Nucleic acid amplification device

Abstract

The present invention provides a nucleic acid amplification device capable of sending a reaction liquid containing a reaction product of PCR without using a pump and performing temperature control, and capable of executing PCR in a short time with a simple device structure. A nucleic acid amplification device (100) is a device that executes PCR and amplifies a nucleic acid, the device comprising: a flow path device (101) having a flow path formed inside the flow path device (101); and a temperature control mechanism (110) capable of controlling the temperatures of multiple regions on the flow path device (101). The flow path device (101) has: a closed ring-shaped chamber (104); an inlet (105) through which fluid can be introduced into the chamber (104); and an outlet (106) through which fluid can be discharged from the chamber (104). The temperatures of the multiple regions on the chamber (104) are controlled to fall within different temperature ranges, and the reaction liquid which contains a nucleic acid and is introduced into the chamber (104) is circulated convectively in the chamber (104), and is subjected to steps configuring PCR in each region, thereby executing PCR in the chamber (104).

Description

Nucleic Acid Amplification Device

[0001] The present invention relates to a nucleic acid amplification device that performs PCR by natural convection of a reaction solution inside a flow path device.

[0002] PCR (Polymerase Chain Reaction) is a reaction that uses polymerase to amplify nucleic acids. PCR can amplify minute amounts of nucleic acids in a short period of time, resulting in large amounts of copies. Therefore, nucleic acids can be detected and analyzed even when the sample contains only minute amounts of nucleic acids. Due to these characteristics, PCR is used in a wide range of fields, including genetic testing, DNA identification, and research and development. Because it can amplify specific regions in a base sequence, it is also widely used as a pretreatment for DNA sequencing.

[0003] Typical PCR is performed using thermal cycling, in which the temperature of the reaction solution is gradually changed to perform each step of PCR. Thermal cycling consists of a denaturation step, in which double-stranded DNA is denatured into single strands, an annealing step, in which primers are bound to the single-stranded DNA, and an extension step, in which a complementary strand is synthesized from the primer. By repeating these steps sequentially, a specific region of the template DNA is amplified.

[0004] Denaturation is generally carried out in a temperature range of about 90 to 96°C. By heating double-stranded DNA, the double-stranded DNA is denatured and dissociated into single strands. Annealing is carried out in a temperature range of about 55 to 65°C. After denaturation, the temperature of the reaction solution is lowered, allowing the primer to bind to the single-stranded DNA template, forming a DNA-primer complex. Extension is carried out in a temperature range of about 70 to 75°C. A complementary strand complementary to the template strand is synthesized using a heat-stable polymerase, starting from the primer.

[0005] When the complementary strand is synthesized by extension, double-stranded DNA is formed again. The region between the forward primer and reverse primer is replicated, and two double-stranded DNAs are generated, doubling the double-stranded DNA before denaturation. Then, denaturation to denature the double-stranded DNA into single strands and the subsequent steps are repeated again. Generally, the thermal cycle is repeated about 30 to 40 times.

[0006] Thermal cyclers have been developed to automatically perform PCR by running thermal cycles. A typical thermal cycler includes an aluminum block thermally connected to a Peltier element. Reaction vessels, such as microtubes containing reaction solutions, are placed in numerous recesses formed on the aluminum block. When the aluminum block is heated or cooled by the Peltier element, the reaction solutions contained in the reaction vessels are regulated to a temperature range appropriate for each step of the PCR.

[0007] PCR is performed by repeating multiple steps in which the reaction solution is adjusted to different temperature ranges. Therefore, PCR typically takes about 1 to 2 hours. Since the technology was developed, increasing the speed of PCR has been a major challenge. One method for shortening the time required for PCR is to reduce the heat capacity of heat transfer members such as reaction vessels and aluminum blocks. Reducing the heat capacity of reaction vessels and heat transfer members makes it possible to quickly change the temperature of the reaction solution, allowing each step to be started in a short period of time and shortening the time required for PCR. However, there is a limit to how much the heat capacity of reaction vessels and heat transfer members can be reduced.

[0008] Patent Document 1 describes a method for ultrafast amplification of nucleic acids in a channel. The device used in this method has a serpentine channel formed therein. The serpentine channel is provided with a temperature zone for DNA denaturation, a temperature zone for annealing, and a temperature zone for extension. A PCR sample solution is transported through the serpentine channel by a pump.

[0009] Patent Document 2 describes a real-time PCR method and device that allows target nucleic acids to be replaced at any time after real-time PCR has been completed. This device has an annular channel formed in a substrate. One or more reaction regions are provided in the annular channel to apply a temperature cycle to the PCR solution. The PCR solution is introduced into and discharged from the annular channel by a pump.

[0010] Patent Document 3 describes a nucleic acid sequence amplification method and device that can shorten PCR reaction time. This device utilizes sample circulation due to thermal convection. Thermal convection in a reaction vessel causes denaturation, annealing, and polymerization processes to occur sequentially and repeatedly. Examples of reaction vessels include a straight tube with one end sealed, and a structure that forms two high-temperature zones and one low-temperature zone.

[0011] JP 2013-055921 A JP 2009-106222 A JP 2009-100761 A

[0012] In the field of nucleic acid amplification devices that automatically perform PCR, there is a demand for technology that can shorten the time required for PCR. There is a need for technology that can quickly change the temperature of a reaction solution containing nucleic acid as a reactant, regardless of the heat capacity of the reaction vessel that contains the reaction solution or the heat capacity of the heat transfer member that mediates heat transfer between the reaction vessel and a temperature control mechanism.

[0013] In Patent Document 1, the temperature of the reaction solution is controlled within a serpentine channel. However, this method requires a long serpentine channel to perform multiple cycles. A problem with a long serpentine channel is that the device size increases. Furthermore, a long channel increases pressure loss, requiring a high-pressure pump to transport the reaction solution. Furthermore, precise control of the transport is required because the reaction solution must be moved according to each step of PCR. Furthermore, components contained in the reaction solution tend to adhere to the inner wall of the serpentine channel, resulting in a problem of a low recovery rate of the amplified product.

[0014] In Patent Document 2, the temperature of the reaction solution is controlled by providing one or more reaction zones in the annular channel. However, the solution is transported through the annular channel by a pump. When a pump is used for transporting the solution, components contained in the reaction solution tend to adhere to the liquid-contacting parts of the pump, which increases the risk of contamination between the reaction solutions. In addition, the need to move the reaction solution precisely makes it difficult to control the transport of the solution.

[0015] In Patent Document 3, the temperature of the reaction solution is controlled by forming a spatial temperature distribution within the reaction vessel. The sample is circulated within the reaction vessel by thermal convection. However, the reaction vessel is typically a straight tube with one end sealed, or a structure that forms two high-temperature zones and one low-temperature zone. The use of such a reaction vessel poses the problem of difficulty in controlling the temperature and thermal convection.

[0016] Therefore, the present invention aims to provide a nucleic acid amplification device that can transport and control the temperature of a reaction solution containing PCR reactants without using a pump, and that can perform PCR in a short time with a simple device structure.

[0017] In order to solve the above problems, the nucleic acid amplification device of the present invention is a nucleic acid amplification device that performs PCR to amplify nucleic acids, and includes a flow path device having a flow path formed therein, and a temperature control mechanism that can control the temperature of multiple regions on the flow path device, wherein the flow path device has a chamber formed in a closed ring shape, an inlet that can introduce fluid into the chamber, and an outlet that can discharge fluid from the chamber, and the reaction solution containing nucleic acid introduced into the chamber is circulated inside the chamber by controlling the temperature of multiple regions on the chamber to different temperature ranges, and is subjected to a step of configuring PCR for each of the regions, thereby performing PCR inside the chamber.

[0018] According to the present invention, it is possible to supply and control the temperature of a reaction solution containing PCR reactants without using a pump, and it is possible to provide a nucleic acid amplification device that can perform PCR in a short time with a simple device structure.

