Nucleic acid amplification method, nucleic acid amplification device and chip for nucleic acid amplification

Abstract

To provide a nucleic acid amplification method using a novel liquid feeding control mechanism.SOLUTION: A nucleic acid amplification method uses a nucleic acid amplification device comprising a chip for nucleic acid amplification and a substrate on which the chip for nucleic acid amplification can be mounted. The method comprises: a step 1 of mounting the chip for nucleic acid amplification on the substrate of the nucleic acid amplification device, the nucleic acid amplification device comprising first and second heaters, a mechanism for liquid feeding, first and second four-way valves, first and second two-way valves, a controller, first and second coupling parts, first to ninth channels, and the chip for nucleic acid amplification comprising first and second curve channels, an intermediate channel, a coupling part, and first and second coupling channels; a step 2 of coupling the coupling part on the chip for nucleic acid amplification with the first and second coupling parts on the nucleic acid amplification device; and a step 3 of causing sample liquid to reciprocate by the mechanism for liquid feeding through the first and second curve channels and the intermediate channel, thereby performing thermal cycling.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a nucleic acid amplification method, a nucleic acid amplification device, and a chip for nucleic acid amplification. [Background technology]

[0002] Detection of nucleic acids is a core technology in various fields, including medicine (pharmaceutical research and development, clinical testing, forensic medicine, etc.), agriculture (agricultural crops, identification of types of pathogenic microorganisms, etc.), etc. PCR, which selectively amplifies a specific region of DNA, is a very useful technique for nucleic acid detection.

[0003] Conventional PCR and real-time PCR thermal cyclers have a problem in that PCR reactions take a long time due to their huge heat capacity. To address this problem, a method has been developed to speed up the thermal cycle by repeatedly sending liquid through multiple temperature zones using a flow path chip (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6226284 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a nucleic acid amplification method using a new liquid feed control mechanism. [Means for solving the problem]

[0006] The inventors have considered using a switching valve instead of a pump to control the sample delivery in the channel chip. However, if the switching valve is opened and closed while the pump is running, the air has no place to escape in the channel directly connected to the pump, and the pressure in the channel becomes excessive, which causes damage and deterioration of the pump. Therefore, the present inventors have found that in a nucleic acid amplification device having one liquid delivery mechanism, by using a two-way valve together with a four-way valve, it is possible to avoid an increase in pressure within the flow channel.

[0007] Furthermore, based on this new finding, the inventors have appropriately modified the arrangement of the flow paths connecting the liquid delivery mechanism and the two types of valves, thereby completing the present invention.

