Channel substrate, cartridge, detection system, and method for manufacturing channel substrate

Abstract

In the present invention, a nucleic acid derived from a detection target subject is amplified by a simple operation such that a liquid is allowed to flow into a flow path. The present invention is provided with: a liquid receiving unit that receives a liquid; and a flow path that is connected to the liquid receiving unit. The flow path has a first region in which a first reagent containing a primer is disposed, and a second region in which a second reagent containing an enzyme for performing the amplification of a nucleic acid is disposed, the second region being positioned at a position different from the first region.

Description

Flow path substrate, cartridge, detection system, and method for manufacturing flow path substrate

[0001] The present disclosure relates to a flow path substrate and the like.

[0002] The dual-chamber reaction vessel of Patent Document 1 includes a first chamber for receiving a liquid sample, a second chamber physically independent from the first chamber, and a channel connecting the first chamber to the second chamber. The first chamber contains amplification reagents, and the second chamber contains an enzyme.

[0003] Japanese Patent Publication No. 11-4678

[0004] A flow path substrate according to one aspect of the present disclosure includes a liquid receiving section that receives a liquid and a flow path connected to the liquid receiving section, and the flow path has a first region in which a first reagent containing a primer is disposed, and a second region in which a second reagent containing an enzyme that amplifies nucleic acid is disposed at a position different from the first region.

[0005] A method for manufacturing a flow path substrate according to one aspect of the present disclosure includes a forming step of forming a liquid receiving portion for receiving a liquid and a flow path connected to the liquid receiving portion on a substrate, a first disposing step of disposing a first reagent containing a primer in the flow path, and a second disposing step of disposing a second reagent containing an enzyme for amplifying nucleic acid at a position different from the first region of the flow path.

[0006] FIG. 1 is a schematic diagram showing an example of a detection system of the present disclosure. FIG. 1 is a schematic diagram showing an example of a configuration of a cartridge of the present disclosure, and a schematic diagram of a window portion of the cartridge of the present disclosure when viewed from above. FIG. 2 is a schematic diagram for explaining an example of use of the cartridge and detection device of the present disclosure. FIG. 3 is a block diagram showing an example of a detection system of the present disclosure. FIG. 4 is a plan view showing an example of a flow path substrate of a cartridge of the present disclosure. FIG. 5 is a schematic diagram showing an example of a first region and a second region arranged in a flow path provided in a flow path substrate according to a first embodiment of the present disclosure. FIG. 6 is a flowchart showing an example of a method for manufacturing a flow path substrate according to the first embodiment of the present disclosure. FIG. 7 is a schematic plan view showing an example of a flow path to which a reagent has been applied, and a schematic diagram showing another example of a method for applying a reagent to a flow path. FIG. 8 is a plan view showing an example of a flow path substrate according to a second embodiment of the present disclosure. FIG. 9 is a schematic diagram showing an example of an arrangement of a detection reagent in a flow path provided in a flow path substrate according to the second embodiment of the present disclosure. FIG. 10 is a schematic diagram for explaining an example of use of a plurality of flow paths provided in a flow path substrate according to the second embodiment of the present disclosure. FIG. 11 is a flowchart showing an example of a method for manufacturing a flow path substrate 4B according to the second embodiment of the present disclosure.

[0007] [Detection System] Fig. 1 is a schematic diagram showing an example of a detection system 1 according to the present disclosure. Fig. 1 is a schematic diagram showing an example of the appearance of a cartridge 2 and an example of the appearance of a detection device 3. Fig. 2 is a block diagram showing an example of the detection system 1. As shown in Fig. 1, the detection system 1 may include the cartridge 2 and the detection device 3.

[0008] The cartridge 2 is a test kit for detecting a target substance contained in a specimen collected from a subject. The cartridge 2 may be a test kit that can amplify nucleic acid derived from the target substance using a reagent when the target substance is contained in a specimen inserted into the cartridge 2. The cartridge 2 may include a main body 21 and a bottle 22.

[0009] The subject is not limited to a human, but may be any living organism that can harbor a virus or bacterium, such as a mammal, bird, reptile, or amphibian.

[0010] The specimen may be saliva, urine, sweat, nasal mucus, blood, cells, etc. collected from the body of a subject, or may be soil and water collected from an object to be inspected, such as the ground, a river, the sea, etc. Alternatively, the specimen may be an attachment collected from the surface of an object to be inspected, such as a handrail, a door, clothing, shoes, or a toilet bowl.

[0011] The detection target may be, for example, a virus or a bacterium. The type of virus or bacterium contained in the sample is not limited to one type, and may be two or more types. When the detection target is a virus, examples of the type of virus include influenza virus, coronavirus (e.g., SARS-CoV-2), respiratory syncytial virus (RS virus), human metapneumovirus, norovirus, HIV (human immunodeficiency virus), herpes virus, streptococcus, and Mycoplasma pominis.

[0012] A specimen collected from a subject may be contained in the bottle portion 22. The main body portion 21 may include a flow path substrate 4 (see FIG. 5 ) that receives a liquid containing the specimen contained in the bottle portion 22 and, when the specimen contains a target substance, contains a reagent that reacts with nucleic acid derived from the target substance. The nucleic acid derived from the target substance may be extracted in the bottle portion 22, as will be described in detail below.

[0013] The detection device 3 is a device that detects nucleic acid derived from a detection target when the nucleic acid is amplified in the cartridge 2. The detection device 3 may have a housing that can receive the cartridge 2.

[0014] The detection device 3 may determine whether nucleic acid derived from the detection target is present at a certain concentration or above. The detection device 3 may determine that the sample is positive when nucleic acid derived from the detection target is present at a certain concentration or above. Alternatively, the detection device 3 may be a measurement device that measures the concentration of nucleic acid derived from the detection target. In this embodiment, the detection device 3 is described as determining whether the sample is positive. However, a determination device separate from the detection device 3 may determine whether the sample is positive by obtaining data corresponding to the amount of nucleic acid detected by the detection device 3 from the detection device 3.

[0015] The specific configurations of the cartridge 2 and the detection device 3 will be described later.

[0016] [NASBA Method] In the flow path substrate 4, nucleic acid derived from the detection target may be amplified by an isothermal nucleic acid amplification method. Examples of the isothermal nucleic acid amplification method include NASBA (Nucieic Acid Sequence-Based Amplification). Other examples include NEAR (Nicking Enzyme Amplification), LAMP (Loop-mediated Isothermal Amplification), and TMA (Transcription-mediated Amplification). In this embodiment, NASBA may be used as the isothermal nucleic acid amplification method.

[0017] The NASBA method is an isothermal nucleic acid amplification method that uses three enzymes (AMV reverse transcriptase, RNase H, and T7 RNA polymerase) and two primers. In the NASBA method, simply adding a NASBA substrate and enzyme to the template RNA results in amplified antisense single-stranded RNA. Because the entire series of steps in the NASBA reaction proceeds isothermally, RNA nucleic acid amplification can be performed without complex temperature control. Furthermore, because the amplification product of the NASBA reaction is single-stranded RNA, sequence-specific detection can be performed using a detection probe without a denaturation step.

[0018] [Specific Configuration of Cartridge] Reference numeral 1101 in Fig. 2 is a schematic diagram showing an example of the configuration of the cartridge 2. Reference numeral 1102 in Fig. 2 is a schematic diagram of the window portion 211 of the cartridge 2 as viewed from above. Fig. 3 is a schematic diagram for explaining an example of how the cartridge 2 and the detection device 3 are used.

[0019] As indicated by reference numeral 1101 in Fig. 2 , the main body 21 may include a flow path substrate 4. The flow path substrate 4 may include a liquid receiving portion 41 that receives liquid. Furthermore, as indicated by reference numeral 1102 in Fig. 2 , the flow path substrate 4 may include a storage portion 46 that stores liquid that has flowed through the flow path substrate 4. 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 portion 211 so that the inside of the main body 21 can be seen.

[0020] The bottle portion 22 is a container capable of holding a liquid. The bottle portion 22 may be made of a transparent material, but may also be made of an opaque material.

[0021] As indicated by the arrow at 1101 in FIG. 2 , the liquid receiving section 41 receives the liquid flowing in from the bottle section 22. The liquid received by the liquid receiving section 41 flows through a flow path 45 (see FIG. 5 ) arranged in the flow path substrate 4 and is then stored in the storage section 46. A reagent may be disposed in the flow path 45. Furthermore, as indicated by 1102 in FIG. 2 , an internal standard (standard section) 47 may be provided near the storage section 46. The internal standard 47 may be used for optical comparison with the storage section 46. The flow path substrate 4 may include four flow paths 45, 45A to 45D (see FIG. 5 ). A mixed fluid containing a sample and a labeling substance that reacts with the target substance contained in the sample may be flowed through at least one of the flow paths 45A to 45D. The internal standard 47 may be located in a region different from the flow path 45 and used for comparison with a mixed fluid containing the sample and a labeling substance that reacts with the target substance contained in the sample. For example, the intensity of light (e.g., fluorescence) emitted by the internal standard 47 may be compared with the intensity of light (e.g., fluorescence) emitted by the sample and a labeling substance that reacts with the target substance contained in the sample.

[0022] As indicated by reference numeral 1102 in Fig. 2 , the main body 21 is formed with a window 211 so that the reservoirs 46 and internal standards 47 arranged on the flow path substrate 4 can be seen through the window 211 when viewed from above. When the flow path substrate 4 includes a plurality of reservoirs 46 and a plurality of internal standards 47, the window 211 may be formed so that all of the reservoirs 46 and all of the internal standards 47 can be seen. In other words, the reservoirs 46 and the internal standards 47 may be arranged on the main body 21 so as to be optically exposed from the window 211.

