10 4 When nucleic acid amplification was performed using a sample containing *Streptococcus mutans* at a concentration of 1 / mL and a fifth primer set, no clear band was observed. Therefore, 10 5 It was found that by using a sample containing *Streptococcus mutans* at a concentration of 1 / mL or higher and a fifth primer set, the nucleic acid sequence of *Streptococcus mutans* Cnm-positive strains could be amplified, and these Cnm-positive strains could be detected.

[0162] In Example 10 (Figure 9), 10 5 nucleic acid amplification was performed using a sample containing *Streptococcus mutans* at a concentration of 10 cells / mL or higher and the 6th primer set, and a clear band was observed. On the other hand, in Comparative Example 5 (Figure 9), 10 4 When nucleic acid amplification was performed using a sample containing *Streptococcus mutans* at a concentration of 1 / mL and a set of primers 6, no clear band was observed. Therefore, 10 5 It was found that by using a sample containing *Streptococcus mutans* at a concentration of 1 / mL or higher and a set of sixth primers, the nucleic acid sequence of *Streptococcus mutans* Cnm-positive strains could be amplified, and these Cnm-positive strains could be detected.

[0163] Furthermore, in Example 15 (Figure 11), nucleic acid amplification was performed using a sample containing *Streptococcus mutans* and the 7th primer set, and a clear band was observed. On the other hand, in Comparative Example 9 (Figure 12), when nucleic acid amplification was performed using a sample without *Streptococcus mutans* and the 7th primer set, no clear band was observed. Therefore, it was found that the nucleic acid sequence of Cnm-positive strains of *Streptococcus mutans* can be amplified using the 7th primer set, and that Cnm-positive strains can be detected.

[0164] Furthermore, in Example 16 (Figure 11), 10 4 Nucleic acid amplification was performed using a sample containing *Streptococcus mutans* at a concentration of 10 cells / mL and the 7th primer set, and a clear band was observed. On the other hand, in Comparative Example 8 (Figure 11), 10 3 When nucleic acid amplification was performed using a sample containing *Streptococcus mutans* at a concentration of 1 / mL and the 7th primer set, no clear band was observed. Therefore, 10 4 It was found that if a sample contains *Streptococcus mutans* at a concentration of 1 / mL or higher, the nucleic acid sequence of *Streptococcus mutans* Cnm-positive strains can be amplified using the seventh primer set, making it possible to detect these Cnm-positive strains.

[0165] Furthermore, in Examples 19-20 (Figure 13), nucleic acid amplification was performed using the seventh primer set, and clear bands were observed. On the other hand, in Comparative Examples 14 and 16 (Figure 13), when nucleic acid amplification was performed without using the primer set, no clear bands were observed. Therefore, it was found that the nucleic acid sequence of the Cnm-positive strain was amplified by using the seventh primer set.

[0166] Furthermore, in Examples 1-3, 6-7, 10, and 13-16 (Figures 7-11), nucleic acid amplification was performed using the first, second, fourth, sixth, or seventh primer sets. As a result, it was observed that the amplification of nonspecific bands was small in these examples. Therefore, it was found that the nucleic acid sequence of Cnm-positive strains of Streptococcus mutans can be amplified with greater accuracy by using the first, second, fourth, sixth, or seventh primer sets.

[0167] Generally, when using lyophilized reagents, the accuracy of nucleic acid amplification tends to be lower than when using non-lyophilized reagents. However, in Examples 11-14 (Figure 10), 15-16 (Figure 11), and 17-18 (Figure 12), nucleic acid amplification was performed using lyophilized reagents, and clear bands were observed. Therefore, it was found that the nucleic acid sequence of Cnm-positive strains of Streptococcus mutans can be amplified with high accuracy by using the first primer set, the second primer set, or the seventh primer set.