[0019] FIG. 1 is a front view of a flow channel device constituting a nucleic acid amplification apparatus according to a first embodiment of the present invention. FIG. 2 is a cross-sectional view of a flow channel device constituting a nucleic acid amplification apparatus according to the first embodiment of the present invention. FIG. 3 is a view showing an example of the configuration of a region on a flow channel device that is the target of temperature control. FIG. 4 is a view showing an example of the configuration of a region on a flow channel device that is the target of temperature control. FIG. 5 is a front view of a flow channel device constituting a nucleic acid amplification apparatus according to a second embodiment of the present invention. FIG. 6 is a cross-sectional view of a flow channel device constituting a nucleic acid amplification apparatus according to the second embodiment of the present invention. FIG. 7 is a front view of a flow channel device constituting a nucleic acid amplification apparatus according to a third embodiment of the present invention. FIG. 8 is a cross-sectional view of a flow channel device constituting a nucleic acid amplification apparatus according to the third embodiment of the present invention. FIG. 9 is a front view of a flow channel device constituting a nucleic acid amplification apparatus according to a fourth embodiment of the present invention.

[0020] Hereinafter, a nucleic acid amplification device according to an embodiment of the present invention will be described. Note that the same reference numerals are used to designate common components in the following drawings, and redundant explanations will be omitted.

[0021] [First embodiment] Fig. 1 is a front view of a flow channel device constituting a nucleic acid amplification apparatus according to a first embodiment of the present invention. Fig. 2 is a cross-sectional view of a flow channel device constituting a nucleic acid amplification apparatus according to a first embodiment of the present invention. Fig. 1 schematically shows a flow channel device 101 and a temperature adjustment mechanism 110 constituting a nucleic acid amplification apparatus 100 according to the first embodiment, as well as a syringe pump 150 used to inject a reaction solution into the flow channel device 101, along with their internal structures. Fig. 2 shows a cross-sectional view taken along line A-A in Fig. 1.

[0022] 1 and 2 , the nucleic acid amplification apparatus 100 according to the first embodiment includes a flow path device 101 and a temperature control mechanism 110. The flow path device 101 includes a substrate 102, a film 103, a chamber 104, an inlet 105, an outlet 106, an inlet channel 107, and an outlet channel 108. The film 103 covering the inlet channel 107 and the outlet channel 108 has a sealing portion 109 that is pressed by a valve (not shown) to seal the chamber 104.

[0023] The nucleic acid amplification device 100 is a device that amplifies nucleic acids by performing PCR using thermal cycling, and performs PCR by natural convection of a reaction solution inside a flow path device 101. The reaction solution used is a buffer in which template DNA containing the base sequence to be amplified, a heat-resistant polymerase, a forward primer, a reverse primer, four types of deoxynucleotide triphosphates (dNTPs), and the like are dissolved.

[0024] The flow channel device 101 is a device having a flow channel formed therein through which a fluid can flow, and provides a reaction field in which the fluid introduced therein undergoes a predetermined reaction. PCR, which amplifies nucleic acids, is performed inside the flow channel device 101. A reaction solution containing nucleic acids, which are PCR reactants, is introduced into the flow channel device 101. Inside the flow channel device 101, the temperature of multiple internal regions is controlled by a temperature control mechanism 110, thereby performing each step of PCR.

[0025] The flow channel device 101 is positioned so that the extension direction of the chamber 104 does not coincide with the horizontal direction when PCR is performed. That is, the flow channel device 101 is positioned upright or tilted so that the extension direction of the chamber 104 is parallel to the vertical direction or tilted relative to the vertical direction. The extension direction of the chamber means the direction parallel to the main surface of the chamber. The main surface means the surface among the top, side, and bottom surfaces that has the greatest length and area.

[0026] The substrate 102 is a member that constitutes the main body of the flow channel device 101. Grooves for forming chambers 104 are machined on the main surface of the substrate 102. The chambers 104 are formed by covering the grooves formed on the substrate 102 with a film 103. In FIG. 1 , an annular groove that has a hexagonal ring shape when viewed from the front is formed on the main surface of the substrate 102.

[0027] The substrate 102 can be formed from a resin such as polypropylene (PP), polyethylene (PE), polystyrene (PS), polycarbonate (PC), polymethylpentene (PMP), polydimethylsiloxane (PDMS), acrylic resin, or fluororesin, a metal such as stainless steel, aluminum, or copper, or ceramics or glass.

[0028] The film 103 is a member that constitutes the main body of the flow channel device 101. The film 103 is bonded to the main surface of the substrate 102 by welding or the like so as to cover the grooves formed in the substrate 102. A flexible thin film, sheet, or the like is used as the film 103. When a flexible film 103 is used, the flow channels formed inside the flow channel device 101 can be opened and closed by pressing the film 103 from the outside of the flow channel device 101.

[0029] The film 103 can be formed from polypropylene (PP), polyethylene (PE), polystyrene (PS), polyester (PE), polymethylpentene (PMP), polydimethylsiloxane (PDMS), acrylic resin, fluororesin, silicone rubber, or the like.

[0030] The chamber 104 is a space formed inside the flow path device 101, and forms a closed annular flow path through which a reaction solution injected therein can circulate. In Fig. 1, the chamber 104 is provided in a hexagonal ring shape when viewed from the front of the flow path device 101. An inlet 105 is connected to one end of the chamber 104 relative to the center of the ring via an inlet channel 107. An outlet 106 is connected to the other end of the chamber 104 relative to the center of the ring via an outlet channel 108.

[0031] The inlet 105 forms an inlet through which a reaction solution can be introduced into the chamber 104. The inlet 105 is formed at the outer end of the inlet channel 107. The inlet channel 107 extends from one end of the chamber 104 toward the outside of the annular center of the chamber 104. The inlet channel 107 is processed as a groove in the main surface of the substrate 102. The inlet 105 passes from the end of the inlet channel 107 through the substrate 102 and communicates with the outside of the flow path device 101.

[0032] The outlet 106 forms an outlet through which the reaction solution can be discharged from the inside of the chamber 104. The outlet 106 is formed at the outer end of the outlet channel 108. The outlet channel 108 extends from the other end side of the chamber 104 toward the outside with respect to the ring center of the chamber 104. The outlet channel 108 is processed as a groove in the main surface of the substrate 102. The outlet 106 passes from the end of the outlet channel 108 through the substrate 102 and communicates with the outside of the flow path device 101.

[0033] 1, the reaction liquid is injected into the flow channel device 101 by a syringe pump 150. The reaction liquid can be injected into the flow channel device 101 manually using various pumps or syringes depending on the volume of the reaction liquid, the required pressure during injection, etc. However, in the flow channel device 101, after the reaction liquid is injected, the liquid is transported by natural convection inside the chamber 104 without using a pump.

[0034] The reaction liquid can be injected into the flow channel device 101 from the inlet 105 side. When PCR is performed, the reaction liquid is injected so that it fills the chamber 104. As the reaction liquid is injected, air bubbles inside the chamber 104 can be discharged to the outside from the outlet 106 side or the like. Removing air bubbles from the chamber 104 and filling it with reaction liquid allows the reaction liquid to circulate smoothly inside the chamber 104 without localized stagnation. Furthermore, the temperature of the reaction liquid can be efficiently controlled.

[0035] The chamber 104 is provided with a structure in which it branches into two flow paths when viewed from the side of the inlet channel 107. The chamber 104 has a first flow path 104a that runs from a branching point on the outlet side of the inlet channel 107 to a junction on the inlet side of the outlet channel 108 via one side of the chamber 104, and a second flow path 104b that runs from the branching point on the outlet side of the inlet channel 107 to the opposite side of the chamber 104 to a junction on the inlet side of the outlet channel 108. The reaction solution can be filled into the chamber 104 by branching the flow from the inlet channel 107 into the first flow path 104a and the second flow path 104b and flowing the reaction solution in parallel through the first flow path 104a and the second flow path 104b.

[0036] In the chamber 104, the pressure loss of the first flow path 104a and the pressure loss of the second flow path 104b are preferably equal to each other. The first flow path 104a and the second flow path 104b are preferably equal to each other in length, width, and volume. Furthermore, the flow paths are preferably equal to each other in flow path resistance, and the flow paths are preferably structured such that the inner walls of the flow paths are smooth or are structured such that the flow paths are line-symmetrical with respect to a plane perpendicular to the main surface of the chamber 104.