[0023] As shown by reference numeral 1112 in FIG. 3 , the sampler 23 may be attached to the main body 21. The sampler 23 may include a specimen collection section 231 and a fixing section 232. The specimen collection section 231 is a section for collecting a specimen. The specimen collection section 231 may be, for example, a pleated resin member. The fixing section 232 may fix the bottle section 22 to the main body 21 when the sampler 23 is inserted into the bottle section 22. The connection section between the main body 21 and the bottle section 22 may have, for example, a screw structure. Furthermore, a lid may be provided at the connection section of the bottle section 22 with the main body 21. This allows the interior of the bottle section 22 to be sealed. The lid may be made of aluminum. The subject may remove the lid and insert the sampler 23 into the bottle section 22.

[0024] As indicated by reference numeral 1111 in Fig. 3 , when collecting saliva as a specimen, for example, the subject inserts the specimen collection portion 231 into the subject's mouth to cause saliva to adhere to the specimen collection portion 231. As indicated by reference numeral 1112 in Fig. 3 , the subject applies saliva to the specimen collection portion 231 and then inserts the sampler 23 into the bottle portion 22.

[0025] The bottle portion 22 may contain a buffer solution 24. An example of the buffer solution 24 is a NASBA solution. The buffer solution 24 may contain, for example, a surfactant and an RNA (ribonucleic acid) degrading enzyme inhibitor. However, the RNA degrading enzyme inhibitor may be disposed on the flow path substrate 4. In this case, the buffer solution 24 may not contain an RNA degrading enzyme inhibitor. An example of a surfactant is Tween-20 (polysorbate 20), which is an example of a nonionic surfactant. When the buffer solution 24 contains a nonionic surfactant, nucleic acids derived from the detection target can be extracted in the bottle portion 22 by mixing the sample and the buffer solution 24.

[0026] When the subject inserts the sampler 23 into the bottle portion 22, the saliva adhering to the specimen collection portion 231 is mixed with the buffer solution 24. The buffer solution 24 mixed with the specimen may be an example of a liquid that the bottle portion 22 can contain.

[0027] As indicated by reference numeral 1113 in Fig. 3 , the detection device 3 may include a receiving section 36 that can receive the cartridge 2. As indicated by reference numeral 1113 in Fig. 3 , the subject attaches the cartridge 2 containing the sample to the receiving section 36, and then turns the detection device 3 upside down, as indicated by reference numeral 1114 in Fig. 3 . As a result, the buffer solution 24 mixed with the sample flows, due to its own weight, down the sampler 23 and into the flow path substrate 4 provided in the main body section 21. The buffer solution 24 mixed with the sample may be an example of a liquid that the liquid receiving section 41 receives.

[0028] As a negative control, only the buffer solution 24 may be poured into the flow path substrate 4. In this case, the buffer solution 24 itself is an example of a liquid that can be accommodated in the bottle portion 22, and an example of a liquid that can be received by the liquid receiving portion 41. Also, a substance that contains a pathogen other than the detection target may be mixed into the buffer solution 24. In this case, the buffer solution 24 mixed with the substance is an example of a liquid that can be accommodated in the bottle portion 22, and an example of a liquid that can be received by the liquid receiving portion 41.

[0029] 4 is a block diagram showing an example of the detection system 1. As shown in Fig. 4, the detection device 3 may be a device that detects nucleic acid amplified in a flow channel 45 provided in a flow channel substrate 4. The detection device 3 may include, for example, a heating unit 31, a pressurizing unit 32, a light irradiating unit 33, an imaging unit 34, and a control unit 35.

[0030] The heating unit 31 may be a member that heats the cartridge 2 inserted into the detection device 3. The heating unit 31 may heat the cartridge 2 to a temperature that promotes the reaction between the nucleic acid and the reagent in the flow path substrate 4. When amplifying nucleic acids by the NASBA method, the heating unit 31 may heat the cartridge 2 to a temperature of 37°C to 41°C. In this embodiment, the heating unit 31 first heats the cartridge 2 at a temperature of 80°C to 95°C for 3 to 10 minutes. This disrupts pathogens and liberates nucleic acid components from the pathogens. It also inactivates DNA (deoxyribonucleic acid) degrading enzymes. Thereafter, the heating unit 31 may maintain the cartridge 2 at a temperature of 37°C to 41°C.

[0031] The heating unit 31 may heat different parts of the cartridge 2 at different temperatures. For example, the heating unit 31 may have a first heating unit that heats the bottle unit 22 to a temperature of 80°C to 95°C, and a second heating unit that heats the main body unit 21 to a temperature of 37°C to 41°C. This allows pathogens to be crushed in the bottle unit 22, liberating nucleic acid components from the pathogens. Furthermore, the DNase can be inactivated in the bottle unit 22. The liquid in the bottle unit 22 then flows into the flow path substrate 4 provided in the main body unit 21, which is maintained at a temperature of 37°C to 41°C, and nucleic acid amplification may be performed in the flow path substrate 4.

[0032] The temperature to which the heating unit 31 heats may be the set temperature of the heating unit 31. Alternatively, it may be the liquid temperature of the liquid whose temperature is to be adjusted by the heating unit 31. When the temperature to which the heating unit 31 heats is the liquid temperature of the liquid whose temperature is to be adjusted by the heating unit 31, for example, the average temperature or central temperature of the liquid 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 2. The detection device 3 may be equipped with a temperature sensor that detects the liquid temperature of the liquid whose temperature is to be adjusted by the heating unit 31. The heating unit 31 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.

[0033] The pressurizing unit 32 may be a member that pressurizes the bottle portion 22 of the cartridge 2 inserted into the detection device 3. When the detection device 3 is configured to pressurize the bottle portion 22 by the pressurizing unit 32, the bottle portion 22 may be made of a material that is deformed by the pressure of the pressurizing unit 32. The bottle portion 22 is deformed by the pressure of the pressurizing unit 32, which makes it easier for the liquid in the bottle portion 22 to flow into the main body portion 21.

[0034] The pressurizing unit 32 may be a member that applies pressure to at least a portion of the side surface of the bottle part 22. The pressurizing unit 32 may be a member that clamps the side surface of the bottle part 22. The pressurizing unit 32 may be disposed in the detection device 3 at a position where it can apply pressure to the side surface on the bottom side of the bottle part 22, for example.

[0035] The light irradiating unit 33 may be a light source that irradiates the storage unit 46 and the internal standard 47 of the flow path substrate 4 with light through the window 211 of the main body 21. The light irradiating unit 33 irradiates, for example, excitation light onto the storage unit 46 and the internal standard 47.

[0036] A labeling substance that specifically binds to the nucleic acid amplified in the flow channel 45 may be disposed in the flow channel 45 of the flow channel substrate 4. The labeling substance may be optically observable, and may include, but is not limited to, a dye, a luminescent substance, or a fluorescent substance. If the labeling substance is a fluorescent substance, when the storage section 46 is irradiated with excitation light, the labeling substance bound to the nucleic acid emits fluorescence having a specific wavelength.

[0037] Examples of fluorescent substances contained in labeling substances include 6-caroxyfluorescein (FAM), Texas Red (TR) (registered trademark), 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.

[0038] The labeling substance may be, for example, a molecular beacon. A molecular beacon is a type of probe DNA. Each molecule of the probe DNA is modified with a fluorescent molecule and a quencher molecule. The quencher molecule is configured to absorb light in a wavelength band corresponding to the fluorescence wavelength of the fluorescent molecule, for example. 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. The base sequence of the probe DNA may be appropriately designed based on the base sequence of the nucleic acid to be detected.

[0039] The internal standard 47 may be any substance that can be optically compared with the storage section 46, and may be, for example, but is not limited to, a dye, a luminescent substance, or a fluorescent substance. A fluorescent substance that emits fluorescence when irradiated with excitation light may be disposed as the internal standard 47. The fluorescent substance as the internal standard 47 and the fluorescent substance contained in the labeling substance may be the same type of fluorescent substance.

[0040] The imaging unit 34 is a component that images the storage unit 46 and the internal standard 47 through the window 211 of the main body 21. By imaging the storage unit 46 with the imaging unit 34, it may be possible to determine whether or not a certain concentration or more of nucleic acid derived from the detection target is present in the liquid stored in the storage unit 46 based on optical information measured from the labeling substance present in the storage unit 46. The optical information may be, for example, information obtained from a dye, a luminescent substance, or a fluorescent substance. The optical information may be, for example, information such as wavelength or luminance value. For example, the control unit 35 may determine whether a certain concentration or more of nucleic acid derived from the detection target is present in the liquid stored in the storage unit 46 based on the intensity of fluorescence emitted from the storage unit 46.

[0041] Furthermore, the image capturing unit 34 may capture an image of the internal standard 47, and the position of the internal standard 47 in the captured image may be identified based on optical information measured from the internal standard 47. The optical information may be, for example, information obtained from a dye, a luminescent substance, or a fluorescent substance. The optical information may be, for example, information such as wavelength or brightness value. For example, the control unit 35 may identify the position of the internal standard 47 in the captured image based on the fluorescence emitted from the internal standard 47. Then, the control unit 35 may identify the position of the flow path 45 in the captured image based on the identified position of the internal standard 47.

[0042] Furthermore, the image capturing unit 34 may analyze an image of the internal standard 47 to determine whether optical information equal to or greater than a predetermined value can be measured from the internal standard 47. The control unit 35 may determine that the image capturing unit 34 is operating normally if optical information equal to or greater than the predetermined value can be measured. For example, the control unit 35 may determine whether fluorescence of equal to or greater than a predetermined intensity can be received from the internal standard 47. The control unit 35 may determine that the image capturing unit 34 is operating normally if fluorescence of equal to or greater than the predetermined intensity can be received.

[0043] The determination of whether optical information equal to or greater than a predetermined value has been measured may be made once before measuring optical information obtained from the specimen after starting the detection device 3. For example, the determination of whether fluorescence equal to or greater than a predetermined intensity has been received, i.e., the measurement of the predetermined fluorescence intensity, may be made once after starting the detection device 3 and before measuring the intensity of fluorescence emitted by the specimen.

[0044] The control unit 35 may comprehensively control each component included in the detection device 3. The control unit 35 may include, for example, an intensity measurement unit 351 and a positive determination unit 352.