[0008] Accordingly, one aspect of the present invention provides the following: Item 1. A method for amplifying nucleic acid using a nucleic acid amplifier (1) including a chip for nucleic acid amplification (2) and a substrate (3) on which the chip for nucleic acid amplification (2) can be mounted, the method comprising the steps of: Step 1: A step of placing a nucleic acid amplification chip (2) on a substrate (3) of a nucleic acid amplification device (1), The nucleic acid amplification device (1), A first heater (4) capable of forming a denaturation temperature zone; A second heater (5) capable of forming an elongation / annealing temperature zone; a liquid transfer mechanism (6) that enables the transfer of a sample liquid (30) between the denaturation temperature zone and the extension / annealing temperature zone; A first four-way valve (7) and a second four-way valve (8), A first two-way valve (9) and a second two-way valve (10), a control device (11) for controlling switching of the first four-way valve, the second four-way valve, the first two-way valve, and the second two-way valve; A first connection part (12) connectable to the nucleic acid amplification chip (2); A second connection part (13) connectable to the nucleic acid amplification chip (2); a first flow path (14) connecting the liquid delivery mechanism to the first four-way valve and the second four-way valve; a second flow path (15) having one end connected to the first four-way valve (7) and the other end connected to the first connection portion (12); a third flow path (16) having one end connected to the second four-way valve (8) and the other end connected to the second connection portion (13); a fourth flow passage (17) having one end connected to the first four-way valve (7) and the other end connected to the first two-way valve (9); a fifth flow path (18) having one end connected to the second four-way valve (8) and the other end connected to the second two-way valve (10); a sixth flow path (19) having one end connected to the first two-way valve (9) and the other end open to the outside of the flow path; a seventh flow path (20) having one end connected to the second two-way valve (10) and the other end open to the outside of the flow path; an eighth flow path (21) having one end connected to the first four-way valve (7) and the other end open to the outside of the flow path; and a ninth flow path (22) having one end connected to the second four-way valve (8) and the other end open to the outside of the flow path; The reciprocal flow type nucleic acid amplification device (1) is characterized in that it performs real-time PCR by measuring the fluorescence intensity for each thermal cycle, a step in which the nucleic acid amplification chip (2) comprises a first curved flow path (23) and a second curved flow path (24) corresponding to the denaturation temperature zone and the extension / annealing temperature zone, respectively, an intermediate flow path (25) connecting the first curved flow path (23) and the second curved flow path (24), two connection parts (26, 27) connectable to the nucleic acid amplification device, a first connection flow path (28) connecting the first curved flow path (23) and the connection part (26), and a second connection flow path (29) connecting the second curved flow path (24) and the connection part (27); Step 2: connecting the connection parts (26, 27) of the nucleic acid amplification chip (2) to the first connection part (12) and the second connection part (13) of the nucleic acid amplification device; Step 3: performing thermal cycling by causing the sample liquid to travel back and forth between the first curved flow path (23) and the second curved flow path (24) via the intermediate flow path (25) using the liquid delivery mechanism; and However, in the case where the sample liquid (30) is moved from the first heater (4) side to the second heater (5) side in step 3, the first four-way valve (7) is controlled to communicate between the first flow path (14) and the second flow path (15) and between the fourth flow path (17) and the eighth flow path (21), the second four-way valve (8) is controlled to communicate between the first flow path (14) and the fifth flow path (18) and between the third flow path (16) and the ninth flow path (22), the second two-way valve (10) is kept in a closed state, and the first two-way valve (9) is kept in a closed or open state, In step 3, when the sample liquid (30) is moved from the second heater (5) side to the first heater (4) side, the second four-way valve (8) is controlled to communicate the first flow path (14) with the third flow path (16) and the fifth flow path (18) with the ninth flow path (22), the first four-way valve (7) is controlled to communicate the first flow path (14) with the fourth flow path (17) and the second flow path (15) with the eighth flow path (21), the first two-way valve (9) is closed, and the second two-way valve (10) is closed or open, When the movement of the sample liquid (30) is stopped in step 3, the first four-way valve (7) is controlled to communicate between the first flow path (14) and the fourth flow path (17) and between the second flow path (15) and the eighth flow path (21), the second four-way valve (8) is controlled to communicate between the first flow path (14) and the fifth flow path (18) and between the third flow path (16) and the ninth flow path (22), and the first two-way valve and / or the second two-way valve are kept open.

[0009] Item 2. The method according to item 1, wherein the nucleic acid amplification device (1) includes the intermediate flow path (25). a fluorescence detector capable of measuring the fluorescence intensity of the sample liquid (30) passing through the The method further comprises step 4: measuring the fluorescence intensity of the sample liquid (30) for each thermal cycle by the fluorescence detector at a predetermined position in the intermediate flow path (25).

[0010] Item 3. The method according to Item 2, wherein in step 3, an electrical signal related to the movement of the sample liquid (30) is sent from the fluorescence detector to the control device, and the control device detects the passage of the sample liquid (30) through the intermediate flow path based on the electrical signal, and controls switching of the first four-way valve, the second four-way valve, the first two-way valve, and the second two-way valve.

