Reactor, cartridge, and nucleic acid amplification method for nucleic acid amplification

Abstract

To provide a nucleic acid amplification reaction vessel, a cartridge, and a nucleic acid amplification method that can suppress the occurrence of false positives.SOLUTION: Provided is a vertically long reaction vessel comprising a lower part in the longitudinal direction provided as a reaction liquid accommodating space, an upper part provided as a reaction liquid supply space, and a reaction liquid supply port and an exhaust port in the reaction liquid supply space. The reaction liquid supply port is located below the exhaust port in the longitudinal direction. A vertically long reaction vessel comprises a lower part in the longitudinal direction provided as a reaction liquid accommodating space, and an upper part provided as a reaction liquid supply space. At least a part of the vicinity of the boundary between the reaction liquid supply space and the reaction liquid accommodating space has a protrusion directed toward the inside of the reaction vessel.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a reaction vessel for nucleic acid amplification, a cartridge, and a nucleic acid amplification method.

Background Art

[0002] With the spread of COVID-19, the demand for technologies to easily and reliably determine the presence or absence of virus infection by collecting, amplifying, and detecting nucleic acids in the virus has been increasing day by day. Also, even after the issues of COVID-19 are resolved, since humanity still faces diseases caused by various viruses and bacteria, the demand for similar technologies still exists.

[0003] Techniques for amplifying and detecting nucleic acids have been almost established as a methodology with the emergence of the PCR method using a thermocycler and the subsequent development of various methods other than the thermocycler PCR method, and have come to be easily performed not only in laboratories but also in hospital laboratories and the like. However, even then, after manually collecting nucleic acids from biological samples such as viruses, nucleic acid amplification and detection were often performed using existing amplification detection devices. However, with the spread of COVID-19, it is clear that existing methods and devices cannot cope with the serious situation when specimens are brought to health centers or testing companies. At present, it is impossible to surely determine the presence or absence of virus infection by simply processing a large amount of specimens regardless of location in a short time.

[0004] Patent Document 1 discloses a portable and small amplification detection device that can more easily perform nucleic acid amplification and detection.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] The apparatus described in Patent Document 1 is a device that separately recovers nucleic acids from a sample, and then amplifies and detects the recovered nucleic acids. However, this method requires the separate recovery of nucleic acids from the sample, and therefore does not allow for the detection of viruses from the sample in a single step.

[0007] In response to this, the inventors devised a device that uses the sample without pretreatment and performs nucleic acid recovery from the sample, amplification of the recovered nucleic acid, and detection of the amplified nucleic acid in a single device. This device is intended to sequentially and automatically carry out the above processes in a chamber connected by a microfluidic channel, but the inventors discovered that false positives occur due to the nucleic acid amplification reaction vessel used to amplify the recovered nucleic acid.

[0008] The problem that the present invention aims to solve is to provide a nucleic acid amplification reactor that can suppress the occurrence of false positives, a nucleic acid amplification cartridge containing the same, and a nucleic acid amplification method using this cartridge. The present invention aims to provide a nucleic acid amplification reactor that can suppress the occurrence of false positives, a nucleic acid amplification cartridge containing the same, and a nucleic acid amplification method using this cartridge.

[0009] When detecting viruses in a sample using a cartridge with multiple chambers connected by microfluidic channels, the smaller the amount of nucleic acid amplification reaction solution, the lower the required amount of enzyme and primer, and the higher the amplified nucleic acid concentration, thus increasing the likelihood of improved sensitivity. On the other hand, the smaller the amount of reaction solution, the more difficult it becomes to position the reaction solution in the desired location, usually at the bottom, due to the interaction between the interfacial tension of the liquid and the inner surface of the reaction vessel. In particular, in methods where the reaction solution is supplied to the reaction solution supply space from the reaction solution supply port by suction from the exhaust port, bubbles may be generated during reaction solution supply, and if bubbles remain, the reaction solution may not be positioned in the desired location. Nucleic acid amplification and detection under these conditions can lead to false positives. This tendency is particularly pronounced when the reaction solution volume is, for example, 10-500 μL or 10-100 μL.