[0045] The intensity measurement unit 351 may measure optical information from the substance contained in the storage unit 46 and the internal standard 47. For example, the intensity measurement unit 351 may measure the intensity of fluorescence emitted by the substance contained in the storage unit 46 and the internal standard 47 upon receiving excitation light.

[0046] The control unit 35 may first control the light irradiation unit 33 to irradiate the storage unit 46 and the internal standard 47 with light. In this state, the control unit 35 may control the imaging unit 34 to capture an image including the storage unit 46 and the internal standard 47. The intensity measurement unit 351 may analyze the image captured by the imaging unit 34 and measure the brightness as optical information obtained from the substances contained in the storage unit 46 and the internal standard 47.

[0047] For example, the control unit 35 controls the light irradiating unit 33 to irradiate the storage unit 46 and the internal standard 47 with excitation light, and then acquires the luminance by analyzing the image captured by the imaging unit 34. In this way, the control unit 35 may measure the intensities of the fluorescence emitted by the substances contained in the storage unit 46 and the internal standard 47, respectively.

[0048] The intensity measurement unit 351 may measure optical information obtained from the reservoir 46 connected to the flow path 45 in which the reagent is disposed as optical information of the detection target. For example, the intensity measurement unit 351 may measure the intensity of fluorescence emitted from the reservoir 46 connected to the flow path 45 in which the reagent is disposed as the intensity of fluorescence of the detection target. Furthermore, the intensity measurement unit 351 may measure optical information emitted from the reservoir 46 connected to the flow path 45 for the negative control as optical information of the negative control. For example, the intensity measurement unit 351 may measure the intensity of fluorescence emitted from the reservoir 46 connected to the flow path 45 for the negative control as the intensity of fluorescence of the negative control. The flow path 45 for the negative control will be described later. The position of the flow path 45 in which the reagent is disposed and the position of the flow path 45 for the negative control may be determined in advance.

[0049] The positive determination unit 352 may determine whether or not a certain concentration or more of nucleic acid derived from the detection target is present in the specimen contained in the liquid stored in the storage unit 46, based on the optical information obtained from the storage unit 46 measured by the intensity measurement unit 351. For example, the positive determination unit 352 may determine whether a certain concentration or more of nucleic acid derived from the detection target is present in the specimen contained in the liquid stored in the storage unit 46, based on the intensity of fluorescence emitted from the storage unit 46 measured by the intensity measurement unit 351. The positive determination unit 352 may determine that the specimen is positive when it determines that a certain concentration or more of nucleic acid derived from the detection target is present.

[0050] For example, the positive determination unit 352 may determine whether a certain concentration or more of nucleic acid derived from the detection target is present by comparing the value indicated by the optical information obtained from the storage unit 46 with a threshold value. For example, the threshold value may be optical information associated with the minimum concentration of nucleic acid derived from the detection target that should be determined as positive. The threshold value may be set in advance through experiments, etc. For example, the positive determination unit 352 may determine whether a certain concentration or more of nucleic acid derived from the detection target is present by comparing the intensity of fluorescence emitted from the storage unit 46 with the threshold value.

[0051] The positive determination unit 352 may calculate a difference value by subtracting the value indicated by the optical information of the negative control from the value indicated by the optical information of the detection target. For example, the positive determination unit 352 may calculate a difference value by subtracting the fluorescence intensity of the negative control from the fluorescence intensity of the detection target. The positive determination unit 352 may compare the difference value with a threshold, and determine that the sample is positive if the difference value is equal to or greater than the threshold, and determine that the sample is negative if the difference value is less than the threshold.

[0052] Alternatively, the positive determination unit 352 may determine whether the sample is positive by comparing the difference value with the value indicated by the optical information obtained from the internal standard 47. For example, two internal standards 47 having different concentrations may be provided. The positive determination unit 352 may determine that the sample is positive if the difference value is within the range of values ​​indicated by the optical information obtained from the two internal standards 47. The optical information obtained from the two internal standards 47 may be optical information associated with the concentration of nucleic acid derived from the detection target to be determined as positive. The optical information obtained from the two internal standards 47 may be set in advance by experiment or the like.

[0053] For example, the positive determination unit 352 may determine whether the sample is positive by comparing the difference value with the intensity of the fluorescence emitted from the internal standard 47. The positive determination unit 352 may determine that the sample is positive if the difference value is within the range of the fluorescence intensities indicated by the two internal standards 47.

[0054] The control unit 35 may also create a calibration curve based on optical information obtained from the multiple internal standards 47. For example, the control unit 35 may create a calibration curve based on the intensities of fluorescence emitted by the multiple internal standards 47. In this case, the control unit 35 may use the calibration curve to calculate the concentration of nucleic acid corresponding to the difference value. The positive determination unit 352 may determine that the sample is positive if the calculated concentration of nucleic acid is equal to or greater than a reference value. The reference value may be a value associated with the concentration of nucleic acid derived from the detection target that should be determined to be positive. The reference value may be set in advance through experiments, etc.

[0055] In the following explanation, as an example, the labeling substance and internal standard 47 are fluorescent substances, and the control unit 35 measures the intensity of fluorescence as the optical information measured from the labeling substance and internal standard 47.

[0056] [First Form of Flow Channel Substrate] Fig. 5 is a plan view showing an example of a flow channel substrate 4A. The flow channel substrate 4A is an example of the flow channel substrate 4. Fig. 6 is a schematic diagram showing an example of a first region 451 and a second region 452 arranged in a flow channel 45 provided in the flow channel substrate 4A.

[0057] 5, the flow path substrate 4A may include a liquid receiving section 41 that receives a liquid, and a flow path 45 that connects to the liquid receiving section 41. The flow path 45 may have a first region 451 in which a first reagent 101 containing a primer is disposed, and a second region 452 in which a second reagent 102 containing an enzyme that amplifies nucleic acid is disposed at a position different from the first region 451. In the flow path 45 that communicates with the liquid receiving section 41, the space in which the first region 451 is located and the space in which the second region 452 is located may communicate with each other.

[0058] In this embodiment, as shown in FIG. 5 , the flow path substrate 4A may include a liquid receiving section 41, a branch flow path 42, a flow path 45, a storage section 46, and an internal standard 47. There may be one or more flow paths 45. There may also be one or more internal standards 47. In this embodiment, the flow paths 45 include four flow paths 45A to 45D, and the storage sections 46 include four storage sections 46A to 46D, but this is not limiting. In addition, the flow path substrate 4A includes five internal standards 47, but this is not limiting.

[0059] 4, the liquid receiving portion 41 is located above the flow path 45 and the storage portion 46. Therefore, the liquid flowing from the bottle portion 22 due to its own weight easily flows from the liquid receiving portion 41 to the storage portion 46 via the flow path 45.

[0060] The liquid receiving portion 41 may be an opening that receives the liquid flowing from the bottle portion 22. The branch flow path 42 may be a flow path that connects the liquid receiving portion 41 to each of the flow paths 45A to 45D. In other words, the flow paths 45A to 45D may branch from the branch flow path 42 that is connected to the liquid receiving portion 41. The branch flow path 42 may be in communication with the liquid receiving portion 41 and each of the flow paths 45A to 45D.

[0061] The flow path substrate 4A may include a filter unit 420. The filter unit 420 may be a portion having a narrower flow path width than other portions. The filter unit 420 may be located on the opposite side of the reservoir unit 46 from the position of the reagent disposed in the flow path 45. That is, the filter unit 420 may be located upstream of the position of the reagent disposed in the flow path 45. When solid impurities are contained in the liquid flowing from the liquid receiving unit 41, the possibility of the impurities flowing downstream of the filter unit 420 can be reduced. Furthermore, the filter unit 420 may be located upstream of the branching positions of the flow paths 45A to 45D. As shown in FIG. 5, the branch flow path 42 may include the filter unit 420. This reduces the possibility of the impurities flowing into the flow paths 45A to 45D when solid impurities are contained in the liquid flowing from the liquid receiving unit 41.

[0062] However, flow path substrate 4A may not be provided with branch flow paths 42. In this case, flow paths 45A to 45D may branch from liquid receiving section 41. When there is only one flow path 45, flow path 45 may be connected to liquid receiving section 41. Furthermore, the filter section may be located in each of flow paths 45A to 45D on the opposite side of reservoir section 46 from the position of the reagent placed in flow path 45. Each of flow paths 45A to 45D may have a filter section upstream of the position where first reagent 101 is placed or the position where first reagent 101 may be placed.

[0063] 5, all of the flow paths 45A to 45D may branch off from one liquid receiving section 41 or from a branch flow path 42 connected to one liquid receiving section 41. However, the flow path substrate 4A may be provided with a plurality of liquid receiving sections 41. In this case, each flow path 45 may be connected to each liquid receiving section 41, or one or more flow paths 45 may be connected to each liquid receiving section 41.

[0064] The flow paths 45A to 45D may be flow paths for flowing a liquid. A mixed fluid containing a specimen and a labeling substance that reacts with the target substance contained in the specimen may be flowed through at least one of the flow paths 45A to 45D. Each of the flow paths 45A to 45D may be a flow path that receives a liquid flowing from the liquid receiving section 41 and flows the liquid to the reservoirs 46A to 46D connected to the flow paths 45A to 45D, respectively. Each of the flow paths 45A to 45D may be in communication from the liquid receiving section 41 to the reservoirs 46A to 46D.

[0065] In at least one of the flow paths 45A to 45D, the reagent may be disposed on the opposite side of the branching position of the flow paths 45A to 45D from the liquid-receiving section 41. In this way, when the liquid received in the liquid-receiving section 41 contains nucleic acid derived from the detection target, the nucleic acid derived from the detection target can be amplified in the flow path 45.

[0066] In this embodiment, one of the flow paths 45A to 45D may have a first region 451 in which a first reagent 101 is disposed, and a second region 452 in which a second reagent 102 is disposed at a position different from the first region 451.