[0037] If the pressure loss in the first flow path 104a and the pressure loss in the second flow path 104b are equal to each other, when the reaction solution is injected into the chamber 104, the linear flow velocity of the reaction solution in the first flow path 104a and the linear flow velocity of the reaction solution in the second flow path 104b will be equal to each other. This allows the reaction solution flowing through the first flow path 104a and the reaction solution flowing through the second flow path 104b to simultaneously reach a junction on the inlet side of the outlet channel 108 and merge. Therefore, injection of the reaction solution from the inlet 105 toward the outlet 106 can reliably push out air from inside the chamber 104. Since air bubbles are less likely to remain inside, smooth circulation of the reaction solution and efficient temperature control are possible.

[0038] Sealed portion 109 is a portion pressed by a valve for opening and closing the flow path, and is set in an area on film 103 that covers the outlet side of inlet channel 107, or in an area on film 103 that covers the inlet side of outlet channel 108. As the valve, an automatic on-off valve or the like can be used, which has a rod-shaped or the like valve element at the tip and is driven to move forward and backward by an actuator or the like.

[0039] When the valve presses the film 103 at the sealing location 109, the film 103 deforms, closing the inlet channel 107 and the outlet channel 108. In this state, the interior of the chamber 104 becomes a liquid-tight and airtight space, forming a closed annular flow path within the chamber 104 through which fluid can circulate without leakage. On the other hand, when the valve is moved away from the film 103 at the sealing location 109, the film 103 becomes free, opening the inlet channel 107 and the outlet channel 108. In this state, fluid can pass from the inlet 105 to the outlet 106.

[0040] The temperature adjustment mechanism 110 is a mechanism that adjusts temperature by heating or cooling. The temperature adjustment mechanism 110 is installed so as to be in contact with the main surface of the flow path device 101, and is capable of adjusting the temperatures of multiple regions on the flow path device 101. When PCR is performed, the temperature adjustment mechanism 110 adjusts the temperatures of the multiple regions on the flow path device 101 to different temperature ranges corresponding to the steps that make up the PCR.

[0041] For example, a Peltier element type temperature adjustment mechanism can be used as the temperature adjustment mechanism 110. The Peltier element type temperature adjustment mechanism includes a Peltier element, a heat transfer member that is thermally connected to the Peltier element and transfers heat between the Peltier element and the device, and a heat sink that dissipates heat from the Peltier element. The heat sink may be a heat dissipation fan or the like.

[0042] 1 and 2, the temperature adjustment mechanism 110 is installed on the rear side of the flow path device 101, in an area that encompasses the chamber 104 when viewed from the front. The temperature adjustment mechanism 110 is installed in contact with the rear side of the substrate 102 and in a state that it is thermally connected to the substrate 102. However, the temperature adjustment mechanism 110 may be installed on the front side of the flow path device 101 as long as it can adjust the temperature of the reaction liquid inside the chamber 104. The temperature adjustment mechanism 110 is preferably installed in an area that encompasses the chamber 104 when viewed from the front.

[0043] 3 and 4 are diagrams showing configuration examples of regions on a flow channel device that are to be temperature-controlled. Fig. 3 is a front view of the flow channel device 101 constituting the nucleic acid amplification apparatus 100 according to the first embodiment, showing a configuration example in which the temperature of two regions on the flow channel device 101 is controlled by a temperature control mechanism 110. Fig. 4 is a front view of the flow channel device 101 constituting the nucleic acid amplification apparatus 100 according to the first embodiment, showing a configuration example in which the temperature of three regions on the flow channel device 101 is controlled by the temperature control mechanism 110.

[0044] 3 and 4 , the temperature of a plurality of regions on the flow channel device 101 adjacent to the chamber 104 can be adjusted to different temperature ranges corresponding to the steps constituting PCR by the temperature adjustment mechanism 110. The temperatures of the plurality of regions on the flow channel device 101 may be adjusted by a single temperature adjustment mechanism 110 or by a plurality of temperature adjustment mechanisms 110.

[0045] 3, in a front view of the flow channel device 101, a region including the linearly extending section of the first flow channel 104a is designated as a denaturation region 201 whose temperature is adjusted to a denaturation temperature range for denaturation by the temperature adjustment mechanism 110. Also, a region including the linearly extending section of the second flow channel 104b is designated as an annealing region 202 whose temperature is adjusted to an annealing temperature range for annealing by the temperature adjustment mechanism 110.

[0046] 4, in a front view of the flow channel device 101, a region including the linearly extending section of the first flow channel 104a is designated as a denaturation region 201 whose temperature is adjusted to a denaturation temperature range for denaturation by the temperature adjustment mechanism 110. A region including the linearly extending section of the second flow channel 104b is designated as an annealing region 202 whose temperature is adjusted to an annealing temperature range for annealing by the temperature adjustment mechanism 110. A region including a curved section connecting the second flow channel 104b to the first flow channel 104a is designated as an extension region 203 whose temperature is adjusted to an extension temperature range for extension by the temperature adjustment mechanism 110.

[0047] The denaturation region 201 is a region where double-stranded DNA is denatured and dissociated into single strands. The temperature of the denaturation region 201 is controlled, for example, to 90°C or higher and 96°C or lower. The annealing region 202 is a region where primers are bound to single-stranded DNA as a template. The temperature of the annealing region 202 is controlled, for example, to 55°C or higher and 65°C or lower. The extension region 203 is a region where a complementary strand complementary to the template strand is synthesized using a thermostable polymerase starting from the primer. The temperature of the extension region 203 is controlled, for example, to 70°C or higher and 75°C or lower.

[0048] 3, in the curved section connecting the second flow path 104b to the first flow path 104a, elongation is performed by utilizing the decrease in temperature of the reaction solution due to natural cooling or thermal convection, without using the temperature adjustment mechanism 110. Since the temperature adjustment mechanism 110 corresponding to the elongation region 203 is not required, the degree of freedom in device layout can be improved. On the other hand, in FIG. 4, the elongation region 203 is formed using the temperature adjustment mechanism 110. Using the temperature adjustment mechanism 110 allows the section between the annealing region 202 and the denaturation region 201 to be temperature-adjusted to a temperature distribution more suitable for elongation.

[0049] In the nucleic acid amplification device 100, a reaction solution containing nucleic acids introduced into the chamber 104 is circulated inside the chamber 104 by adjusting the temperature of multiple regions on the chamber 104 to different temperature ranges, and PCR is performed inside the chamber 104 by performing a PCR step for each of the regions adjusted to different temperature ranges. In the nucleic acid amplification device 100, one section of the chamber 104 extending along the vertical direction is adjusted to a higher temperature than the other section extending along the vertical direction. PCR using the nucleic acid amplification device 100 can be performed by the following procedure.

[0050] First, the flow channel device 101 is positioned so that the direction in which the chambers 104 extend does not coincide with the horizontal direction. That is, the flow channel device 101 is positioned upright or tilted so that the direction in which the chambers 104 extend is parallel to the vertical direction, or so that the direction in which the chambers 104 extend is tilted relative to the vertical direction. When the direction in which the chambers 104 extend is tilted, it is preferable that the angle between the direction in which the chambers 104 extend and the vertical direction is a small angle of 45 degrees or less.

[0051] Next, a reaction solution containing nucleic acids, which are the PCR reactants, is injected into the chamber 104 to fill the chamber 104. The reaction solution can be injected through the inlet 105 using a syringe pump 150 or the like. Any air bubbles remaining inside the chamber 104 are pushed out toward the outlet channel 108 by the injection of the reaction solution and are then discharged to the outside through the outlet 106.

[0052] Next, with the reaction solution filled inside chamber 104, the inlet and outlet sides of chamber 104 are sealed. A valve is used to press film 103 at sealing point 109 on the inlet 105 side to close inlet channel 107, and a valve is used to press film 103 at sealing point 109 on the outlet 106 side to close outlet channel 108. When the inlet and outlet sides of chamber 104 are sealed, a closed annular flow path is formed inside chamber 104 through which fluid can circulate without leakage.