[0010] Figure 4 shows a vertically elongated reaction vessel 50 having an exhaust port 51 at the top and a reaction liquid supply port 52 near the bottom, where the reaction liquid RS is stagnating on the walls inside the reaction vessel and cannot be contained in the reaction liquid storage space at the bottom in the longitudinal direction. In this reaction vessel 50, the reaction liquid is transferred by creating negative pressure inside the vessel through suction from the exhaust port 51 and sucking the reaction liquid from a separate chamber to the supply port near the bottom. However, as shown in Figure 4, the reaction liquid may splatter inside the reaction vessel 50, causing it to adhere to the walls inside the vessel and form a foamy substance.

[0011] In order to eliminate foamy reaction liquid adhering to the walls inside the reaction vessel and to store the reaction liquid in a predetermined location inside the vessel, in one embodiment of the present invention, the reaction vessel has a reaction liquid supply port and an exhaust port in the reaction liquid supply space, and the reaction liquid supply port is located below the exhaust port in the longitudinal direction of the reaction vessel. Because the reaction liquid supply port is located below the exhaust port, the reaction liquid supplied from the reaction liquid supply port flows downward, and the foam can easily escape upward.

[0012] Furthermore, in order to eliminate foamy reaction liquid adhering to the walls inside the reaction vessel and to store the reaction liquid in a predetermined location inside the vessel, in another embodiment of the present invention, projections facing inward are provided at least a portion of the boundary between the lower and upper parts of the reaction vessel. Foam is more easily eliminated at the tip of the projection, and as a result, the liquid tends to accumulate at the bottom.

[0013] Furthermore, in this invention, we have found that if the reaction liquid supply port is located below the exhaust port and above the protrusion, rather than near the bottom, foam generation can be further suppressed, the reaction liquid can be more easily stored in the space below the protrusion, and as a result, the occurrence of false positives due to the reaction liquid not being in the predetermined position can be further suppressed. [Means for solving the problem]

[0014] The present invention is as follows: [1] A vertically elongated reaction vessel, with the lower part in the longitudinal direction being a space for containing the reaction liquid, and the upper part being a space for supplying the reaction liquid, having a reaction liquid supply port and an exhaust port in the reaction liquid supply space, The reaction solution supply port is located below the longitudinal exhaust port in the nucleic acid amplification reaction vessel. [2] The reaction vessel according to [1], for use in a method in which the reaction liquid is supplied from the reaction liquid supply port to the reaction liquid supply space by suction to the outside from the exhaust port. [3] A reaction vessel for nucleic acid amplification, having a vertically elongated shape, wherein the lower part in the longitudinal direction is a space for containing the reaction solution, the upper part is a space for supplying the reaction solution, and at least a portion of the area near the boundary between the reaction solution supply space and the reaction solution containment space has a projection that extends inward into the reaction vessel. [4] The reaction vessel according to [1] or [2], having at least a portion of the area near the boundary between the reaction liquid supply space and the reaction liquid containment space that extends inward into the reaction vessel. [5] The reaction vessel according to [3] or [4], wherein the projection is present along the entire boundary. [6] A reaction vessel according to any one of items [3] to [5], wherein, when the horizontal cross-sectional area of ​​the reaction vessel is set to 100, the ratio of the horizontal cross-sectional area of ​​the location having the protrusion is in the range of 30 to 95. [7] The area of ​​the opening in the horizontal cross-section at the location of the protrusion is 1 to 10 mm². 2 A reaction vessel described in any one of items [3] to [6], which falls within the range of [3] to [6]. [8] When the reaction vessel has an exhaust port in the space for supplying the reaction liquid, The reaction vessel according to any one of items [4] to [7], wherein the reaction liquid supply port is located below the exhaust port and above the protrusion. [9] The reaction vessel, according to any one of items [1] to [8], has a reaction solution containment space with a volume in the range of 10 to 500 μL.

[10] When the reaction vessel has an exhaust port in the space for supplying the reaction liquid, The ratio L1:L2:L3 of the distance L1 between the exhaust port and the reaction liquid supply port, the distance L2 between the protrusion and the reaction liquid supply port, and the distance L3 between the protrusion and the bottom of the reaction liquid storage space can be, for example, in the range of 1:0.1 to 1:0.1 to 1. When L1 + L2 + L3 is set to 1, L2 + L3 is, for example, in the range of 0.3 to 0.7. The reaction tank according to any one of [4] to [9].

[11] The reaction liquid storage space is the reaction tank according to any one of [1] to

[10] , wherein at least a part of the side wall has light transmissibility.