[0067] The liquid received in liquid receiving section 41 flows through each of flow paths 45A to 45D and is stored in reservoirs 46A to 45D. In this embodiment, as shown in FIG. 5 , flow path 45B has a first region 451 and a second region 452. Therefore, the liquid flowing through flow path 45B comes into contact with first reagent 101 and second reagent 102. If the sample contains a detection target, nucleic acid derived from the detection target can be amplified by first reagent 101 and second reagent 102.

[0068] Therefore, nucleic acid derived from the detection target can be amplified by a simple operation of flowing a liquid through the flow channel 45 without using a special tool such as a pipette.

[0069] The first reagent 101 may contain, for example, a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from the target nucleic acid molecule. The primer may be added as needed as long as it is capable of amplifying the target nucleic acid molecule, and may be, for example, one type or three or more types. The primers may be, for example, a first primer and a second primer. For example, the first primer may be a forward primer that amplifies the target nucleic acid molecule in the sense direction, and the second primer may be a reverse primer that amplifies the nucleic acid molecule in the antisense direction.

[0070] In addition, the first reagent 101 may contain, for example, deoxynucleoside triphosphates (dNTPs), nucleoside triphosphates (NTPs), trehalose, and an RNase inhibitor. However, if the RNase inhibitor is contained in the bottle portion 22, the first reagent 101 does not need to contain the RNase inhibitor.

[0071] As shown in FIG. 6 , the first reagent 101 may be arranged in the first region 451 as a plurality of first attachments. A dried solution containing the above-described reagent may be arranged in the first region 451 as an attachment of the first reagent 101. This allows the first reagent 101 to dissolve in the liquid when the liquid flowing into the flow path 45 comes into contact with the first attachment. Furthermore, when the first reagent 101 is arranged in the flow path 45 independently as a plurality of attachments, the surface area of ​​the first attachment can be increased. This increases the solubility of the first reagent 101 in the liquid. The first reagent 101 may be arranged in the first region 451 as a plurality of films. The number of first attachments may be a plurality, for example, five or more, or ten or more.

[0072] As shown in FIG. 6 , the multiple first attachments may be arranged in one direction. This allows the first reagent 101 to be dissolved in the liquid sequentially along the flow of the liquid. The multiple first attachments may be arranged in one direction along the flow path 45. In this embodiment, the multiple first attachments 101 are arranged in one row, but they may also be arranged in two or more rows. Furthermore, the multiple first attachments may be arranged on the bottom 454 of the flow path 45 so as not to come into contact with the inner wall 455 of the flow path 45. This makes it even easier to dissolve the first reagent 101 in the liquid.

[0073] The first attachment may have a dot shape when viewed in a plan view, as shown in Fig. 6. Alternatively, the first attachment may have a linear shape, an elliptical shape, or a polygonal shape such as a square, a hexagon, or a star shape when viewed in a plan view.

[0074] The second reagent 102 may be, for example, an enzyme such as AMV-RT (Avian Myeloblastosis Virus), RNase H (Ribonuclease H), or T7 RNA polymerase. In addition, the second reagent 102 may contain, for example, trehalose and a surfactant.

[0075] As shown in FIG. 6 , the second reagent 102 may be disposed in the second region 452 as a plurality of second attachments. A dried product of a solution containing the above-described reagent may be disposed in the second region 452 as an attachment of the second reagent 102. This allows the second reagent 102 to dissolve in the liquid when the liquid flowing into the flow path 45 comes into contact with the second attachment. When the second reagent 102 is disposed in the flow path 45 as a plurality of independent attachments, the surface area of ​​the second attachment can be increased. This increases the solubility of the second reagent 102 in the liquid. The second reagent 102 may be disposed in the second region 452 as a plurality of films. The number of second attachments may be any number, for example, five or more, or ten or more.

[0076] As shown in FIG. 6 , the plurality of second attachments may be arranged in one direction. This allows the second reagent 102 to be dissolved in the liquid sequentially along the flow of the liquid. The plurality of second attachments may be arranged in one direction along the flow path 45. In this embodiment, the second reagent 102 is arranged in one row as the plurality of second attachments, but may be arranged in two or more rows. Furthermore, the plurality of second attachments may be arranged on the bottom 454 of the flow path 45 so as not to come into contact with the inner wall 455 of the flow path 45. This makes it even easier to dissolve the second reagent 102 in the liquid.

[0077] The second attachment may have a dot shape when viewed in a plan view, as shown in Fig. 6. Alternatively, the second attachment may have a linear shape, an elliptical shape, or a polygonal shape such as a square, a hexagon, or a star shape when viewed in a plan view.

[0078] 5, the second region 452 may be disposed on the opposite side of the liquid receiving section 41 from the position where the first region 451 is disposed. In other words, the second region 452 may be disposed downstream of the first region 451. Therefore, the liquid flowing through the flow path 45B can be brought into contact with the first reagent 101 and the second reagent 102 in that order.

[0079] 5 , the second region 452 may be disposed closer to the storage portion 46 than the first region 451. In other words, the first region 451, the second region 452, and the storage portion 46 may be disposed in this order along the flow path 45. Therefore, the liquid flowing through the flow path 45B can be brought into contact with the first reagent 101 and the second reagent 102 in this order, and then stored in the storage portion 46.

[0080] When the sample contains a target substance, the liquid containing nucleic acid derived from the target substance can be first brought into contact with the first reagent 101. This allows the nucleic acid derived from the target substance to anneal with the first primer and the second primer. Thereafter, the liquid flowing through the flow path 45B comes into contact with the enzyme contained in the second reagent 102, thereby amplifying the nucleic acid derived from the target substance.

[0081] It is sufficient that a first primer and a second primer are disposed in the first region 451 as the first reagent 101. Therefore, for example, dNTP and NTP may be disposed in the second region 452 as the second reagent 102. Even with such an arrangement, nucleic acid derived from the detection target contained in the liquid flowing through the flow channel 45 can be amplified.

[0082] 6 , the second attachment may be larger than the first attachment. For example, the first attachment and the second attachment may be disposed on the flow path 45 by applying a solution of the first reagent 101 and a solution of the second reagent 102 to the flow path 45. The second reagent 102 may contain a surfactant. Therefore, the solution of the second reagent 102 spreads more easily on the bottom surface of the flow path 45 than the solution of the first reagent 101.

[0083] The total area of ​​the plurality of first deposits can be determined by the amount of solution containing the first reagent 101. The total area of ​​the plurality of second deposits can be determined by the amount of solution containing the second reagent 102. For example, the total area of ​​the plurality of first deposits may be approximately twice the total area of ​​the plurality of second deposits.

[0084] The width of the flow path 45 may be, for example, 220 μm. In this case, if the first deposits are dot-shaped, their diameter may be smaller than the width of the flow path 45. The diameter of the first deposits may be, for example, 50 to 100 μm. The spacing between the first deposits may be 100 to 200 μm. If the second deposits are dot-shaped, their diameter may be smaller than the width of the flow path 45. The diameter of the second deposits may be, for example, 80 to 200 μm. The spacing between the second deposits may be 200 to 250 μm.

[0085] For example, if the volume of the liquid containing the specimen stored in the bottle 22 is approximately 50 nL, approximately 48 nL of the first reagent 101 solution and approximately 24 nL of the second reagent 102 solution are required to amplify the nucleic acid contained in the liquid. In this case, if the application volume per attachment is approximately 0.7 nL, the number of first attachments will be 69 and the number of second attachments will be 34. These liquid and solution volumes are smaller than when using a PCR (Polymerase Chain Reaction) tube. In other words, by using the flow path substrate 4A, the volume of the liquid containing the specimen and the application volume (printing volume) of the first reagent 101 and the second reagent 102 applied to the flow path 45 can be reduced.

[0086] The amounts of the first reagent 101 and the second reagent 102 may vary depending on the detection target. Therefore, the amount of the solution of the first reagent 101, the number of first attachments, the amount of the solution of the second reagent 102, and the number of second attachments may be changed depending on the detection target. Furthermore, the size of the first attachments, the size of the second attachments, the spacing between the first attachments, and the spacing between the second attachments may be adjusted depending on the length of the flow channel 45 in the extension direction and the width of the flow channel 45.

[0087] As described above, nucleic acids contained in the liquid flowing from the liquid receiving section 41 may first react with the first reagent 101 disposed in the first region 451 and then react with the second reagent 102 disposed in the second region 452. This is because the first reagent 101 anneals to the nucleic acids contained in the liquid in order to amplify the nucleic acids contained in the liquid with the second reagent 102. Furthermore, as described above, if the cartridge 2 is once heated to a high temperature and then cooled to a temperature that promotes nucleic acid amplification, the temperature may be lowered before the nucleic acids that have reacted with the first reagent 101 react with the second reagent 102. The distance between the first region 451 and the second region 452 may be determined taking these points into consideration. Consider a case in which the heating section 31 heats the bottle section 22 to a temperature of 80°C to 95°C and the main body section 21 to a temperature of 37°C to 41°C. In this case, the second region 452 may be positioned so that the liquid that has flowed in from the bottle portion 22 via the liquid receiving portion 41 comes into contact with the second region 452 after the temperature of the liquid has sufficiently dropped.

[0088] The first region 451 and the second region 452 may be disposed in a linear portion of the flow path 45. In this case, the first reagent 101 and the second reagent 102 can be easily disposed in the flow path 45. When it is not possible to dispose the reagents in one linear portion of the flow path 45, like the first region 451 shown in FIG. 6 , the reagents may be disposed in two or more linear portions.

[0089] A labeling substance that specifically binds to the amplified nucleic acid may further be disposed in the flow path 45. If the labeling substance is a fluorescent substance, the labeling substance emits fluorescence when irradiated with excitation light. Therefore, the detection device 3 can measure the intensity of fluorescence emitted by the nucleic acid derived from the detection target by irradiating the storage section 46 with excitation light.