[0053] Next, the temperatures of the multiple regions on the flow channel device 101 are adjusted to temperature ranges corresponding to the steps constituting PCR. By operating the temperature adjustment mechanism 110, at least a denaturation region 201 and an annealing region 202 are formed, as shown in Fig. 3. Alternatively, a denaturation region 201, an annealing region 202, and an extension region 203 are formed, as shown in Fig. 4. In the configurations of Figs. 3 and 4, one section of the annular flow channel extending along the vertical direction is adjusted to a higher temperature than the other section of the annular flow channel extending along the vertical direction.

[0054] When the flow channel device 101 is positioned so that the direction in which the chamber 104 extends does not coincide with the horizontal direction, and when the temperature of multiple regions on the chamber 104 is controlled to different temperature ranges, the reaction solution convects inside the chamber 104. When the denaturation region 201, which is controlled to a relatively high temperature, is formed on the lower side in the vertical direction, and the annealing region 202, which is controlled to a relatively low temperature, is formed on the upper side in the vertical direction, the reaction solution inside the chamber 104 can be naturally convected in a unidirectional circular manner, as shown by the arrows in Figures 3 and 4 .

[0055] The reaction solution circulating inside the chamber 104 is subjected to each step of PCR in each region on the chamber 104. In the denaturation region 201, which is regulated to a denaturation temperature range, double-stranded DNA is denatured into single strands. In the annealing region 202, which is regulated to an annealing temperature range, a primer binds to the single-stranded DNA template. In the extension region 203, which is regulated to an extension temperature range, and in the region between the annealing region 202 and the denaturation region 201, a complementary strand complementary to the template strand is synthesized starting from the primer.

[0056] One PCR cycle is performed when the reaction solution circulates around the chamber 104. As the circulation of the reaction solution continues, each step constituting the PCR is automatically performed in each region of the chamber 104, allowing the PCR cycle to be automatically repeated. The user of the nucleic acid amplification device 100 can set the time for circulating the reaction solution based on the time required for the reaction solution to circulate around the chamber 104, etc., so that the desired number of cycles is performed.

[0057] According to the nucleic acid amplification device of the first embodiment, the reaction solution containing the PCR reactants, i.e., nucleic acids, can be circulated by natural convection in the annular channel formed inside the channel device. Therefore, the reaction solution containing the PCR reactants can be transported and temperature-controlled without using a pump. Once the reaction solution is temperature-controlled to a temperature range corresponding to each step of the PCR in a predetermined region on the channel device, it is transported by natural convection to the region where the next step is performed. Therefore, heat transfer is less affected by the thermal capacity of the reaction vessel or the thermal capacity of the heat transfer member that mediates heat transfer between the reaction vessel and the temperature control mechanism. Therefore, PCR can be performed in a short time with a simple device structure. Furthermore, a microreactor with a fine channel can be used as the channel device. The annular channel can be provided with a simple structure or a short channel. Therefore, PCR can be performed with high accuracy with a simple device structure and a compact device.

[0058] The technology of Patent Document 1 suffers from problems such as a large device size, the need for a high-pressure pump to deliver the reaction solution, the need for precise control of the delivery, and a low recovery rate of the amplified product. In contrast, the nucleic acid amplification device and nucleic acid amplification method according to the present embodiment can utilize a fine channel or a small annular channel, thereby enabling a smaller device size. Furthermore, since the reaction solution circulates by natural convection, a pump is not required for delivery. Because an annular channel with a short channel length can be used, various pumps can be used to inject the reaction solution. Furthermore, since the reaction solution circulates by natural convection, precise control of the delivery is not required. Furthermore, since an annular channel with a simple shape can be used, residual reaction solution in the annular channel and adhesion of components contained in the reaction solution to the inner walls of the annular channel are reduced, thereby suppressing contamination between reaction solutions and a decrease in the recovery rate of the amplified product.

[0059] Furthermore, the technology of Patent Document 2 has problems such as increased likelihood of contamination between reaction solutions and difficulty in controlling the liquid delivery. In contrast, with the nucleic acid amplification device and nucleic acid amplification method according to the present embodiment, the reaction solution circulates by natural convection, eliminating the need to use a pump to deliver the reaction solution. Contamination between reaction solutions can be avoided because components contained in the reaction solution do not adhere to the liquid-contacting parts of the pump. Furthermore, because the reaction solution circulates by natural convection, precise control of the liquid delivery is not required.

[0060] Furthermore, the technology of Patent Document 3 has the problem that it is difficult to control the temperature of the reaction solution and control thermal convection. In contrast, the nucleic acid amplification device and nucleic acid amplification method according to the present embodiment use a channel device with an annular channel formed therein, which allows the use of a microreactor or the like with a fine channel, thereby enabling efficient and stable natural convection of the reaction solution. The regions corresponding to each step of PCR can be formed as more clearly separated regions, preventing mixing of reaction solutions undergoing different steps.

[0061] [Second Embodiment] Fig. 5 is a front view of a flow channel device constituting a nucleic acid amplification apparatus according to a second embodiment of the present invention. Fig. 6 is a cross-sectional view of a flow channel device constituting a nucleic acid amplification apparatus according to the second embodiment of the present invention. Fig. 5 schematically shows the flow channel device 101A and temperature adjustment mechanism 110 constituting a nucleic acid amplification apparatus 100A according to the second embodiment, as well as a syringe pump 150 used to inject a reaction solution into the flow channel device 101A, along with their internal structures. Fig. 6 shows a cross-sectional view taken along line B-B in Fig. 5.

[0062] 5 and 6 , a nucleic acid amplification apparatus 100A according to the second embodiment includes a flow path device 101A and a temperature control mechanism 110. The flow path device 101A includes a substrate 102, a film 103, a chamber 104A, an inlet 105, an outlet 106, an inlet channel 107, and an outlet channel 108. The film 103 covering the inlet channel 107 and the outlet channel 108 includes a sealing portion 109 that is pressed by a valve (not shown) to seal the chamber 104A.

[0063] The nucleic acid amplification device 100A is an apparatus that amplifies nucleic acids by performing PCR using thermal cycling, similar to the nucleic acid amplification device 100 described above, and performs PCR by natural convection of a reaction solution inside a flow path device 101 A. The nucleic acid amplification device 100A according to the second embodiment differs from the nucleic acid amplification device 100 described above in that a chamber 104A is formed as a non-circular space, and the interior of the chamber 104A is partitioned by a partition plate 120.

[0064] Like the above-described flow channel device 101, the flow channel device 101A is a device having a flow channel formed therein through which a fluid can flow, and provides a reaction field in which a fluid introduced therein undergoes a predetermined reaction. PCR, which amplifies nucleic acids, is performed inside the flow channel device 101A. A reaction solution containing nucleic acids, which are PCR reactants, is introduced into the flow channel device 101A. Inside the flow channel device 101A, the temperature of multiple internal regions is controlled by a temperature control mechanism 110, thereby performing each step of PCR.

[0065] The flow channel device 101A is positioned so that the extension direction of the chambers 104A does not coincide with the horizontal direction during PCR. That is, the flow channel device 101A is positioned upright or tilted so that the extension direction of the chambers 104A is parallel to the vertical direction or tilted relative to the vertical direction. When the extension direction of the chambers 104A is tilted, it is preferable to tilt the extension direction of the chambers 104A so that the angle between the extension direction of the chambers 104A and the vertical direction is a small angle of 45 degrees or less.

[0066] The flow channel device 101 is configured so that the reaction solution circulates in a direction parallel to the direction in which the chamber 104 extends. However, from the viewpoint of device layout, usability, etc., a structure in which the reaction solution circulates in a direction perpendicular to the direction in which the chamber 104 extends may be desirable. In such cases, a flow channel device 101A such as that shown in Figures 5 and 6 can be used.

[0067] The chamber 104A is formed as a non-closed annular space inside the flow channel device 101A. The interior of the chamber 104A is partitioned by a partition plate 120, thereby forming a closed annular flow channel through which a reaction solution injected into the interior can circulate. In FIG. 5 , the chamber 104A is provided in a hexagonal shape that is long in one axial direction when viewed from the front of the flow channel device 101A. An inlet 105 is connected to one end of the chamber 104A in the longitudinal direction via an inlet channel 107. An outlet 106 is connected to the other end of the chamber 104A in the longitudinal direction via an outlet channel 108.