[12] The horizontal cross-sectional area of the reaction liquid storage space is smaller than the horizontal cross-sectional area of the reaction liquid supply space. The reaction tank according to any one of [1] to

[11] .

[13] The surface roughness Ra of at least a part or all of the inner surface of the reaction tank is 25 nm or less. The reaction tank according to any one of [1] to

[12] .

[14] At least a part or all of the inner surface of the reaction tank is coated with wax. The reaction tank according to any one of [1] to

[12] .

[15] A nucleic acid amplification cartridge (10) having chambers connected by microchannels and including one or more reaction tanks (20) for nucleic acid amplification connected to the chambers via the microchannels (30 and 31), The reaction tank (20) is the reaction tank according to any one of [1] to

[14] . Cartridge.

[16]

[15] A nucleic acid amplification method using the cartridge according to [[ID= \25]]

[16] By sucking from the exhaust port to make the inside of the reaction tank negative pressure, a reaction liquid containing the nucleic acid to be amplified is supplied from the reaction liquid supply port to the reaction liquid supply space,

[16] The reaction liquid that has spontaneously moved from the reaction liquid supply space to the reaction liquid storage space is subjected to a nucleic acid amplification reaction. A nucleic acid amplification method including this.

[16]

Advantages of the Invention

[16]

[16]

[0015]

[16]

[16] ​According to the present invention, the occurrence of false positives in detection after nucleic acid amplification can be suppressed. [Brief explanation of the drawing]

[0016] [Figure 1] This is a schematic diagram illustrating a portion of the cartridge of the present invention that includes an example of a reaction vessel. [Figure 2] This is a schematic diagram illustrating an example of a reaction vessel according to the present invention. [Figure 3-1] This is a schematic diagram illustrating an example of a reaction vessel according to the present invention. [Figure 3-2] This is a schematic diagram illustrating an example of a reaction vessel according to the present invention. [Figure 4] This is a schematic diagram illustrating a reaction vessel other than the one described in the present invention. [Figure 5] This is a schematic diagram illustrating an example of a reaction vessel according to the present invention. [Modes for carrying out the invention]

[0017] <Reactor for nucleic acid amplification> The first aspect of the present invention relates to a nucleic acid amplification reaction vessel, which is vertically elongated, with the lower part in the longitudinal direction being a space for containing the reaction solution, the upper part being a space for supplying the reaction solution, and the reaction solution supply space having a reaction solution supply port and an exhaust port, the reaction solution supply port being located below the exhaust port in the longitudinal direction.

[0018] A second aspect of the present invention relates to a nucleic acid amplification reaction vessel, which is vertically elongated, wherein the lower part in the longitudinal direction is a space for containing the reaction solution, the upper part is a space for supplying the reaction solution, and at least a portion near the boundary between the reaction solution supply space and the reaction solution containment space has a projection that extends inward into the reaction vessel.

[0019] The preferred nucleic acid amplification reaction vessel of the present invention is a vertically elongated reaction vessel, which is a common part of the first and second embodiments, wherein the lower part in the longitudinal direction is a space for containing the reaction solution, the upper part is a space for supplying the reaction solution, the space for supplying the reaction solution has a reaction solution supply port and an exhaust port, the reaction solution supply port is located below the exhaust port in the longitudinal direction, and at least a part of the vicinity of the boundary between the reaction solution supply space and the reaction solution containment space has a projection toward the inside of the reaction vessel.

[0020] Figures 1 and 2 are schematic diagrams illustrating an example of a nucleic acid amplification reactor according to the present invention. The nucleic acid amplification reactor of the present invention will be described based on Figures 1 and 2.

[0021] The nucleic acid amplification reaction vessel 20 of the present invention is a vertically elongated reaction vessel, with the lower part in the longitudinal direction being a space 21 for containing the reaction solution and the upper part being a space 22 for supplying the reaction solution. The reaction solution supply space 22 has a reaction solution supply port 32 and an exhaust port 24. At least a portion of the area near the boundary between the reaction solution supply space 22 and the reaction solution containment space 21 has a projection 25 that extends into the inside of the reaction vessel. Figure 2A shows a cross-section from the same plane as Figure 1, and Figure 2B shows a cross-section of the reaction vessel 20 when rotated 90° with respect to the vertical axis (longitudinal axis). From this figure, it can be seen that the space from the reaction solution supply space 22 to the projection 25 is a rectangular parallelepiped. However, the reaction solution supply space 22 may also be cylindrical, and the projection 25 may be circular in shape with a circular opening in the center. The reaction solution supply space 22 may have a columnar portion 23 extending from the side wall inside the reaction vessel 20 near the reaction solution supply port 32 or the projection 25.