[0011] Item 4. A substrate (3) on which the nucleic acid amplification chip (2) can be mounted; A first heater (4) capable of forming a denaturation temperature zone; A second heater (5) capable of forming an elongation / annealing temperature zone; a liquid transfer mechanism (6) that enables the transfer of a sample liquid (30) between the denaturation temperature zone and the extension / annealing temperature zone; A first four-way valve (7) and a second four-way valve (8), A first two-way valve (9) and a second two-way valve (10), a control device (11) for controlling switching of the first four-way valve, the second four-way valve, the first two-way valve, and the second two-way valve; A first connection part (12) connectable to the nucleic acid amplification chip (2); A second connection part (13) connectable to the nucleic acid amplification chip (2); a first flow path (14) connecting the liquid delivery mechanism (6) to the first four-way valve and the second four-way valve; a second flow path (15) having one end connected to the first four-way valve (7) and the other end connected to the first connection part (12); a third flow path (16) having one end connected to the second four-way valve (8) and the other end connected to the second connection portion (13); a fourth flow path (17) having one end connected to the first four-way valve (7) and the other end connected to the first two-way valve (9); a fifth flow path (18) having one end connected to the second four-way valve (8) and the other end connected to the second two-way valve (10); a sixth flow path (19) having one end connected to the first two-way valve (9) and the other end open to the outside of the flow path; a seventh flow path (20) having one end connected to the second two-way valve (10) and the other end open to the outside of the flow path; an eighth flow path (21) having one end connected to the first four-way valve (7) and the other end open to the outside of the flow path; and a ninth flow path (22) having one end connected to the second four-way valve (8) and the other end open to the outside of the flow path; The reciprocal flow type nucleic acid amplifier (1) is characterized in that it performs real-time PCR by measuring the fluorescence intensity for each thermal cycle.

[0012] The present inventors have also discovered that in a nucleic acid amplification device having one liquid delivery mechanism, a three-way valve can be used to adjust the sample speed, etc., of the pump, thereby preventing an increase in pressure within the flow channel.

[0013] Furthermore, based on this new finding, the inventors have completed one of the present inventions by setting the arrangement of the flow path connecting the liquid delivery mechanism and the valve, the sample speed by the pump, and the conditions for stopping the pump. Accordingly, one aspect of the present invention provides the following: Item 5. A method for amplifying nucleic acid using a nucleic acid amplifier (1') including a chip for nucleic acid amplification (2') and a substrate (3') on which the chip for nucleic acid amplification (2') can be mounted, the method comprising the steps of: Step 1: A step of placing a nucleic acid amplification chip (2') on a substrate (3') of a nucleic acid amplification device (1'), The nucleic acid amplification device (1'), A first heater (4') capable of forming a denaturation temperature zone; A second heater (5') capable of forming an elongation / annealing temperature zone; a liquid transfer mechanism (6') that enables the transfer of a sample liquid (30') between the denaturation temperature zone and the extension / annealing temperature zone; A first three-way valve (7') and a second three-way valve (8'), a control device (11') for controlling the operation of the first three-way valve (7'), the second three-way valve (8'), and the liquid delivery mechanism (6'); A first connection part (12') connectable to the nucleic acid amplification chip (2'); A second connection part (13') connectable to the nucleic acid amplification chip (2'); a first flow path (14') connecting the liquid delivery mechanism to a first three-way valve (7') and a second three-way valve (8'); a second flow path (15') having one end connected to the first three-way valve (7') and the other end connected to the first connection part (12'); a third flow path (16') having one end connected to the second three-way valve (8') and the other end connected to the second connection part (13'); a fourth flow path (21"), one end of which is connected to the first three-way valve (7') and the other end of which is open to the outside of the flow path; and a fifth flow path (22"), one end of which is connected to the second three-way valve (8') and the other end of which is open to the outside of the flow path; A reciprocal flow type nucleic acid amplifier (1') characterized in that it performs real-time PCR by measuring the fluorescence intensity for each thermal cycle, a step in which the nucleic acid amplification chip (2') comprises a first curved channel (23') and a second curved channel (24') corresponding to the denaturation temperature zone and the extension / annealing temperature zone, respectively, an intermediate channel (25') connecting the first curved channel (23') and the second curved channel (24'), two connection parts (26', 27') connectable to the nucleic acid amplification device, a first connection channel (28') connecting the first curved channel (23') and the connection parts, and a second connection channel (29') connecting the second curved channel (24') and the connection part; Step 2: connecting the connection parts (26', 27') in the nucleic acid amplification chip (2') to the first connection part (12') and the second connection part (13') in the nucleic acid amplification device (1'); step 3: performing thermal cycling by using the liquid delivery mechanism to move the sample liquid (30') back and forth between the first curved flow path (23') and the second curved flow path (24') via the intermediate flow path (25'); and, in step 3, when the sample liquid (30') is moved from the first heater (4') side to the second heater (5') side, controlling the first three-way valve (7') so that the first flow path (14') and the second flow path (15') are communicated and the fourth flow path (21") is not communicated with the first flow path (14') and the second flow path (15') and controlling the second three-way valve (8') so that the third flow path (16) and the fifth flow path (22") are communicated and the first flow path (14') is not communicated with the third flow path (16') and the fifth flow path (22"); and controlling the liquid delivery mechanism (6') so that the speed at which the sample liquid passes through the intermediate flow path (25') is 100 mm / s or less; In step 3, when the sample liquid (30') is moved from the second heater (5') side to the first heater (4') side, the second three-way valve (8') is controlled so that the first flow path (14') and the third flow path (16') are communicated with each other and the fifth flow path (22") is not communicated with the first flow path (14') and the third flow path (16') and the first three-way valve (7') is controlled so that the second flow path (15') and the fourth flow path (21") are communicated with each other and the first flow path (14') is not communicated with the second flow path (15') and the fourth flow path (21"); and the liquid sending mechanism (6') is controlled so that the speed at which the sample liquid (30') passes through the intermediate flow path (25') is 100 mm / s or less. When the movement of the sample liquid (30') is stopped in step 3, the first three-way valve (7') is controlled so that the second flow path (15') and the fourth flow path (21") are connected and the first flow path (14') is not connected to the second flow path (15') and the fourth flow path (21"), the second three-way valve (8') is controlled so that the third flow path (16') and the fifth flow path (22") are connected and the first flow path (14') is not connected to the third flow path (16') and the fifth flow path (22"), and the liquid delivery mechanism (6') is stopped within two seconds after both the first three-way valve (7') and the second three-way valve (8') are controlled to the above-mentioned states.