[0090] The labeling substance may be disposed downstream of the first region 451. For example, the labeling substance may be disposed in the second region 452 as the second reagent 102. This allows the labeling substance to efficiently bind to nucleic acid amplified by the enzyme contained in the second reagent 102. The labeling substance may be a molecular beacon.

[0091] The liquid flowing through one of the channels 45A to 45D may function as a negative control. Figure 5 shows an example in which the channel 45A functions as a channel for negative control.

[0092] The flow path 45A may not include the first region 451, but may include at least a labeling substance. In this embodiment, as shown in FIG. 5 , the flow path 45A may include at least a second region 452 including a labeling substance. This prevents the nucleic acid from reacting with a primer for amplifying the nucleic acid, even if the liquid flowing through the flow path 45A contains the nucleic acid. Therefore, the detection device 3 can detect the intensity of fluorescence emitted by substances other than the nucleic acid derived from the target nucleic acid by irradiating the liquid flowing through the flow path 45A and stored in the storage section 46A with excitation light. Therefore, the detection device 3 can accurately detect the intensity of fluorescence emitted by the nucleic acid derived from the target nucleic acid amplified by reaction with the first reagent 101 and the second reagent 102 in the flow path 45B, using the intensity of the fluorescence as a reference.

[0093] In this way, in the flow path substrate 4A, both the first reagent 101 and the second reagent 102 may not be arranged, and a flow path 45 that is not intended for detecting nucleic acids derived from the detection target may be connected to the branch flow path 42. In addition to the flow path 45A, the flow paths 45C and 45D may also be flow paths that are not intended for detecting nucleic acids derived from the detection target.

[0094] Each of the flow channels 45A to 45D may have a bending region 453A to 453D downstream of the flow channel 45. Each of the flow channels 45A to 45D may have a bending region 453A to 453D downstream of the position where the reagent is disposed or the position where the reagent can be disposed. This allows a liquid in which the reagent is dissolved to flow through the bending region 453. As this liquid flows through the bending region 453, the reagent and the liquid can be mixed. Therefore, the nucleic acid derived from the detection target can react with the reagent efficiently.

[0095] It is sufficient that the bent region 453 is formed in the flow channel 45 in which the first reagent 101 and the second reagent 102 are disposed. In this embodiment, it is sufficient that the bent region 453B is formed in at least the flow channel 45B.

[0096] The reservoir 46 may be connected to the flow path 45. As shown in Fig. 5, the reservoirs 46A to 46D may be connected to the flow paths 45A to 45D, respectively. The reservoir 46 may store the liquid that has flowed from the flow path 45. The reservoir 46 may be part of the flow path.

[0097] When the flow channel 45 has the first region 451 and the second region 452, the reservoir 46 may be located downstream of the second region 452 and may store a liquid containing amplified nucleic acid. In the present embodiment, the flow channel 45B has the first region 451 and the second region 452. Therefore, the reservoir 46B may be located downstream of the second region 452B and may store a liquid containing amplified nucleic acid.

[0098] The reservoir 46 can store the liquid flowing from the flow path 45. When the liquid contains nucleic acid derived from the detection target, the reservoir 46 can store the liquid containing the amplified nucleic acid, which is connected to the flow path 45 having the first region 451 and the second region 452. Therefore, the detection device 3 can stably image the amplified nucleic acid.

[0099] The width of the storage portion 46 may be larger than the width of the flow path 45. This can improve the measurement accuracy of the intensity of fluorescence emitted from the storage portion 46. The volume of the storage portion 46 may be a volume equivalent to the amount of liquid received by the liquid receiving portion 41. The volume of the storage portion 46 may be, for example, approximately 50 nL. In this way, the storage portion 46 may be formed on the flow path substrate 4A so that the volume of the storage portion 46 is relatively small.

[0100] 5, the first distance D1 between two adjacent reservoirs 46 among the reservoirs 46A to 46D may be greater than the second distance D2 between the two flow paths 45 connected to each of the reservoirs 46. This allows the spacing between the two adjacent reservoirs 46 to be relatively wide. Therefore, the detection device 3 can easily obtain the intensity of the fluorescence emitted from each reservoir 46 through image analysis.

[0101] A reagent may be placed in the reservoir 46. A reagent used for amplifying nucleic acid derived from the detection target may be placed in the reservoir 46. For example, either the first reagent 101 or the second reagent 102 may be placed in the reservoir 46. Also, for example, a portion of the first reagent 101 and / or the second reagent 102 may be placed in the reservoir 46.

[0102] When either the first reagent 101 or the second reagent 102 is placed in the reservoir 46, a first reagent 101 or a second reagent 102 different from the reagent placed in the reservoir 46 may be placed in a flow path 45 located upstream from the reservoir 46. For example, when the second reagent 102 is placed in the reservoir 46, the first reagent 101 may be placed in a flow path 45 located upstream from the reservoir 46. That is, the first region 451 may be located in the flow path 45 located upstream from the reservoir 46, and the second region 452 may be located in the reservoir 46. This allows the liquid flowing through the flow path 45B to come into contact with the first reagent 101 and then the second reagent 102.

[0103] (Other Configurations) Additionally, the flow path substrate 4A may have second storage portions 48 downstream of the storage portions 46A to 46D, each connected to one of the storage portions 46A to 46D. In other words, the second storage portion 48 may be disposed at a position farther away from the liquid receiving portion 41 than the storage portion 46. In this case, when a volume of liquid equal to or greater than the capacity of the storage portion 46 flows into the storage portion 46, the liquid leaking from the storage portion 46 can be stored in the second storage portion 48. This reduces the possibility of the liquid flowing back from the storage portion 46 into the flow path 45.

[0104] The second reservoir 48 may also contain a substance that functions as a positive control. For example, a substance that functions as a positive control may be placed only in the second reservoir 48 of the flow path substrate 4A used as a positive control. The substance may be optically compared with the substance in the reservoir 46, and may be, but is not limited to, a dye, a luminescent substance, or a fluorescent substance. To verify whether the detection system 1 is functioning properly, a sample intentionally containing the target substance may be passed through the liquid receiving section 41 into the flow path 45 before measuring the optical information of the target sample, and the optical information corresponding to the nucleic acid derived from the target sample may be measured in the second reservoir 48. As shown by reference numeral 1102 in FIG. 2 , the second reservoir 48 can be viewed through the window 211 of the main body 21.

[0105] Furthermore, in the flow path substrate 4A, a sensing substance that detects the passage of liquid flowing through the flow path 45 may be located at a position farther from the liquid receiving section 41 than the storage section 46 in at least one of the branched flow paths 45. The flow path 45 may be a space that branches at the branch flow path 42 and then communicates with the second storage section 48 via the storage section 46. For example, the position farther from the liquid receiving section 41 than the storage section 46 refers to the position where the liquid received by the liquid receiving section 41 reaches after flowing through the flow path 45 and passing through the storage section 46. The sensing substance may also be a fluorescent substance that is soluble in the liquid flowing through the flow path 45. In this configuration, when the liquid flowing through the flow path 45 passes the position where the sensing substance is located, the sensing substance dissolves. Therefore, the brightness of the sensing substance decreases. The progress of the liquid can be confirmed by confirming the decrease in brightness of the sensing substance. In other words, whether the liquid flowing through the flow path 45 is normal can be determined based on whether the brightness of the sensing substance decreases. The detection substance is not particularly limited as long as it can confirm the passage of the liquid flowing through the flow channel 45 .

[0106] The second reservoir 48 may be connected to at least one of the reservoirs 46 A to 46 D. However, the flow path substrate 4 A may not necessarily have the second reservoir 48 .

[0107] Furthermore, the flow path substrate 4A may have an outlet 49 connected to the flow path 45 downstream of the flow path 45. The outlet 49 may be connected to the storage portion 46 downstream of the storage portion 46. As shown in FIG. 5 , when the flow path substrate 4A has a second storage portion 48, the outlet 49 may be connected to the second storage portion 48 downstream of the second storage portion 48. The outlet 49 is a vent that discharges air or liquid in a space located between the liquid receiving portion 41 and the outlet 49 to the outside of the flow path substrate 4A. The space located between the liquid receiving portion 41 and the outlet 49, i.e., the space located within the flow path substrate 4A including the flow path 45, may be referred to as a flow path.

[0108] (Position of Reagents) In the present embodiment, when the first reagent 101 and the second reagent 102 are placed in the flow path 45, it is sufficient that at least a primer and an enzyme are placed in the flow path 45. Other reagents may be placed at positions other than the flow path 45. For example, dNTP, NTP, RNase inhibitor, trehalose, and surfactant may be placed upstream of the flow path 45. For example, these reagents may be placed at branching positions of the flow paths 45A to 45D. For example, these reagents may be placed in the liquid receiving section 41 and / or the branch flow path 42. Furthermore, for example, a standard substance may be placed downstream of the flow path 45. For example, the standard substance may be placed in the reservoir 46.

[0109] (Number of Flow Channels, etc.) In this embodiment, the flow channel substrate 4A has four flow channels 45, but this is not limited thereto. In this embodiment, the flow channel substrate 4A only needs to have at least one flow channel 45 having a first region 451 and a second region 452. In this embodiment, when the flow channel substrate 4A has a plurality of flow channels 45 each having a first region 451 and a second region 452, these flow channels 45 may function as flow channels used to detect the same target substance. That is, the first reagent 101 disposed in the plurality of flow channels 45 may be the same reagent, and the second reagent 102 disposed in the plurality of flow channels 45 may be the same reagent. Furthermore, when the flow channel substrate 4A has a plurality of flow channels 45 each having a first region 451 and a second region 452, these flow channels 45 may function as flow channels used to detect different target substances. That is, the first reagent 101 disposed in the plurality of flow channels 45 may be different reagents, and the second reagent 102 disposed in the plurality of flow channels 45 may be different reagents.