[0022] The reaction solution containment space is a space for containing the reaction solution and amplifying the nucleic acids contained in it. There are no particular limitations on its volume, but from the viewpoint of amplifying nucleic acids in minute amounts of sample and detecting them with high sensitivity, it is, for example, in the range of 10 to 500 μL, preferably in the range of 10 to 100 μL. However, this is not intended to be the sole numerical range.

[0023] The protrusions 25 near the boundary between the reaction liquid supply space 22 and the reaction liquid containment space 21 are present in part or all of the boundary, preferably all of it. The presence of the protrusions 25 makes it easier to eliminate bubbles generated during reaction liquid supply, and facilitates the storage of the reaction liquid in the reaction liquid containment space 21. Furthermore, the opening 26 formed by the protrusions 25 serves as an entrance for the reaction liquid supplied to the reaction liquid supply space 22 to move into the reaction liquid containment space 21, and also has the function of determining the area in which the liquid surface of the reaction liquid after moving into the reaction liquid containment space 21 comes into contact with the air in the reaction liquid supply space 22. By setting the amount of reaction liquid so that the liquid surface of the reaction liquid is at approximately the same height as the opening 26, the liquid surface of the reaction liquid becomes approximately equal to the area of ​​the opening 26, which is smaller than when there are no protrusions 25, and as a result, the reaction liquid is more likely to remain in the reaction liquid containment space 21. As shown in Figure 3-1, when the protrusion 25 is present, the reaction liquid tends to remain in the reaction liquid containment space 21, minimizing its surface area through interfacial tension.

[0024] When the horizontal cross-sectional area of ​​the reaction vessel (for example, the cross-sectional area at the location of the reaction liquid supply port 32) is set to 100, the ratio of the horizontal cross-sectional area at the location of the projection, i.e., the area of ​​the opening 26, is preferably in the range of 30 to 95, and more preferably in the range of 60 to 80. The area of ​​the opening 26 in the horizontal cross-sectional area at the location of the projection 25 depends on the horizontal cross-sectional area of ​​the reaction vessel, but for example, it is 1 to 10 mm. 2 The horizontal cross-sectional area of ​​the reaction vessel can be, for example, 2 to 20 mm². 2 It can be within this range; however, it is not intended to be limited to these numerical ranges.

[0025] From the viewpoint of facilitating the storage of the reaction liquid in the reaction liquid containment space 21 as a single mass, it is preferable that the reaction liquid supply port 32 be located below the exhaust port 24 and above the projection 25. If the reaction liquid supply port 32 is located within the reaction liquid containment space 21, the exhaust port 24 is located near the top, and as shown in Figure 5, the reaction liquid supplied from the reaction liquid supply port 32 tends to remain in a space other than the reaction liquid containment space 21, making it difficult to store it as a single mass within the reaction liquid containment space 21. As shown in Figure 2, the ratio L1:L2:L3 of the distance L1 between the exhaust port 24 and the reaction liquid supply port 32, the distance L2 between the projection 25 and the reaction liquid supply port 32, and the distance L3 between the projection 25 and the bottom of the reaction liquid containment space 21 can be, for example, in the range of 1:0.1 to 1:0.1 to 1, and in the range of 1:0.3 to 0.8:0.3 to 0.8. When L1 + L2 + L3 equals 1, L2 + L3 can be, for example, in the range of 0.3 to 0.7. However, it is not intended to be limited to these numerical ranges.

[0026] From the viewpoint of detecting the amplified nucleic acid directly after or during nucleic acid amplification, it is preferable that at least a portion of the side wall of the reaction solution containment space is light-transmitting. Furthermore, the horizontal cross-sectional area of ​​the reaction solution containment space (e.g., the maximum cross-sectional area) can be smaller than the horizontal cross-sectional area of ​​the reaction solution supply space. In particular, when at least a portion of the side wall is light-transmitting, it is preferable to make the horizontal cross-sectional area of ​​the reaction solution containment space smaller than the horizontal cross-sectional area of ​​the reaction solution supply space by shortening the distance from the light-transmitting side wall to the inner surface of the wall facing it. This reduces the amount of reaction solution to be contained in the reaction solution containment space and allows for efficient use of detection light (excitation light) for the reaction solution containing the amplified nucleic acid.