[0014] Item 6. The method according to item 5, wherein the nucleic acid amplifier (1') comprises a fluorescence detector capable of measuring a fluorescence intensity of the sample liquid (30') passing through the intermediate flow path (25'), The method includes a step of measuring the fluorescence intensity of the sample liquid for each thermal cycle by the fluorescence detector at a predetermined position in the intermediate flow path (25').

[0015] Item 7. The method according to Item 6, wherein in step 3, an electrical signal related to the movement of the sample liquid (30') is sent from the fluorescence detector to the control device (11'), and the control device detects the passage of the sample liquid (30') through the intermediate flow path (25') based on the electrical signal, and controls the operation of the first three-way valve (7'), the second three-way valve (8'), and the liquid delivery mechanism (6').

[0016] Item 8. The method according to any one of Items 5 to 7, wherein the first flow path (14') has an internal cross section larger than internal cross sections of the first curved flow path (23'), the second curved flow path (24') and the intermediate flow path (25'). [Brief description of the drawings]

[0017] [Figure 1] 1 shows an embodiment of a nucleic acid amplification device (1) and a nucleic acid amplification chip (2) according to the present invention. [Diagram 2] An outline of a four-way valve is shown. [Diagram 3] 1 shows a schematic of a four-way valve in another embodiment. [Figure 4] 4 shows an outline of the flow of the sample liquid and air in step 3. [Diagram 5] 4 shows an outline of the flow of the sample liquid and air in step 3. [Figure 6] 4 shows an outline of the flow of the sample liquid and air in step 3. [Figure 7] The outline of a two-way valve is shown. (a) shows a valve with two ports on different faces, and (b) shows a valve with two ports on the same face. [Figure 8] 1 shows an embodiment of a nucleic acid amplification device (1') and a nucleic acid amplification chip (2') according to the present invention. [Figure 9] The outline of a three-way valve is shown. (a) shows a valve with two ports on one side and one port on the opposite side, and (b) shows a valve with three ports on the same side. [Figure 10] 4 shows an outline of the flow of the sample liquid and air in step 3'. [Figure 11] 4 shows an outline of the flow of the sample liquid and air in step 3'. [Figure 12] 4 shows an outline of the flow of the sample liquid and air in step 3'. [Figure 13] 1 shows an embodiment of a channel chip. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below with reference to the accompanying drawings, but the present invention is not limited to the specific embodiments embodied in these drawings.