[0110] The flow path substrate 4A may include at least one flow path 45 for a positive control. That is, the flow path 45 may include a flow path 45 for flowing a liquid intentionally containing a detection target. The flow path 45 for the positive control may have a first region 451 and a second region 452 arranged from the upstream side. A liquid intentionally containing a detection target that reacts with the first reagent 101 and the second reagent may be flowed through the flow path 45 for the positive control. However, the flow path 45 having the first region 451 and the second region 452 and used for flowing a liquid containing a specimen may be substituted for the flow path 45 for the positive control.

[0111] The number of reservoirs 46 may be determined depending on the number of flow paths 45 to be connected.

[0112] (Position of Internal Standard) The internal standard 47 may be located in a region different from the branch channel 42 , the channel 45 , and the reservoir 46 .

[0113] In this embodiment, the internal standard 47 is disposed on the flow path substrate 4A, but this is not limiting. The internal standard 47 may be disposed on the surface of the housing of the cartridge 2, instead of on the flow path substrate 4A. In this case, the internal standard 47 may be disposed near the window 211 of the main body 21.

[0114] [Method of Manufacturing Flow Channel Substrate According to First Embodiment] Fig. 7 is a flowchart showing an example of a method of manufacturing the flow channel substrate 4A. This manufacturing method may be performed by a manufacturing apparatus that manufactures the flow channel substrate 4A.

[0115] 7 , first, a liquid receiving section 41 and a flow path 45 may be formed on a substrate (S1; forming step). In this embodiment, in addition to the liquid receiving section 41 and the flow path 45, a branch flow path 42, a storage section 46, a second storage section 48, and an outlet 49 may be formed on the substrate.

[0116] The liquid receiving portion 41, the flow path 45, etc. may be formed, for example, as follows. For example, a template having the shapes of the liquid receiving portion 41, the flow path 45, etc. patterned thereon is placed on a substrate, and then a resin is poured into the substrate. After the resin has hardened, the template is removed. After the template is removed, a lid is placed on the hardened resin, thereby forming the liquid receiving portion 41, the flow path 45, etc. on the substrate.

[0117] Next, a reagent may be placed in the flow path 45. In this embodiment, a first reagent 101 may be placed in the flow path 45B (S2; first placing step). The first reagent 101 may be placed in the flow path 45B by applying the first reagent 101 to the flow path 45B. Furthermore, in this embodiment, a second reagent 102 may be placed in the flow paths 45A and 45B (S3; second placing step). The second reagent 102 may be placed in the flow paths 45A and 45B by applying the second reagent 102 to the flow paths 45A and 45B.

[0118] Next, an internal standard 47 may be placed on the substrate (S4). The internal standard 47 may be placed on the substrate by applying a fluorescent substance as the internal standard 47 to the substrate. Next, the fluorescent substance may be applied to the second reservoir 48, thereby placing the fluorescent substance in the second reservoir 48 (S5). The order of the processes from S2 to S5 does not matter. The processes from S2 to S5 may be performed in parallel.

[0119] Fig. 8 is a schematic diagram showing an example of a method for applying the reagent 100 to the flow path 45 provided in the flow path substrate 4A. Reference numeral 1131 in Fig. 9 is a schematic plan view showing an example of the flow path 45 to which the reagent 100 has been applied, and reference numeral 1132 in Fig. 9 is a schematic diagram showing another example of a method for applying the reagent 100 to the flow path 45. The reagent 100 in Figs. 8 and 9 may be the first reagent 101 or the second reagent 102.

[0120] The application of the reagent 100 may be performed using, for example, an inkjet printer. Use of an inkjet printer enables fine application of the reagent 100. The inkjet printer may include a head 201 that ejects the reagent 100. As shown by reference numeral 1121 in FIG. 8 , the inkjet printer may eject the reagent 100 from the head 201, thereby adhering the reagent 100 to the flow path 45 as shown by reference numeral 1122 in FIG. 8 . The reagent 100 that has adhered to the flow path 45 may be allowed to dry naturally.

[0121] The inkjet printer may apply the reagent 100 while moving the head 201 along the extension direction of the flow path 45, thereby adhering multiple reagents 100 to the bottom 454 of the flow path 45, as shown by reference numeral 1131 in Figure 9.

[0122] The inkjet printer may control the movement of the head 201 and the ejection of the reagent 100 so that the multiple deposits are disposed independently (separately) on the bottom 454. The inkjet printer may also control the movement of the head 201 and the ejection of the reagent 100 so that the deposits do not come into contact with the inner wall 455 of the flow channel 45.

[0123] As indicated by reference numeral 1131 in Fig. 9 , the inkjet printer may control the movement of the head 201 and the ejection of the reagent 100 so that a plurality of deposits are arranged independently side by side in a direction different from the extension direction of the flow channel 45. The plurality of deposits may be arranged side by side, for example, in the width direction of the flow channel 45. Furthermore, as indicated by reference numeral 1132 in Fig. 9 , new reagent 100 may be applied on top of the dried reagent 100 arranged in the flow channel 45.

[0124] [Second Form of Flow Channel Substrate] Fig. 10 is a plan view showing an example of a flow channel substrate 4B. Fig. 11 is a schematic diagram showing an example of the arrangement of detection reagents in the flow channels 45 provided in the flow channel substrate 4B. The flow channel substrate 4B is an example of the flow channel substrate 4.

[0125] As shown in FIG. 10 , the flow path substrate 4B may include a plurality of flow paths 45A-45D through which liquid flows, and a plurality of reservoirs 46A-46D connected to the plurality of flow paths 45, respectively. The plurality of flow paths 45 may include a first flow path in which at least a portion of a first detection reagent, which is a detection reagent for detecting a detection target, is located, and a second flow path in which at least a portion of a second detection reagent, which is a detection reagent for detecting a detection target different from the first detection reagent, is located. The detection reagent may be an example of a reagent that reacts with the detection target. The detection target may be, for example, a nucleic acid derived from the detection target. This allows multiple different detection targets to be detected at once by the simple operation of introducing liquid into the flow path substrate 4B.

[0126] 10 , the flow path substrate 4B may include a liquid receiving section 41, a branch flow path 42, a plurality of flow paths 45A to 45D, a plurality of storage sections 46A to 46D, and an internal standard 47. The flow path substrate 4B may include a second storage section 48 and an outlet 49.

[0127] The flow paths 45A to 45D may branch from a branch flow path 42 connected to the liquid receiving section 41. However, the flow path substrate 4B does not have to be provided with a branch flow path 42. In this case, the flow paths 45A to 45D may branch from the liquid receiving section 41. By having the flow paths 45A to 45D branch from the liquid receiving section 41 or the branch flow path 42, the liquid received in the liquid receiving section 41 can be introduced into the plurality of flow paths 45A to 45D. Therefore, it is possible to detect a plurality of different detection targets at once without needing to introduce a liquid into each of the plurality of flow paths 45A to 45D.

[0128] Furthermore, all of the flow paths 45A to 45D may branch off from a single liquid receiving section 41 or from a branch flow path 42 connected to a single liquid receiving section 41. This allows the liquid received in a single liquid receiving section 41 to be introduced into multiple flow paths 45A to 45D. Therefore, multiple different detection targets can be detected at the same time by introducing the liquid once.

[0129] Furthermore, the detection reagents may be disposed on the opposite side of the liquid receiving section 41 from the branching positions of the flow paths 45A to 45D, thereby reducing the possibility of different detection reagents mixing together, for example, reducing the possibility of the first detection reagent and the second detection reagent mixing together.

[0130] The flow path substrate 4B differs from the flow path substrate 4A in that the multiple flow paths 45 have a first flow path and a second flow path. Hereinafter, only the differences between the flow path substrate 4B and the flow path substrate 4A will be described. In other words, the same content as that described for the flow path substrate 4A will not be described in this embodiment.

[0131] In the present embodiment, the flow path substrate 4B may include a flow path 45B as the first flow path and a flow path 45C as the second flow path. As shown in Fig. 10 , a first region 451B and a second region 452B may be arranged in the flow path 45B, and a first region 451C and a second region 452C may be arranged in the flow path 45C.

[0132] 11 , a first reagent 1011 may be placed in the first region 451B as a first detection reagent. A first reagent 1012 different from the first reagent 1011 may be placed in the first region 451C as a second detection reagent. The first reagents 1011 and 1012 are examples of the first reagent 101. The first reagent 1011 may be the second detection reagent, and the first reagent 1012 may be the first detection reagent.

[0133] At least a portion of the detection reagent may be located in the flow path 45, or all of the detection reagent may be located therein. For example, a portion of the detection reagent may be located in the flow path 45, and the remainder of the detection reagent may be located in the reservoir 46. Here, "at least a portion of the detection reagent" may refer to some of the types of reagents when the detection reagent contains multiple types of reagents. For example, the first reagents 1011 and 1012 may be located in the flow path 45, and the second reagents 1021 and 1022 may be located in the reservoir 46. Furthermore, substantially the same components of the detection reagent may be located both in the flow path 45 and at a position other than the flow path 45. For example, substantially the same components of the detection reagent may be located in the flow path 45 and the reservoir 46.

[0134] The first reagents 1011 and 1012 may be primers having a sequence complementary to the sequence of a nucleic acid molecule derived from the detection target. The primers may be added as needed as long as they are capable of amplifying the target nucleic acid molecule, and may be, for example, one type or three or more types. In this embodiment, the first reagents 1011 and 1012 may be, for example, a first primer and a second primer. By arranging primers as detection reagents on the flow path substrate 4B, when the liquid received in the liquid receiving section 41 contains nucleic acid derived from the detection target, the nucleic acid derived from the detection target can be amplified in the flow path 45.

[0135] However, the first reagent 1012 may be a primer having a different sequence from that of the first reagent 1011. The first reagent 1012 may be a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from a detection target different from that of the first reagent 1011. For example, the first reagent 1011 may be a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from a first detection target, and the first reagent 1012 may be a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from a second detection target different from the first detection target. This allows a second detection target different from the first detection target that reacts with the first reagent 1011 to react with the first reagent 1012. The first detection target and the second detection target are examples of detection targets.