[0068] The inlet 105 forms an inlet through which a reaction solution can be introduced into the chamber 104. The inlet 105 is formed at the outer end of the inlet channel 107. The inlet channel 107 extends from one end of the chamber 104 toward the outside of the chamber 104. The inlet channel 107 is processed as a groove in the main surface of the substrate 102. The inlet 105 passes from the end of the inlet channel 107 through the substrate 102 and communicates with the outside of the flow path device 101A.

[0069] The outlet 106 forms an outlet through which the reaction solution can be discharged from the inside of the chamber 104A. The outlet 106 is formed at the outer end of the outlet channel 108. The outlet channel 108 extends outward from the other end of the chamber 104A. The outlet channel 108 is processed as a groove in the main surface of the substrate 102. The outlet 106 passes from the end of the outlet channel 108 through the substrate 102 and communicates with the outside of the flow path device 101A.

[0070] 5, the reaction liquid is injected into the flow path device 101A by a syringe pump 150. The reaction liquid can be injected into the flow path device 101A manually using various pumps or syringes depending on the volume of the reaction liquid, the required pressure during injection, etc. However, in the flow path device 101A, after the reaction liquid is injected, the liquid is transported by natural convection inside the chamber 104A without using a pump.

[0071] The reaction liquid can be injected into the flow channel device 101A from the inlet 105 side. When PCR is performed, the reaction liquid is injected so that it fills the chamber 104A. As the reaction liquid is injected, air bubbles inside the chamber 104A can be discharged to the outside from the outlet 106 side or the like. Removing the air bubbles and filling the chamber 104A with reaction liquid allows the reaction liquid to circulate smoothly inside the chamber 104A without localized stagnation. Furthermore, the temperature of the reaction liquid can be efficiently controlled.

[0072] The partition plate 120 is formed in a plate shape and is fixed inside the chamber 104A in a direction parallel to the main surface of the chamber 104A. The partition plate 120 separates the interior of the chamber 104A into a first flow path 104c that runs from the outlet side of the inlet channel 107 to the inlet side of the outlet channel 108 via the front side of the chamber 104A, and a second flow path 104d that runs from the outlet side of the inlet channel 107 to the inlet side of the outlet channel 108 via the back side of the chamber 104A. The reaction solution can be filled inside the chamber 104A by dividing the flow from the inlet channel 107 into the first flow path 104c and the second flow path 104d and flowing the reaction solution in parallel through the first flow path 104c and the second flow path 104d.

[0073] In the chamber 104A, the pressure loss of the first flow path 104c and the pressure loss of the second flow path 104d are preferably equal to each other. The first flow path 104c and the second flow path 104d are preferably equal to each other in length, width, and volume. Furthermore, the flow paths are preferably equal to each other in flow path resistance, and the flow paths are preferably structured such that the inner walls of the flow paths are smooth or the flow paths are structured such that they are line-symmetrical with respect to a plane parallel to the main surface of the chamber 104A.

[0074] If the pressure loss in the first flow path 104c and the pressure loss in the second flow path 104d are equal to each other, when the reaction solution is injected into the chamber 104A, the linear flow velocity of the reaction solution in the first flow path 104c and the linear flow velocity of the reaction solution in the second flow path 104d will be equal to each other. This allows the reaction solution flowing through the first flow path 104c and the reaction solution flowing through the second flow path 104d to simultaneously reach the inlet side of the outlet channel 108 and merge. Therefore, injection of the reaction solution from the inlet 105 toward the outlet 106 can reliably expel air from inside the chamber 104A. Since air bubbles are less likely to remain inside, smooth circulation of the reaction solution and efficient temperature control are possible.

[0075] 6, a plurality of temperature adjustment mechanisms 110 are thermally connected to the flow path device 101A. The temperature adjustment mechanisms 110 are installed so as to be in contact with the main surface of the flow path device 101A and are capable of adjusting the temperatures of a plurality of regions on the flow path device 101A. When PCR is performed, the temperature adjustment mechanisms 110 adjust the temperatures of the plurality of regions on the flow path device 101A to different temperature ranges corresponding to the steps constituting the PCR.

[0076] 6, the temperature adjustment mechanisms 110 include a first temperature adjustment mechanism 110a installed on the front side of the flow path device 101A and a second temperature adjustment mechanism 110b installed on the back side of the flow path device 101A. The first temperature adjustment mechanism 110a is installed so as to contact an area close to the first flow path 104c on the main surface on the front side of the flow path device 101A. The second temperature adjustment mechanism 110b is installed so as to contact an area close to the second flow path 104d on the main surface on the back side of the flow path device 101A.

[0077] The first temperature adjustment mechanism 110a adjusts the temperature of the flow path device 101A from the front side, mainly adjusting the temperature of the linearly extending section of the first flow path 104c. In FIG. 6, the first temperature adjustment mechanism 110a adjusts the temperature of the linearly extending section of the first flow path 104c to a relatively high temperature. The temperature of the first flow path 104c is adjusted to, for example, a denaturation temperature range. By adjusting the temperature to a relatively high temperature, the reaction solution flowing through the first flow path 104c has a relatively low density. As a result, an upward flow is formed that rises from the bottom to the top of the first flow path 104c.

[0078] On the other hand, the second temperature adjustment mechanism 110b adjusts the temperature of the flow channel device 101A from the rear side, mainly adjusting the temperature of the linearly extending section of the second flow channel 104d. In FIG. 6, the second temperature adjustment mechanism 110b adjusts the temperature of the linearly extending section of the second flow channel 104d to a relatively low temperature. The temperature of the second flow channel 104d is adjusted to, for example, an annealing temperature range. The reaction solution flowing through the second flow channel 104d has a relatively high density due to the temperature being adjusted to a relatively low temperature. As a result, a downward flow is formed that flows downward from above to below through the second flow channel 104d.

[0079] In the nucleic acid amplifier 100A, a reaction solution containing nucleic acids introduced into the chamber 104A is circulated through the chamber 104A by adjusting the temperature of multiple regions on the chamber 104A to different temperature ranges, and PCR is performed within the chamber 104A by performing steps to configure PCR for each of the regions adjusted to different temperature ranges. In the nucleic acid amplifier 100A, one section of the chamber 104A extending along the vertical direction is adjusted to a higher temperature than the other section extending along the vertical direction. PCR using the nucleic acid amplifier 100A can be performed using the same procedure as the nucleic acid amplifier 100 described above.

[0080] When the flow channel device 101A is arranged so that the direction in which the chamber 104A extends does not coincide with the horizontal direction, and when the temperature of multiple regions on the chamber 104A is controlled to different temperature ranges, the reaction solution convects inside the chamber 104A. When one section extending along the vertical direction of the chamber 104A separated by the partition plate 120 is controlled to a higher temperature than the other section extending along the vertical direction, the reaction solution inside the chamber 104A can be naturally convected in a unidirectional circular manner, as shown by the arrows in FIG. 6 .

[0081] The reaction solution circulating inside chamber 104A is subjected to each step of PCR in each region above chamber 104A. In the denaturation region, which is regulated to a denaturation temperature range, double-stranded DNA is denatured into single strands. In the annealing region, which is regulated to an annealing temperature range, primers bind to the single-stranded DNA template. In the extension region, which is regulated to an extension temperature range, and in the region between the annealing region and the denaturation region, a complementary strand complementary to the template strand is synthesized starting from the primer.

[0082] When the reaction solution circulates around chamber 104A, one PCR cycle is performed. As the circulation of the reaction solution continues, each step constituting the PCR is automatically performed in each region of chamber 104A, allowing the PCR cycle to be automatically repeated. The user of nucleic acid amplification device 100A can set the time for circulating the reaction solution based on the time required for the reaction solution to circulate around chamber 104, etc., so that the desired number of cycles is performed.

[0083] According to the nucleic acid amplification device of the second embodiment described above, similar to the nucleic acid amplification device of the first embodiment, PCR can be performed in a short time with a simple device structure. Furthermore, a microreactor or the like with a thin and wide channel can be used as the channel device. The annular channel can be provided with a simple structure or a short channel. Therefore, PCR can be performed with high accuracy with a simple device structure or a compact device. Because a thin and wide annular channel can be used, a large heat transfer area can be easily ensured between the temperature adjustment mechanism 110 and the reaction solution.