[0019] [I] Nucleic acid amplification method using a four-way valve FIG. 1 shows an embodiment of a nucleic acid amplification device (1) and a nucleic acid amplification chip (2) used in the nucleic acid amplification of the present invention. As shown in FIG. 1, the nucleic acid amplification device (1) is A substrate (3) on which a nucleic acid amplification chip can be mounted; A first heater (4) capable of forming a denaturation temperature zone; A second heater (5) capable of forming an elongation / annealing temperature zone; a liquid transfer mechanism (6) that enables the transfer of a sample liquid (30) between the denaturation temperature zone and the extension / annealing temperature zone; A first four-way valve (7) and a second four-way valve (8), A first two-way valve (9) and a second two-way valve (10), a control device (11) that controls switching of the first four-way valve (7), the second four-way valve (8), the first two-way valve (9), and the second two-way valve (10); A first connection part (12) connectable to the nucleic acid amplification chip; A second connection part (13) connectable to the nucleic acid amplification chip; a first flow path (14) connecting the liquid delivery mechanism to the first four-way valve and the second four-way valve; a second flow path (15) having one end connected to the first four-way valve and the other end connected to the first connection portion; a third flow path (16) having one end connected to the second four-way valve and the other end connected to the second connection portion; a fourth flow path (17) having one end connected to the first four-way valve and the other end connected to the first two-way valve; a fifth flow path (18) having one end connected to the second four-way valve and the other end connected to the second two-way valve; a sixth flow path (19) having one end connected to the first two-way valve and the other end open to the outside of the flow path, a seventh flow path (20) having one end connected to the second two-way valve and the other end open to the outside of the flow path, an eighth flow path (21) having one end connected to the first four-way valve and the other end open to the outside of the flow path, and and a ninth flow passage (22) having one end connected to the second four-way valve and the other end open to the outside of the flow passage.

[0020] In the present invention, the first heater (4) is not particularly limited as long as it can form a denaturation temperature zone. The second heater (5) is not particularly limited as long as it can form an extension / annealing temperature zone. Specific examples of the first heater (4) and the second heater (5) include a cartridge heater, a film heater, a Peltier heater, and the like. The denaturation temperature zone is maintained at a temperature required for DNA denaturation reaction in PCR. The temperature of the denaturation temperature zone is preferably about 90 to 100°C, more preferably about 93 to 98°C. The extension / annealing temperature zone is maintained at a temperature required for DNA annealing reaction and extension reaction in PCR. The temperature of the extension / annealing temperature zone is preferably about 40 to 75°C, more preferably about 55 to 65°C.

[0021] The liquid delivery mechanism (6) is not particularly limited as long as it can move the sample liquid via the movement of air in the flow channel and the chip flow channel, and examples of the mechanism include a microblower, a fan, and a syringe pump.

[0022] A microblower (also called a piezoelectric microblower) is a known device that draws in and discharges air, and is characterized by not having a sealed structure (having no check valve). In a typical microblower, the diaphragm is bent and deformed by applying a voltage to a piezoelectric element, thereby achieving the drawing in and discharge of air. As a microblower, for example, those manufactured by Murata Manufacturing Co., Ltd. can be used (MZB1001T02, MZB3004T04, MZB3005T06, MZB4001T05).

[0023] A fan is a device that blows air by the rotational motion of an impeller. Due to the structural characteristics of the impeller, the flow path is not a closed system.

[0024] In the present invention, a four-way valve means a valve that connects two sets of two flow paths (four flow paths in total) both before and after valve switching. For example, as shown in Fig. 2, when a four-way valve connects flow paths A, B, C, and D, the four-way valve can be controlled to switch from a state in which flow paths A and B, and flow paths C and D are connected, to a state in which flow paths A and D, and flow paths B and C are connected, respectively. In the present invention, the four-way valve is not particularly limited as long as it connects two sets of two flow paths both before and after valve switching, and for example, a valve that connects flow paths by a mechanism as shown in Fig. 3 is also included in the "four-way valve".

[0025] Preferably, it is a four-way solenoid valve having four or five ports.

[0026] In the present invention, a two-way valve refers to a valve that connects two flow paths and can be controlled to open or close. For example, a two-way valve as shown in FIG. 7 can be mentioned.