[0027] It is even more preferable, from the viewpoint of further suppressing false positives, that the surface roughness Ra of at least part or all of the inner surface of the reaction solution containment space 21 is 25 nm or less. It is even more preferable, from the viewpoint of further suppressing false positives, that the surface roughness Ra of at least part or all of the inner surface of the reaction solution supply space 22 is 25 nm or less. However, this is not intended to be the only numerical range.

[0028] It is preferable that at least part or all of the inner surface of the reaction solution containment space 21 is coated with wax, from the viewpoint of controlling surface roughness and further suppressing the occurrence of false positives. It is also preferable that at least part or all of the inner surface of the reaction solution supply space 22 is coated with wax, from the viewpoint of further suppressing the occurrence of false positives.

[0029] <Cartridge for nucleic acid amplification> The present invention relates to a nucleic acid amplification cartridge 10 having a chamber connected by microchannels, and comprising one or more nucleic acid amplification reaction vessels 20 connected to the chamber via microchannels 30 and 31, wherein the reaction vessel 20 is the reaction vessel of the present invention.

[0030] There are no particular restrictions on the chambers connected by microchannels, the microchannels themselves, or the method and structure of communication between each microchannel. The number of reaction vessels 20 in the cartridge of the present invention can be one or more, and the number of more than two can be 3, 4, 5, 6, 7, 8, 9, or 10, but is not limited to these numbers. Figure 1 shows an example of a cartridge having four reaction vessels 20. A microchannel 30 from the upstream chamber branches into two to become microchannels 31, and each microchannel 31 further branches into two to form a total of four channels, which are connected to the respective reaction liquid supply ports 32 of the reaction vessels. The supply of reaction liquid to the reaction vessels 20 via microchannels 30 and 31 is carried out by applying negative pressure to the reaction liquid supply space 22 from an exhaust port 24 located within the reaction liquid supply space 22. Negative pressure can be applied, for example, by connecting a vacuum pump to the exhaust port 24. The vacuum pump can be provided separately from the cartridge.

[0031] Even if the cartridge of the present invention has two or more reaction tanks 20, the reaction solution can be stored in a predetermined position in any of the reaction tanks, as long as the reaction tanks 20 are the reaction tanks of the present invention. For example, even in a cartridge having four reaction tanks as shown in Figure 3-2, the reaction solution can be stored in a predetermined position in any of the reaction tanks.

[0032] <Nucleic acid amplification method> The present invention encompasses a nucleic acid amplification method using the cartridge of the present invention described above. This method includes (1) supplying a reaction solution containing the nucleic acid to be amplified from the reaction solution supply port to the reaction solution supply space by creating negative pressure inside the reaction vessel by drawing in suction from the exhaust port, and (2) subjecting the reaction solution that has spontaneously moved from the reaction solution supply space to the reaction solution containment space to the nucleic acid amplification reaction.

[0033] In step (1), the reaction solution containing the nucleic acid to be amplified is supplied from the reaction solution supply port to the reaction solution supply space. The reaction solution is supplied by suction from the exhaust port to create negative pressure inside the reaction vessel. By supplying the reaction solution to the reaction solution supply space from the reaction solution supply port 32, which is located below the exhaust port 24 and above the projection 25, the generation of bubbles in the reaction solution can be suppressed, and the occurrence of false positives in detection after nucleic acid amplification can be further suppressed. Furthermore, it is preferable that the amount of reaction solution supplied to the reaction solution supply space be equal to the volume of the reaction solution containment space. This makes it easier for the reaction solution to be contained as a single mass in the reaction solution containment space due to the action of the projection, and the occurrence of false positives in detection after nucleic acid amplification can be further suppressed.

[0034] In step (2), the reaction solution that has spontaneously moved from the reaction solution supply space to the reaction solution containment space is subjected to the nucleic acid amplification reaction. The reaction solution contains the nucleic acid to be amplified, and in addition, it contains a nucleic acid amplification enzyme and a nucleic acid amplification primer or probe. The enzyme and primer or probe can be added to the reaction solution in advance before being supplied to the reaction vessel, or they can be added in the reaction vessel. Addition in the reaction vessel can be performed by placing water-soluble beads containing the nucleic acid amplification enzyme and water-soluble beads containing the nucleic acid amplification primer or probe between the reaction solution supply port and the projection of the reaction solution supply space.