[0136] Therefore, when the liquid flowing from liquid-receiving unit 41 into flow path 45B contains a first target substance, the first target substance can be amplified in flow path 45B. When the liquid flowing from liquid-receiving unit 41 into flow path 45C contains a second target substance, the second target substance can be amplified in flow path 45C. In other words, the liquid received in liquid-receiving unit 41 can be introduced into multiple flow paths 45, and multiple different targets can be used as target substances for nucleic acid amplification at the same time.

[0137] The first reagents 1011 and 1012 may contain, for example, dNTPs, NTPs, trehalose, and an RNase inhibitor. However, if the RNase inhibitor is contained in the bottle portion 22, the first reagents 1011 and 1012 may not contain the RNase inhibitor.

[0138] 11 , a second reagent 1021 may be disposed as the first detection reagent in the second region 452B. A second reagent 1022 different from the second reagent 1021 may be disposed as the second detection reagent in the second region 452C. The second reagents 1021 and 1022 are examples of the second reagent 102. The second reagent 1021 may be the second detection reagent, and the second reagent 1022 may be the first detection reagent.

[0139] The second reagents 1021 and 1022 may be, for example, enzymes such as AMV-RT (Avian Myeloblastosis Virus), RNase H (Ribonuclease H), and T7 RNA polymerase. However, the second reagent 1022 may be an enzyme with a different sequence from that of the second reagent 1021.

[0140] This allows a second detection target, which is different from the first detection target that reacts with second reagent 1021, to react with second reagent 1022. Therefore, when the liquid flowing from liquid receiving unit 41 into flow path 45B contains the first detection target, the first detection target can be amplified in flow path 45B. When the liquid flowing from liquid receiving unit 41 into flow path 45C contains the second detection target, the second detection target can be amplified in flow path 45C.

[0141] The second reagents 1021 and 1022 may contain, for example, trehalose and a surfactant.

[0142] A labeling substance may further be disposed in the flow channel 45 as a detection reagent. When the substances to be detected in each flow channel 45 are different from each other, the labeling substance may have a sequence corresponding to the different detection targets. In this embodiment, the labeling substance disposed in flow channel 45B and the labeling substance disposed in flow channel 45C may be different from each other. This allows the labeling substance to bind to the detection target in accordance with the type of detection target. In this embodiment, the labeling substance may be disposed in the second regions 452B and 452C.

[0143] Furthermore, the flow path substrate 4B may include a flow path 45 that does not function as the first flow path or the second flow path. That is, in the flow path substrate 4B, a detection reagent may not be disposed, and a flow path 45 that is not intended for detecting a detection target substance may be connected to the branch flow path 42.

[0144] The flow path substrate 4B may include a negative control flow path 45 in which at least a labeling substance is disposed, but at least one element used in nucleic acid amplification, such as an enzyme for amplifying nucleic acids, is not disposed. In FIG. 10 , the flow path 45A may be a negative control flow path in which at least a labeling substance is disposed. The flow path 45A may have a second region 452A in which a second reagent 102 containing a labeling substance is disposed. The flow path substrate 4B may also include a flow path 45 in which no detection reagent is disposed. In FIG. 10 , the flow path 45D may be a flow path in which no detection reagent is disposed.

[0145] When the types of substances to be detected in each reservoir 46 are different, it is sufficient that at least the sequences of the primers and labeling substances in the detection reagents placed in each of the flow paths 45 connected to these reservoirs 46 are different from each other. In other words, some of the detection reagents placed in each of the flow paths 45 may be the same reagent. By using the same reagent for different detection targets, the flow path substrate 4B can be manufactured more inexpensively. For example, the enzymes placed in each of the flow paths 45 may be the same reagent. Furthermore, the dNTPs and NTPs placed in each of the flow paths 45 may also be the same reagent. Furthermore, the RNase inhibitor, trehalose, and surfactant may also be the same reagent.

[0146] The same reagent may be located on the opposite side of the liquid-receiving section 41 in the multiple flow paths 45 in which different detection reagents are located. That is, the same reagent may be located downstream of the position in the flow path 45 where the different detection reagents are located. For example, as described above, in flow paths 45B and 45C, an enzyme as the same reagent may be located in second regions 452B and 452C located downstream of first regions 451B and 451C in which primers are located. Furthermore, in flow paths 45B and 45C, dNTP and NTP as the same reagent may also be located in second regions 452B and 452C, for example. However, dNTP, NTP, RNase inhibitor, trehalose, and surfactant as the same reagent may be located in the liquid-receiving section 41 and / or branch flow path 42.

[0147] Furthermore, in each of the multiple flow paths 45, the length of the multiple flow paths 45 from the end opposite the storage section 46 of the detection reagent along the liquid feed direction to the end on the storage section 46 side may be approximately the same. That is, in each flow path 45, the length of the region in which the detection reagent is disposed along the extension direction of the flow path 45 may be approximately the same. This makes it possible to make the time it takes for the liquid to pass through the region in which the detection reagent is disposed approximately the same in each flow path 45. Therefore, it is possible to make the reaction time between the detection target and the detection reagent approximately the same in each flow path 45.

[0148] As shown in FIG. 11 , in this embodiment, the lengths LB and LC of the flow paths 45B and 45C may be substantially the same. The length LB is the length from the end of the first region 451B opposite the reservoir 46B to the reservoir 46B side of the second region 452B. The length LC is the length from the end of the first region 451C opposite the reservoir 46C to the reservoir 46C side of the second region 452C. The length L1B of the first region 451B in which the first reagent 1011 is disposed and the length L1C of the first region 451C in which the first reagent 1012 is disposed may be substantially the same or different. Furthermore, in the flow paths 45B and 45C, the length L2B of the second region 452B in which the second reagent 1021 is disposed and the length L2C of the second region 452C in which the second reagent 1022 is disposed may be substantially the same or different.

[0149] FIG. 12 is a schematic diagram illustrating an example of use of multiple flow channels 45. As shown in FIG. 12, for example, flow channel 45B may have a first region 451B in which a first reagent 1011 is disposed and a second region 452B in which a second reagent 1021 is disposed. Flow channel 45C may have a first region 451C in which a first reagent 1012 is disposed and a second region 452C in which a second reagent 1022 is disposed. Flow channel 45D may have a first region 451D in which a first reagent 1013 is disposed and a second region 452D in which a second reagent 1023 is disposed. The first reagents 1011, 1012, and 1013 may be different detection reagents. The second reagents 1021, 1022, and 1023 may be different detection reagents. Furthermore, the channel 45A may be a channel for negative control in which at least a labeling substance is disposed, but at least one of the elements used for nucleic acid amplification, such as an enzyme for amplifying nucleic acid, is not disposed.

[0150] In this way, different detection reagents may be placed in each of the flow paths 45A to 45D. However, the same detection reagent, including the primer and the labeling substance, may be placed in each of some of the multiple flow paths 45. For example, the first reagent 1012 and the second reagent 1022 placed in the flow path 45C may be placed in the flow path 45D instead of the first reagent 1013 and the second reagent 1023.

[0151] That is, the flow path substrate 4B only needs to include at least one first flow path in which a first detection reagent is disposed and at least one second flow path in which a second detection reagent is disposed, as the flow paths 45. This makes it possible to detect multiple substances by the simple operation of introducing liquid into the multiple flow paths 45.

[0152] In this embodiment, liquid introduced from liquid receiving section 41 is branched by branch flow path 42, flows through each of flow paths 45A to 45D, and is stored in each of storage sections 46A to 46D. If the liquid contains a first detection target, the first detection target reacts with the first detection reagent as the liquid flows through the first flow path in which the first detection reagent is disposed. If the liquid contains a second detection target, the second detection target reacts with the second detection reagent as the liquid flows through the second flow path in which the second detection reagent is disposed.

[0153] Therefore, the detection device 3 can detect a first detection target by measuring the intensity of fluorescence emitted from the reservoir 46 connected to the first flow path. The detection device 3 can detect a second detection target by measuring the intensity of fluorescence emitted from the reservoir 46 connected to the second flow path. In other words, by arranging detection reagents capable of detecting different detection targets in different flow paths 45, the detection device 3 can detect multiple different detection targets at once with the simple operation of introducing liquid into the flow path substrate 4B.

[0154] The flow path substrate 4B may include at least two or more flow paths 45 that function as a first flow path and a second flow path. As shown in FIG. 12 , the flow path substrate 4B may include a flow path 45X having a first region 451X in which a first reagent 1014 is disposed and a second region 452X in which a second reagent 1024 is disposed. The first reagent 1014 may be different from the first reagents 1011, 1012, and 1013. The second reagent 1024 may be different from the second reagents 1021, 1022, and 1023. Therefore, the detection device 3 can simultaneously detect a plurality of different detection targets, the number of which corresponds to the number of flow paths 45 in which different detection reagents are disposed.

[0155] The first reagents 1011 to 1014 do not all have to be different from one another. Any two of the first reagents 1011 to 1014 may be detection reagents that are different from one another. That is, some of the first reagents 1011 to 1014 may be first detection reagents, and another part may be second detection reagents. Furthermore, the second reagents 1021 to 1024 do not all have to be different from one another. Any two of the second reagents 1021 to 1024 may be detection reagents that are different from one another. That is, some of the second reagents 1021 to 1024 may be first detection reagents, and another part may be second detection reagents.

[0156] The flow path substrate 4B may include at least one positive control flow path 45. A detection reagent that reacts with a liquid that has been intentionally made to contain a detection target may be placed in the positive control flow path 45. However, the flow path 45 that functions as the first flow path and / or the second flow path used to flow a liquid containing a specimen may be substituted for the positive control flow path 45. In this case, a liquid that has been intentionally made to contain a detection target that reacts with the detection reagent placed in the flow path 45 may be flowed in the flow path 45.

[0157] 13 is a flowchart showing an example of a method for manufacturing the flow path substrate 4B. This manufacturing method may be performed by a manufacturing apparatus for manufacturing the flow path substrate 4B.