[0168] Furthermore, generally, when using a sample containing artificial saliva containing the target organism, the accuracy of nucleic acid amplification tends to be lower than when using only a bacterial cell treatment solution containing the target organism. In Examples 19-20 (Figure 13) and 21 (Figure 14), nucleic acid amplification was performed using samples containing artificial saliva, and clear bands were observed. Therefore, it was found that by adopting the second primer set or the seventh primer set, the nucleic acid sequence of the Cnm-positive strain of Streptococcus mutans can be amplified with even greater accuracy. Also, in Examples 19 and 20 (Figure 13), 10 4 A clear band was observed in samples containing artificial saliva with a concentration of the target organism at nucleotides / mL, indicating that the use of the seventh primer set allows for accurate amplification of smaller amounts of the target organism.

[0169] In Examples 6 (Figure 8) and 16 (Figure 11), nucleic acid amplification was performed using samples containing the TW295 strain as the Cnm-positive strain, and clear bands were observed. Furthermore, in Examples 8-10 (Figure 9) and 18 (Figure 12), nucleic acid amplification was performed using samples containing a mixed strain as the Cnm-positive strain, and clear bands were observed. Therefore, it was found that nucleic acid sequences can be amplified regardless of the type of Cnm-positive strain by using the 4th, 5th, 6th, or 7th primer sets. Additionally, since the 1st to 3rd primer sets also contain sequences complementary to some of the common nucleic acid sequences of Cnm-positive strains, it is considered that they can amplify nucleic acid sequences regardless of the type of Cnm-positive strain, similar to the 4th to 7th primer sets.

[0170] In Examples 17-18 (Figure 12) and 19-20 (Figure 13), clear bands were observed as a result of nucleic acid amplification. On the other hand, in Comparative Examples 9-12 (Figure 12) and 13-16 (Figure 13), nucleic acids were not amplified, and no clear bands were observed. Furthermore, as shown in Figures 12 and 13, fluorescence observation of the second solution showed fluorescence in Examples 17-18 (Figure 12) and 19-20 (Figure 13), but not in Comparative Examples 9-12 (Figure 12) and 13-16 (Figure 13). Thus, the electrophoresis results and the fluorescence observation results were consistent. Therefore, it was found that amplified nucleic acid sequences can be detected by using the probe according to this disclosure.

[0171] As described above, it was found that the primer set relating to this disclosure can effectively amplify and detect the common nucleic acid sequence of Cnm-positive strains. Furthermore, it was found that the second primer set and the seventh primer set, particularly the seventh primer set, can amplify the target nucleic acid sequence with greater accuracy. In addition, it was confirmed that the nucleic acid sequence amplified using the primer set relating to this disclosure can be detected by using the probe relating to this disclosure. [Explanation of Symbols]

[0172] 10 cartridges (amplifiers) 11 1st preparation part 12. First Incubation Section 13 2nd Preparation Section 14. Second Incubation Department 15 3rd preparation section 16 4th Preparation Department 31, 71 Installation part 22A, 22B Temperature control area 30 Detection device 32 Temperature control unit 33 Detection unit 70 Temperature control device 100 nucleic acid amplification systems 1000 detection systems S1 Pretreatment process S2 1st preparation process S3 First Incubation Process S4 Step to lower the temperature of the first liquid S5 2nd preparation process S6 Second incubation process S7 Detection process

Claims

1. A set of primers represented by SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 10, or SEQ ID NOs: 9 and 12.

2. The primer set according to claim 1, represented by SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 11 and 10, or SEQ ID NOs: 9 and 12.

3. The primer set according to claim 1, represented by SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or SEQ ID NO: 9 and SEQ ID NO:

12.

4. The primer set according to claim 1, represented by SEQ ID NOs: 3 and 4, or SEQ ID NOs: 9 and 12.

5. The primer set according to claim 1, as represented by Sequence ID No. 9 and Sequence ID No.

12.

6. A probe that specifically recognizes a portion of the nucleic acid sequence commonly found in Cnm-positive Streptococcus mutans bacteria.

7. The probe according to claim 6, represented by sequence number 13.

8. A detection kit comprising the primer set according to claim 1 and the probe according to claim 6.

Images