[0035] The nucleic acid to be amplified in the reaction solution is not particularly limited and can be appropriately selected according to the intended use of the cartridge of the present invention. Furthermore, there are no particular limitations on the nucleic acid amplification enzyme, nucleic acid amplification primer, and probe; appropriate materials can be used depending on the nucleic acid amplification method.

[0036] The nucleic acid to be amplified is not particularly limited, but it can be, for example, RNA or DNA. The nucleic acid amplification reaction can be an isothermal amplification reaction or a thermocycle amplification reaction. An isothermal amplification reaction is, for example, the LAMP method or the SmartAmp method. A thermocycle amplification reaction can be a PCR amplification reaction.

[0037] The nucleic acid amplification enzyme is not particularly limited, but can be, for example, an enzyme for isothermal amplification of nucleic acids or an enzyme for thermocycle amplification. The isothermal amplification of nucleic acids can be, for example, the LAMP method or SmartAmp method, and can be an enzyme for nucleic acid amplification using a chain substitution reaction.

[0038] Polymerases, which are nucleic acid amplification enzymes having strand displacement activity, can be known enzymes. For example, the polymerase described in International Publication No. 2004 / 040019 can be cited, but is not intended to be limited to. The polymerase having strand displacement activity can also be DNA polymerase (Aac), which is disclosed in International Publication No. 2009 / 054510 (Japanese Patent No. 4450867).

[0039] Polymerases used in nucleic acid amplification reactions with strand displacement activity can be suitably those that are thermothermally, mesothermally, or thermostable. Furthermore, these polymerases may be either naturally occurring or artificially mutated mutants. Examples of such polymerases include DNA polymerases. Examples of such DNA polymerases include mutants of DNA polymerases derived from thermophilic Bacillus bacteria such as Bacillus stearothermophilus (hereinafter referred to as "B.st") and Bacillus caldotenax (hereinafter referred to as "B.ca") that lack 5'→3' exonuclease activity, and Klenow fragments of DNA polymerase I derived from Escherichia coli (E. coli). DNA polymerases used in nucleic acid amplification reactions include Vent DNA polymerase, Vent(Exo-)DNA polymerase, DeepVent DNA polymerase, DeepVent(Exo-)DNA polymerase, Φ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase, Pfu DNA polymerase, Pfu turbo DNA polymerase, KOD DNA polymerase, 9°Nm DNA polymerase, Therminator DNA polymerase, and Taq DNA polymerase.

[0040] When the nucleic acid to be amplified is RNA, a reverse transcriptase can be used in combination with DNA polymerase, or a DNA polymerase that also possesses reverse transcription activity can be used. The reverse transcriptase is not particularly limited as long as it has cDNA synthesis activity using RNA as a template, and examples include reverse transcriptases from various origins, such as reverse transcriptase derived from avian myeloblastosis virus (AMVRTase), reverse transcriptase from Rouss-associated virus 2 (RAV-2RTase), and reverse transcriptase derived from Moloney's mouse leukemia virus (MMLV RTase). Examples of DNA polymerases that also possess reverse transcription activity include BcaBEST DNA polymerase, Bca(exo-)DNA polymerase, and Tth DNA polymerase.

[0041] Primers are appropriately selected depending on the enzyme used for the nucleic acid amplification reaction. If the enzyme used for the nucleic acid amplification reaction is one that utilizes a chain substitution reaction, examples of primers can be found in International Publication No. 2004 / 040019, Japanese Patent Publication No. 2009-171935, Japanese Patent Publication No. 2011-50380, and others.

[0042] The method of the present invention may further include a step of optically detecting, electrically detecting, or detecting the nucleic acid amplified in the reaction vessel after the nucleic acid amplification operation. Detection of the amplified nucleic acid can be performed using a fluorogenic primer as the primer and labeling of the fluorogenic primer. Detection of the amplified nucleic acid can be performed by using an exciton primer or exciton probe in the amplification reaction and utilizing the exciton effect. The method of optically detecting nucleic acid may also be a method using an intercalating dye.