[0084] [Third Embodiment] Fig. 7 is a front view of a flow channel device constituting a nucleic acid amplification apparatus according to a third embodiment of the present invention. Fig. 8 is a cross-sectional view of a flow channel device constituting a nucleic acid amplification apparatus according to the third embodiment of the present invention. Fig. 7 schematically shows a flow channel device 101B and a temperature adjustment mechanism 110 constituting a nucleic acid amplification apparatus 100B according to the third embodiment, as well as a syringe pump 150 used to inject a reaction solution into the flow channel device 101B, along with their internal structures. Fig. 8 shows a cross-sectional view taken along line CC in Fig. 7.

[0085] 7 and 8, a nucleic acid amplification apparatus 100B according to the third embodiment includes a flow path device 101B and a temperature control mechanism 110. The flow path device 101B includes a substrate 102, a film 103, a chamber 104B, an inlet 105, an outlet 106, an inlet channel 107, and an outlet channel 108. The film 103 covering the inlet channel 107 and the outlet channel 108 includes a sealing portion 109 that is pressed by a valve (not shown) to seal the chamber 104B.

[0086] Similar to the nucleic acid amplification device 100 described above, the nucleic acid amplification device 100B is an apparatus that amplifies nucleic acids by performing PCR using thermal cycling, and performs PCR by natural convection of the reaction solution inside the flow-path device 101B. The nucleic acid amplification device 100B according to the third embodiment differs from the nucleic acid amplification device 100 described above in that the nucleic acid amplification device 100B is provided with a structure suitable for injecting the reaction solution into the device from the horizontal direction, and the inlet and outlet sides of the closed-ring chamber 104B are provided with different flow-path volumes.

[0087] Like the above-described flow channel device 101, the flow channel device 101B is a device having a flow channel formed therein through which a fluid can flow, and provides a reaction field in which a fluid introduced therein undergoes a predetermined reaction. PCR, which amplifies nucleic acids, is performed inside the flow channel device 101B. A reaction solution containing nucleic acids, which are PCR reactants, is introduced into the flow channel device 101B. Inside the flow channel device 101B, the temperature of multiple internal regions is controlled by a temperature control mechanism 110, thereby executing each step of PCR.

[0088] The flow channel device 101B is positioned so that the extension direction of the chambers 104B does not coincide with the horizontal direction during PCR. That is, the flow channel device 101B is positioned upright or tilted so that the extension direction of the chambers 104B is parallel to the vertical direction or tilted relative to the vertical direction. When the extension direction of the chambers 104B is tilted, it is preferable to tilt the extension direction of the chambers 104B so that the angle between the extension direction of the chambers 104B and the vertical direction is a small angle of 45 degrees or less.

[0089] To utilize natural convection, the flow channel device 101 must be positioned so that the direction in which the chamber 104 extends does not coincide with the horizontal direction. Meanwhile, the flow channel device 101 is configured so that the reaction liquid is injected into the interior from the lower or upper side when the flow channel device 101 is upright. However, from the viewpoint of device layout, usability, and the like, it may be desirable to inject or discharge the reaction liquid into or from the side of the flow channel device 101 when the flow channel device 101 is upright. In such cases, a flow channel device 101B such as that shown in FIGS. 7 and 8 can be used.

[0090] The chamber 104B is formed as a closed annular space inside the flow channel device 101B. The chamber 104B forms a closed annular flow channel through which the reaction solution injected therein can circulate. In FIG. 7 , the chamber 104B is provided in a rectangular ring shape with curved corners when viewed from the front of the flow channel device 101B. An inlet 105 is connected to one end of the chamber 104B relative to the center of the ring via an inlet channel 107. An outlet 106 is connected to the other end of the chamber 104B relative to the center of the ring via an outlet channel 108.

[0091] The inlet 105 forms an inlet through which a reaction solution can be introduced into the chamber 104B. The inlet 105 is formed at the outer end of the inlet channel 107. In FIG. 7, the inlet channel 107 extends from one end of the chamber 104B toward the outside of the chamber 104B in the horizontal direction, then bends upward on the outside and extends above the chamber 104B. The inlet channel 107 is processed as a groove in the main surface of the substrate 102. The inlet 105 passes from the end of the inlet channel 107 through the substrate 102 and communicates with the outside of the flow path device 101B.

[0092] The outlet 106 forms an outlet through which the reaction solution can be discharged from the inside of the chamber 104B. The outlet 106 is formed at the outer end of the outlet channel 108. In FIG. 7, the outlet channel 108 extends from the other end of the chamber 104B toward the outside of the chamber 104B in the horizontal direction, bends upward on the outside, and extends above the chamber 104B. The outlet channel 108 is processed as a groove in the main surface of the substrate 102. The outlet 106 extends from the end of the outlet channel 108 through the substrate 102 and communicates with the outside of the flow path device 101B.

[0093] 7, the reaction liquid is injected into the flow path device 101B by a syringe pump 150. The reaction liquid can be injected into the flow path device 101B manually using various pumps or syringes depending on the volume of the reaction liquid, the required pressure during injection, etc. However, in the flow path device 101B, after the reaction liquid is injected, the liquid is transported by natural convection inside the chamber 104B without using a pump.

[0094] The reaction liquid can be injected into the flow channel device 101B from the side of the inlet 105, which is arranged horizontally outward from the chamber 104B. When PCR is performed, the reaction liquid is injected so as to fill the interior of the chamber 104B. As the reaction liquid is injected, air bubbles inside the chamber 104B can be discharged to the outside from the side of the outlet 106, which is arranged horizontally outward from the chamber 104B. Removing the air bubbles and filling the chamber with the reaction liquid allows the reaction liquid to circulate smoothly inside the chamber 104B without localized stagnation. Furthermore, the temperature of the reaction liquid can be efficiently controlled.

[0095] Chamber 104B is provided with a structure in which it branches into two flow paths when viewed from the side of inlet channel 107. Chamber 104B has a first flow path 104e that runs from a branching point on the outlet side of inlet channel 107 to a junction on the inlet side of outlet channel 108 via the lower side of chamber 104B, and a second flow path 104f that runs from a branching point on the outlet side of inlet channel 107 to a junction on the inlet side of outlet channel 108 via the upper side of chamber 104B. The reaction solution can be filled into chamber 104B by branching from inlet channel 107 to first flow path 104e and second flow path 104f and flowing the reaction solution in parallel through first flow path 104e and second flow path 104f.

[0096] In the chamber 104B, the pressure loss of the first flow path 104e and the pressure loss of the second flow path 104f are preferably equal to each other. The first flow path 104e and the second flow path 104f are preferably equal to each other in length, width, and volume. Furthermore, the flow paths are preferably equal to each other in flow path resistance, and the flow paths are preferably structured such that the inner walls of the flow paths are smooth or the flow paths are structured such that they are line-symmetrical with respect to a plane perpendicular to the main surface of the chamber 104B.

[0097] If the pressure loss in the first flow path 104e and the pressure loss in the second flow path 104f are equal to each other, when the reaction solution is injected into the chamber 104B, the linear flow velocity of the reaction solution in the first flow path 104e and the linear flow velocity of the reaction solution in the second flow path 104f become equal to each other. This allows the reaction solution flowing through the first flow path 104e and the reaction solution flowing through the second flow path 104f to simultaneously reach the inlet side of the outlet channel 108 and merge. Therefore, injection of the reaction solution from the inlet 105 toward the outlet 106 can reliably expel air from inside the chamber 104B. Since air bubbles are less likely to remain inside, smooth circulation of the reaction solution and efficient temperature control become possible.

[0098] The chamber 104B is formed in a rectangular ring shape with curved corners when viewed from the front of the flow path device 101B, and has a lower section arranged parallel to the horizontal direction on the lower side of the flow path device 101B, an upper section arranged parallel to the horizontal direction on the upper side of the flow path device 101B, an inlet side section arranged parallel to the vertical direction on the inlet side of the flow path device 101B, and an outlet side section arranged parallel to the vertical direction on the outlet side of the flow path device 101B.