[0158] 13 , first, a flow path 45 and a storage portion 46 may be formed on a substrate (S11). In this embodiment, in addition to the flow path 45 and the storage portion 46, a liquid receiving portion 41, a branch flow path 42, a second storage portion 48, and an outlet 49 may be formed on the substrate.

[0159] Next, a detection reagent may be placed in the flow path 45. In this embodiment, a first detection reagent may be placed as a detection reagent in a first flow path, which is any one of the flow paths 45A to 45D (S12). A second detection reagent different from the first detection reagent may be placed in a second flow path, among the flow paths 45A to 45D, different from the first flow path in which the first detection reagent is placed (S13). Next, a fluorescent substance may be placed on the substrate as an internal standard 47 (S14). Next, a fluorescent substance may be placed in the second reservoir 48 (S15). The order of the processes of S12 to S15 does not matter. The processes of S12 to S15 may be performed in parallel.

[0160] [Summary] The flow path substrate according to aspect 1 of the present disclosure comprises a liquid receiving section that receives a liquid and a flow path connected to the liquid receiving section, and the flow path has a first region in which a first reagent containing a primer is disposed, and a second region in which a second reagent containing an enzyme that amplifies nucleic acid is disposed at a position different from the first region.

[0161] A flow path substrate according to a second aspect of the present disclosure is the same as that of the first aspect, wherein the second region is disposed on the opposite side of the liquid receiving section relative to the position where the first region is disposed.

[0162] A flow path substrate according to a third aspect of the present disclosure is the flow path substrate of the first or second aspect, wherein the flow path further includes a reservoir downstream of the second region for storing a liquid containing amplified nucleic acid.

[0163] A flow path substrate according to Aspect 4 of the present disclosure is any one of Aspects 1 to 3, wherein the first reagent is disposed in the first region as a plurality of first attachments.

[0164] A flow path substrate according to a fifth aspect of the present disclosure is the same as in the fourth aspect, wherein the plurality of first attachments are arranged as a film.

[0165] A flow path substrate according to a sixth aspect of the present disclosure is the same as that of the fourth or fifth aspect, wherein the plurality of first attachments are aligned in one direction.

[0166] A flow path substrate according to aspect 7 of the present disclosure is any one of aspects 4 to 6, wherein the plurality of first attachments are arranged at the bottom of the flow path so as not to contact the inner wall of the flow path.

[0167] A flow path substrate according to an eighth aspect of the present disclosure is any one of the first to seventh aspects, wherein the second reagent is disposed in the second region as a plurality of second attachments.

[0168] A flow path substrate according to Aspect 9 of the present disclosure is the same as Aspect 8, wherein the second attachment is arranged as a film.

[0169] A flow path substrate according to aspect 10 of the present disclosure is the same as aspect 8 or 9, wherein the plurality of second attachments are aligned in one direction.

[0170] A flow path substrate according to an eleventh aspect of the present disclosure is any one of aspects 8 to 10, wherein the plurality of second attachments are arranged at the bottom of the flow path so as not to contact the inner wall of the flow path.

[0171] A flow path substrate according to aspect 12 of the present disclosure is any one of aspects 4 to 7, in which the second reagent is arranged in the second region as a plurality of second attachments, and the second attachments are larger than the first attachments.

[0172] A flow channel substrate according to Aspect 13 of the present disclosure is any one of Aspects 1 to 11, wherein a labeling substance that specifically binds to the amplified nucleic acid is further disposed in the flow channel.

[0173] A flow path substrate according to Aspect 14 of the present disclosure is the same as Aspect 13, wherein the labeling substance is disposed downstream of the first region.

[0174] A flow path substrate according to Aspect 15 of the present disclosure is the flow path substrate of Aspect 13 or 14, wherein the second reagent includes the labeling substance.

[0175] A flow path substrate according to Aspect 16 of the present disclosure is the flow path substrate of any one of Aspects 13 to 15, wherein the labeling substance is a molecular beacon.

[0176] A cartridge according to a seventeenth aspect of the present disclosure includes the flow path substrate according to any one of the first to sixteenth aspects and a container capable of containing a liquid.

[0177] A detection system according to an eighteenth aspect of the present disclosure includes the cartridge according to the seventeenth aspect and a detection device that detects nucleic acid amplified in the flow channel.

[0178] A method for manufacturing a flow path substrate according to aspect 19 of the present disclosure includes a forming step of forming a liquid receiving portion for receiving a liquid and a flow path connected to the liquid receiving portion on a substrate, a first disposing step of disposing a first reagent containing a primer in the flow path, and a second disposing step of disposing a second reagent containing an enzyme for amplifying nucleic acid at a position on the flow path different from the position where the first reagent is disposed.

[0179] In the method for manufacturing a flow path substrate according to aspect 20 of the present disclosure, in the first placement step, the first reagent is placed in the flow path by applying a solution containing the first reagent to the flow path, and in the second placement step, the second reagent is placed in the flow path by applying a solution containing the second reagent to the flow path.

[0180] A flow path substrate according to aspect 22 of the present disclosure is any one of aspects 1 to 16, wherein the flow paths are a plurality of flow paths, and further includes a plurality of reservoirs connected to each of the plurality of flow paths, and the plurality of flow paths have a first flow path in which at least a portion of a first detection reagent, which is a detection reagent for detecting a detection target, is located, and a second flow path in which at least a portion of a second detection reagent, which is a detection reagent for detecting a detection target different from the first detection reagent, is located.

[0181] A flow path substrate according to aspect 22 of the present disclosure is any of aspects 1 to 16, wherein the flow path is a flow path through which a mixed fluid containing a sample and a labeled substance that reacts with a target substance contained in the sample flows, and further includes a standard section located in a region different from the flow path and for comparison with the mixed fluid.

[0182] [Additional Notes] 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 could 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.

[0183] DESCRIPTION OF SYMBOLS 1 Detection system 2 Cartridge 3 Detection device 4, 4A Flow path substrate 22 Bottle part (container) 41 Receiving part 45 Flow path (first flow path, second flow path) 46 Reservoir part 47 Internal standard (standard part) 101, 1011 to 1014 First reagent (detection reagent, first detection reagent, second detection reagent) 102, 1021 to 1024 Second reagent (detection reagent, first detection reagent, second detection reagent) 451 First region 452 Second region 454 Bottom 455 Inner wall

Claims

1. A flow path substrate comprising: a liquid receiving section for receiving a liquid; and a flow path connected to the liquid receiving section, the flow path having a first region in which a first reagent containing a primer is disposed, and a second region in which a second reagent containing an enzyme for amplifying nucleic acid is disposed at a position different from the first region.

2. The flow path substrate according to claim 1, wherein the second region is disposed on the opposite side of the liquid receiving portion with respect to a position where the first region is disposed.

3. A flow path substrate according to claim 1 or 2, wherein the flow path further comprises a reservoir section downstream of the second region for storing a liquid containing amplified nucleic acid.

4. A flow path substrate according to any one of claims 1 to 3, wherein the first reagent is disposed in the first region as a plurality of first attachments.

5. The flow path substrate according to claim 4, wherein the plurality of first deposits are arranged as a coating.

6. The flow path substrate according to claim 4 or 5, wherein the plurality of first attachments are arranged in one direction.

7. A flow path substrate according to any one of claims 4 to 6, wherein the plurality of first attachments are arranged on the bottom of the flow path so as not to contact an inner wall of the flow path.

8. A flow path substrate according to any one of claims 1 to 7, wherein the second reagent is disposed in the second region as a plurality of second attachments.

9. The flow path substrate according to claim 8, wherein the second deposit is disposed as a coating.

10. The flow path substrate according to claim 8 or 9, wherein the plurality of second attachments are arranged in one direction.

11. A flow path substrate according to any one of claims 8 to 10, wherein the plurality of second attachments are arranged on the bottom of the flow path so as not to contact an inner wall of the flow path.

12. A flow path substrate as claimed in any one of claims 4 to 7, wherein the second reagent is arranged in the second region as a plurality of second attachments, and the second attachments are larger than the first attachments.

13. A flow path substrate according to any one of claims 1 to 11, wherein a labeling substance that specifically binds to the amplified nucleic acid is further disposed in the flow path.

14. The flow path substrate according to claim 13, wherein the labeling substance is disposed downstream of the first region.

15. The flow path substrate according to claim 13 or 14, wherein the second reagent includes the labeling substance.

16. The flow channel substrate according to any one of claims 13 to 15, wherein the labeling substance is a molecular beacon.

17. A cartridge comprising: a flow path substrate according to any one of claims 1 to 16; and a container capable of containing a liquid.

18. A detection system comprising the cartridge of claim 17 and a detection device for detecting amplified nucleic acid in the flow path.

19. A method for manufacturing a flow path substrate, comprising: a forming step of forming a liquid receiving section for receiving liquid and a flow path connected to the liquid receiving section in a substrate; a first disposing step of disposing a first reagent containing a primer in the flow path; and a second disposing step of disposing a second reagent containing an enzyme that amplifies nucleic acid in a position in the flow path different from the position where the first reagent is disposed.

20. A method for manufacturing a flow path substrate as described in claim 19, wherein in the first placement step, the first reagent is placed in the flow path by applying a solution containing the first reagent to the flow path, and in the second placement step, the second reagent is placed in the flow path by applying a solution containing the second reagent to the flow path.

21. A flow path substrate as described in claim 1, wherein the flow path is a plurality of flow paths, and further comprising a plurality of storage sections connected to each of the plurality of flow paths, the plurality of flow paths having: a first flow path in which at least a portion of a first detection reagent which is a detection reagent for detecting a detection target is located; and a second flow path in which at least a portion of a second detection reagent which is a detection reagent for detecting a detection target different from the first detection reagent is located.

22. The flow path substrate of claim 1, wherein the flow path is a flow path through which a mixed fluid containing a specimen and a labeling substance that reacts with the target substance contained in the specimen flows, and further comprising a standard portion located in an area different from the flow path and for comparing with the mixed fluid.

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