[0043] In the method of the present invention, it is preferable to perform the nucleic acid amplification reaction by the SmartAmp method or the LAMP method and to label and detect it with an exciton primer or exciton probe. Alternatively, it is preferable to perform the nucleic acid amplification reaction by the PCR method and to label and detect it with an exciton primer or exciton probe.

[0044] Following the nucleic acid amplification reaction, a nucleic acid melting curve can be drawn, and the properties of the amplified product, such as false positives and true positives, can be determined from the melting curve. [Industrial applicability]

[0045] This invention is useful in fields related to nucleic acid amplification and detection. [Explanation of Symbols]

[0046] 10 cartridges 20 reaction vessels 21 Space for containing reaction solution 22 Space for supplying reaction solution 23 Columnar part 24 Exhaust vents 25 Protrusion 26 Aperture 30,31 Channel 32 Reaction liquid supply port

Claims

1. A vertically elongated reaction vessel, with the lower part in the longitudinal direction being a space for containing the reaction liquid, and the upper part being a space for supplying the reaction liquid, having a reaction liquid supply port and an exhaust port in the reaction liquid supply space, The reaction liquid supply port is located below the longitudinal exhaust port, The reaction solution containment space has side walls that are at least partially light-transmitting to detection light, so that the amplified nucleic acid can be detected after or during nucleic acid amplification. A nucleic acid amplification reaction vessel having a projection extending inward towards the inside of the reaction vessel, at least a portion of the vicinity of the boundary between the reaction solution supply space and the reaction solution containment space.

2. The reaction vessel according to claim 1, for use in a method in which the reaction liquid is supplied to the reaction liquid supply space from the reaction liquid supply port by suction to the outside from the exhaust port.

3. A reaction vessel for nucleic acid amplification, having a vertically elongated shape, wherein the lower part in the longitudinal direction is a space for containing the reaction solution, the upper part is a space for supplying the reaction solution, and at least a portion of the area near the boundary between the reaction solution supply space and the reaction solution containment space has a projection that extends inward into the reaction vessel.

4. The reaction vessel according to claim 1, wherein the reaction liquid supply port is located above the projection.

5. The reaction vessel according to claim 1 or 4, wherein the ratio L1:L2:L3 of the distance L1 between the exhaust port and the reaction liquid supply port, the distance L2 between the projection and the reaction liquid supply port, and the distance L3 between the projection and the bottom of the reaction liquid containment space is in the range of 1:0.1 to 1:0.1 to 1, or when L1 + L2 + L3 is set to 1, L2 + L3 is in the range of 0.3 to 0.

7.

6. The reaction vessel according to any one of claims 1 to 5, wherein the projection is present along the entire boundary.

7. The reaction vessel according to any one of claims 1 to 6, wherein, when the horizontal cross-sectional area of ​​the reaction vessel is set to 100, the ratio of the horizontal cross-sectional area of ​​the position having the protrusion is in the range of 30 to 95.

8. The area of ​​the opening in the horizontal cross-section at the location of the protrusion is 1 to 10 mm ２ A reaction vessel according to any one of claims 1 to 7, which is within the range of [the specified range].

9. The reaction vessel according to any one of claims 1 to 8, wherein the space for containing the reaction solution has a volume in the range of 10 to 500 μL.

10. The reaction vessel according to any one of claims 1 to 9, wherein the horizontal cross-sectional area of ​​the space for containing the reaction liquid is smaller than the horizontal cross-sectional area of ​​the space for supplying the reaction liquid.

11. The reaction vessel according to any one of claims 1 to 10, wherein the surface roughness Ra of at least a part or all of the inner surface of the reaction vessel is 25 nm or less.

12. The reaction vessel according to any one of claims 1 to 10, wherein at least part or all of the inner surface of the reaction vessel is coated with wax.

13. A nucleic acid amplification cartridge (10) having a chamber connected by a microchannel, and including one or more nucleic acid amplification reaction vessels (20) connected to the chamber via microchannels (30 and 31), The cartridge wherein the reaction vessel (20) is the reaction vessel according to any one of claims 1 to 12.

14. A nucleic acid amplification method using the cartridge described in claim 13, By drawing air in through the exhaust port to create negative pressure inside the reaction vessel, the reaction solution containing the nucleic acid to be amplified is supplied from the reaction solution supply port into the reaction solution supply space. A nucleic acid amplification method comprising subjecting a reaction solution that has spontaneously moved from a reaction solution supply space to a reaction solution containment space to a nucleic acid amplification reaction.

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