[0099] Chamber 104B is preferably provided so that the pressure loss in the inlet section and the pressure loss in the outlet section are different from each other. The inlet section and the outlet section are preferably provided with different flow path lengths, different flow path widths, and different flow path volumes. They may also be provided with different flow path resistances.

[0100] When the pressure loss in the inlet section and the pressure loss in the outlet section are different, the reaction liquid easily flows from the section with the larger pressure loss to the section with the smaller pressure loss. It becomes easier to flow a reaction liquid that has been adjusted to a relatively high temperature into the section with the larger pressure loss. When a high-temperature reaction liquid flows into the section with the larger pressure loss, an upward flow of the reaction liquid, which is high-temperature and has a relatively small density, is easily formed. This makes it possible to efficiently form a circulating flow by natural convection.

[0101] 8, a temperature adjustment mechanism 110 can be thermally connected to the flow path device 101B. The temperature adjustment mechanism 110 is installed so as to be in contact with the main surface of the flow path device 101B and is capable of adjusting the temperatures of multiple regions on the flow path device 101B. When PCR is performed, the temperature adjustment mechanism 110 adjusts the temperatures of the multiple regions on the flow path device 101B to different temperature ranges corresponding to the steps constituting the PCR.

[0102] 8, the temperature adjustment mechanisms 110 include a first temperature adjustment mechanism 110c installed below the flow path device 101B and a second temperature adjustment mechanism 110d installed above the flow path device 101B. The first temperature adjustment mechanism 110c is installed so as to contact an area of ​​the main surface of the flow path device 101B that is close to the first flow path 104e. The second temperature adjustment mechanism 110d is installed so as to contact an area of ​​the main surface of the flow path device 101B that is close to the second flow path 104f.

[0103] The first temperature adjustment mechanism 110c mainly adjusts the temperature of the lower section, which is located at the lower side of the flow path device 101B and is arranged parallel to the horizontal direction. In FIG. 8, the first temperature adjustment mechanism 110c adjusts the temperature of the lower section to a relatively high temperature. The temperature of the first flow path 104e is adjusted to, for example, a denaturation temperature range. The reaction solution flowing through the lower section has a relatively low density due to the temperature being adjusted to a relatively high temperature. As a result, an upward flow is formed, rising from the lower section to the upper section.

[0104] On the other hand, the second temperature adjustment mechanism 110d mainly adjusts the temperature of the upper section, which is located on the upper side of the flow path device 101B and is arranged parallel to the horizontal direction. In FIG. 8, the second temperature adjustment mechanism 110d adjusts the temperature of the upper section to a relatively low temperature. The temperature of the second flow path 104f is adjusted to, for example, an annealing temperature range. The reaction solution flowing through the upper section has a relatively high density due to its temperature being adjusted to a relatively low temperature. As a result, a downward flow is formed, which descends from the upper section to the lower section.

[0105] In the nucleic acid amplifier 100B, a reaction solution containing nucleic acids introduced into the chamber 104B is circulated through the chamber 104B by adjusting the temperature of multiple regions of the chamber 104B to different temperature ranges, and PCR is performed within the chamber 104B by performing steps to configure PCR for each of the regions adjusted to different temperature ranges. In the nucleic acid amplifier 100B, the temperature of the section located on the lower side of the chamber 104B in the vertical direction is adjusted to a higher temperature than the section located on the upper side of the chamber 104B. PCR using the nucleic acid amplifier 100B can be performed using the same procedure as the nucleic acid amplifier 100 described above.

[0106] When the flow path device 101B is arranged so that the direction in which the chamber 104B extends does not coincide with the horizontal direction, and when the temperature of a plurality of regions on the chamber 104B is adjusted to different temperature ranges, the reaction solution convects inside the chamber 104B. In a structure in which the pressure loss in the inlet section arranged parallel to the vertical direction on the inlet side of the flow path device 101B and the pressure loss in the outlet section arranged parallel to the vertical direction on the outlet side of the flow path device 101B are different from each other, when the section located on the lower side in the vertical direction of the chamber 104B is adjusted to a higher temperature than the section located on the upper side in the vertical direction, the reaction solution inside the chamber 10BA can be naturally convected in a unidirectional circular manner, as shown by the arrows in FIG.

[0107] The reaction solution circulating inside chamber 104B is subjected to each step of PCR in each region above chamber 104B. In the denaturation region, which is regulated to a denaturation temperature range, double-stranded DNA is denatured into single strands. In the annealing region, which is regulated to an annealing temperature range, primers bind to the single-stranded DNA template. In the extension region, which is regulated to an extension temperature range, and in the region between the annealing region and the denaturation region, a complementary strand complementary to the template strand is synthesized starting from the primer.

[0108] When the reaction solution circulates around chamber 104B, one PCR cycle is performed. As the circulation of the reaction solution continues, each step constituting the PCR is automatically performed in each region of chamber 104B, allowing the PCR cycle to be automatically repeated. The user of nucleic acid amplification device 100B can set the time for circulating the reaction solution based on the time required for the reaction solution to circulate around chamber 104, etc., so that the desired number of cycles is performed.

[0109] The nucleic acid amplification device according to the third embodiment, as with the nucleic acid amplification device according to the first embodiment, can perform PCR in a short time with a simple device structure. Furthermore, a microreactor or the like having a thin, wide channel can be used as the flow path device. The annular channel can be provided in a simple structure or a short channel. Therefore, PCR can be performed with high accuracy using a simple device structure and a compact device. Because the reaction solution can be injected and discharged from the inside of the flow path device on the side when the flow path device is upright, after the flow path device is installed in an upright or tilted position, the reaction solution can be easily injected into and discharged from the fixed flow path device.

[0110] 9 is a front view of a flow channel device constituting a nucleic acid amplification apparatus according to a fourth embodiment of the present invention, and schematically shows a flow channel device 101C constituting a nucleic acid amplification apparatus 100C according to the fourth embodiment, together with its internal structure.

[0111] 9 , a nucleic acid amplification apparatus 100C according to the fourth embodiment includes a flow path device 101C and a temperature control mechanism 110. The flow path device 101C includes a substrate 102, a film 103, a chamber 104, an inlet 105, an outlet 106, an inlet channel 107, and an outlet channel 108. The film 103 covering the inlet 105 and the outlet 106 includes a sealing portion 109 that is pressed by a valve (not shown) to seal the chamber 104.

[0112] The nucleic acid amplifier 100C is an apparatus that amplifies nucleic acids by performing PCR using thermal cycling, similar to the nucleic acid amplifier 100 described above, and performs PCR by natural convection of the reaction solution inside the flow path device 101C. The nucleic acid amplifier 100C according to the fourth embodiment differs from the nucleic acid amplifier 100 described above in that a plurality of chambers 104 are formed on the substrate 102, and a common temperature control mechanism 110 can be used for the plurality of chambers 104.

[0113] Like the above-described flow channel device 101, the flow channel device 101C is a device having a flow channel formed therein through which a fluid can flow, and provides a reaction field in which a fluid introduced therein undergoes a predetermined reaction. Inside the flow channel device 101C, PCR is performed in each chamber 104 to amplify nucleic acids. A reaction solution containing nucleic acids, which are PCR reactants, is introduced into each chamber 104 inside the flow channel device 101C. Inside the flow channel device 101C, each step constituting PCR is performed by adjusting the temperature of multiple internal regions using a temperature adjustment mechanism 110.

[0114] The flow channel device 101C is arranged so that the extension direction of the multiple chambers 104 does not coincide with the horizontal direction during PCR. In the flow channel device 101C, the multiple chambers 104 extend in the same direction. The flow channel device 101C can be arranged upright or tilted so that the extension direction of the multiple chambers 104 is parallel to the vertical direction, or so that the extension direction of the multiple chambers 104 is tilted relative to the vertical direction. When the extension direction of the chambers 104 is tilted, it is preferable to tilt the extension direction of the chambers 104 so that the angle between the extension direction of the chambers 104 and the vertical direction is a small angle of 45 degrees or less.

[0115] Each of the plurality of chambers 104 is provided in a structure in which it branches into two flow paths when viewed from the side of the inlet channel 107. The plurality of chambers 104 has a first flow path 104a that extends from a branching point on the outlet side of the inlet channel 107 to a joining point on the inlet side of the outlet channel 108 via one side of the chamber 104 located outside the flow path device 101C, and a second flow path 104b that extends from the branching point on the outlet side of the inlet channel 107 to a joining point on the inlet side of the outlet channel 108 via the opposite side of the chamber 104 located inside the flow path device 101C.

[0116] 9, a temperature adjustment mechanism 110 can be thermally connected to the flow path device 101C. The temperature adjustment mechanism 110 is installed so as to be in contact with the main surface of the flow path device 101C and is capable of adjusting the temperatures of multiple regions on the flow path device 101C. When PCR is performed, the temperature adjustment mechanism 110 adjusts the temperatures of the multiple regions on the flow path device 101C to different temperature ranges corresponding to the steps constituting the PCR.

[0117] 9 , the temperature adjustment mechanism 110 is installed on the rear side of the flow channel device 101C at a position where it can adjust the temperature of an area that includes multiple chambers 104 when viewed from the front. The temperature adjustment mechanism 110 is installed in contact with the rear side of the substrate 102 and is thermally connected to the substrate 102. However, the temperatures of the multiple chambers 104 on the flow channel device 101C may be adjusted by a single temperature adjustment mechanism 110, or may be adjusted by multiple temperature adjustment mechanisms 110. The temperature adjustment mechanism 110 may be installed on the front side of the flow channel device 101C as long as it can adjust the temperatures of the chambers 104. The temperature adjustment mechanism 110 is preferably arranged to include the chambers 104 when viewed from the front.

[0118] 9, for each of the plurality of chambers 104, a region including the linearly extending section of the first flow path 104a is regulated to a relatively high temperature and serves as a denaturation region 201, which is regulated to a temperature range for denaturation by the temperature regulation mechanism 110. Also, a region including the linearly extending section of the second flow path 104b is regulated to a relatively low temperature and serves as an annealing region 202, which is regulated to a temperature range for annealing by the temperature regulation mechanism 110.

[0119] 9, in the curved section connecting the second flow path 104b to the first flow path 104a, extension is performed by utilizing the decrease in temperature of the reaction solution due to natural cooling or thermal convection, without using the temperature adjustment mechanism 110. However, this section may be formed with an extension region 203 whose temperature is adjusted to a temperature range suitable for extension by the temperature adjustment mechanism 110. Using the temperature adjustment mechanism 110 allows the section between the annealing region 202 and the denaturation region 201 to be adjusted to a temperature distribution more suitable for extension.

[0120] 9, among the multiple chambers 104 formed on the substrate 102, sections forming adjacent second flow paths 104b are regulated to the same annealing temperature range. The temperature of the annealing regions 202 of each of the multiple chambers 104 is regulated by a common temperature regulation mechanism 110. However, sections forming first flow paths 104a may be arranged to be adjacent to each other. Sections forming adjacent first flow paths 104a may be regulated to the same denaturation temperature range by the common temperature regulation mechanism 110.

[0121] 9, two chambers 104 are formed on the substrate 102. However, any number of chambers 104 greater than or equal to two may be formed on one substrate 102. Of the multiple chambers 104 formed on the substrate 102, any number of the chambers 104 may be provided in a structure in which the temperature is controlled by a common temperature control mechanism 110.

[0122] According to the nucleic acid amplification device of the fourth embodiment, similar to the nucleic acid amplification device of the first embodiment, PCR can be performed in a short time with a simple device structure. Furthermore, a microreactor with a fine channel or a microreactor with a thin and wide channel can be used as the channel device. The annular channel can be provided in a simple structure or a short channel. Therefore, PCR can be performed with high accuracy with a simple device structure or a compact device. Since multiple chambers are formed on the substrate, the PCR throughput can be increased. Furthermore, since a common temperature control mechanism can be used, the flexibility of the device layout is improved and the device cost can be reduced.

[0123] Although the embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the present invention. For example, the present invention is not necessarily limited to those having all of the configurations of the above-described embodiments. It is possible to replace part of the configuration of an embodiment with another configuration, add part of the configuration of an embodiment to another form, or omit part of the configuration of an embodiment.

[0124] For example, a plurality of chambers having the structure according to the first embodiment may be provided on a substrate, a plurality of chambers having the structure according to the second embodiment may be provided on a substrate, or a plurality of chambers having the structure according to the third embodiment may be provided on a substrate. The chambers, inlet channels, and outlet channels inside the flow channel device may be provided in any shape.

[0125] Furthermore, in the nucleic acid amplification device, the sealed portion is provided as a portion pressed by a valve for opening and closing the flow path, and the valve is configured to use an automatic on-off valve with a rod-shaped or similar valve element at the tip, but the inlet and outlet sides of the chambers of the flow path device may be opened and closed by valves with other mechanisms. For example, a dial-type valve may be built into the inlet and outlet sides of the chambers of the flow path device. The dial-type valve can be opened and closed by rotating it with a motor or the like.

[0126] 100 Nucleic acid amplification device 101 Flow channel device 102 Substrate 103 Film 104 Chamber 105 Inlet 106 Outlet 107 Inlet channel 108 Outlet channel 109 Sealing point 110 Temperature control mechanism 120 Partition plate 150 Syringe pump 201 Denaturation region 202 Annealing region 203 Elongation region

Claims

1. A nucleic acid amplification device that performs PCR to amplify nucleic acids, comprising: a flow path device having a flow path formed therein; and a temperature control mechanism capable of controlling the temperature of multiple regions on the flow path device, wherein the flow path device has a chamber formed in a closed ring shape, an inlet through which a fluid can be introduced into the chamber, and an outlet through which the fluid can be discharged from the chamber, and the nucleic acid amplification device performs PCR inside the chamber by controlling the temperature of multiple regions on the chamber to different temperature ranges, causing a reaction solution containing nucleic acid introduced into the chamber to circulate inside the chamber, and subjecting the reaction solution to a step of configuring PCR for each of the regions.

2. A nucleic acid amplification device as described in claim 1, wherein the flow path device is arranged so that the direction in which the chamber extends is parallel to the vertical direction, or so that the direction in which the chamber extends is inclined relative to the vertical direction.

3. A nucleic acid amplification device according to claim 2, wherein one section of the chamber extending along the vertical direction is controlled to a temperature higher than that of the other section extending along the vertical direction.

4. A nucleic acid amplification device according to claim 2, wherein the temperature of the section of the chamber located on the vertically lower side is controlled to a higher temperature than the section located on the vertically upper side.

5. A nucleic acid amplification device according to claim 1, wherein the chamber is configured so that the pressure loss in a first flow path that runs from the inlet through one side of the chamber to the outlet and the pressure loss in a second flow path that runs from the inlet through the opposite side of the chamber to the outlet are equal to each other.

6. A nucleic acid amplification device according to claim 1, wherein the flow path device is formed by a substrate on which an annular groove forming the chamber is formed, and a flexible film covering the annular groove.

7. A nucleic acid amplification device according to claim 6, comprising an inlet valve that can freely open and close the inlet of the flow path device, and an outlet valve that can freely open and close the outlet of the flow path device, wherein the inlet valve and the outlet valve open and close the inlet and the outlet by pressing the film to close the inlet and the outlet with the film, respectively, and by moving away from the film to open the inlet and the outlet.

8. A nucleic acid amplification apparatus according to claim 7, wherein the inside of the flow path device is sealed liquid-tight and airtight when the inlet and outlet are closed, forming a circular flow path through which the reaction liquid circulates by natural convection.

9. A nucleic acid amplification device as described in claim 1, wherein the regions whose temperature is controlled by the temperature control mechanism are a denaturation region in which double-stranded nucleic acid is denatured and an annealing region in which a primer is annealed to single-stranded nucleic acid, and the nucleic acid to which the primer has annealed is elongated between the annealing region and the denaturation region.

10. A nucleic acid amplification device as described in claim 1, wherein the regions whose temperatures are controlled by the temperature control mechanism are a denaturation region in which double-stranded nucleic acid is denatured, an annealing region in which a primer is annealed to a single-stranded nucleic acid, and an extension region in which the nucleic acid to which the primer has annealed is extended.

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