[0027] Preferably, it is a two-way solenoid valve, and more preferably, it is a two-way solenoid valve such as LVM09R3 (manufactured by SMC Corporation) that is normally closed and opens when energized.

[0028] The control device (11) may be a CPU or the like for controlling switching of the first four-way valve (7), the second four-way valve (8), the first two-way valve (9), and the second two-way valve (10). The control device (11) may further control the liquid delivery mechanism (6).

[0029] In this embodiment, the first flow path (14), the second flow path (15), the third flow path (16), the fourth flow path (17), the fifth flow path (18), the sixth flow path (19), the seventh flow path (20), the eighth flow path (21), and the ninth flow path (22) are tubular members connecting the various members in the nucleic acid amplifier (1). The materials constituting these flow paths are not particularly limited, but examples thereof include silicon and various plastics. The cross-sectional shape of each of these flow paths is not particularly limited, and may be a semicircular shape, a circular shape, a rectangular shape, a trapezoid shape, a wedge shape, a polygonal shape, or the like. When the cross section of each flow path is approximately circular, the diameter is not particularly limited, but may be designed to be, for example, in the range of 1 to 6 mm, preferably 2 to 5 mm.

[0030] The nucleic acid amplification chip (2) includes a first curved channel (23) and a second curved channel (24) corresponding to the denaturation temperature zone and the extension / annealing temperature zone, respectively, an intermediate channel (25) connecting the first curved channel and the second curved channel, two connection parts (26, 27) connectable to the nucleic acid amplification device (1), a first connection channel (28) connecting the first curved channel and the connection parts, and a second connection channel (29) connecting the second curved channel and the connection parts. In this specification, the first curved channel (23), the second curved channel (24), the intermediate channel (25), the first connection channel (28), and the second connection channel (29) in the nucleic acid amplification chip (2) are collectively referred to simply as chip channels.

[0031] The material constituting these chip channels is not particularly limited, and examples thereof include various thermosetting or photosetting resins such as glass, quartz, silicon, and cycloolefin polymer (COP). The shape of the cross section of these chip channels is not particularly limited, and may be semicircular, circular, rectangular, trapezoidal, wedge, polygonal, or the like. The cross section of the chip channel may be, for example, a rectangular or trapezoidal shape with a width of about 100 to 1000 μm and a depth of about 100 to 1000 μm. Preferably, the cross section is a rectangular or trapezoidal shape with a width of about 100 to 1000 μm and a depth of about 100 to 1000 μm. The width and depth of the chip channel may be constant, or the width or depth may vary partially. Therefore, in this specification, unless otherwise specified, the "width" of each chip channel refers to the average width of the chip channel. Similarly, in this specification, unless otherwise specified, the "depth" of each chip channel refers to the depth of the chip channel. The average value of the thickness is shown.

[0032] The length of each of the first curved flow path (23) corresponding to the denaturation temperature range and the second curved flow path (24) corresponding to the extension / annealing temperature range is preferably 20 mm or more.

[0033] Next, a step is performed to connect the connection parts (26, 27) of the nucleic acid amplification chip to the first connection part (12) and the second connection part (13) of the nucleic acid amplification device (1). This step enables the air flow from the nucleic acid amplification device (1) to be sent to the nucleic acid amplification chip. Note that the connection between the nucleic acid amplification chip (2) and the nucleic acid amplification device (1) may be performed simultaneously with the placement of the nucleic acid amplification chip (2) on the substrate (3) of the nucleic acid amplification device (1).

[0034] Then, a step of thermal cycling is performed by moving the sample liquid (30) back and forth between the first curved flow path (23) and the second curved flow path (24) by the liquid delivery mechanism (6). As shown in FIG. 4, in step 3, when the sample liquid (30) is moved from the first heater (4) side to the second heater (5) side, the first four-way valve (7) is controlled to communicate the first flow path (14) with the second flow path (15) and the fourth flow path (17) with the eighth flow path (21). In this case, the first two-way valve (9) may be opened or closed. On the other hand, the second four-way valve (8) is controlled to communicate the first flow path (14) with the fifth flow path (18) and the third flow path (16) with the ninth flow path (22), and the second two-way valve (10) is closed. Air sent out from the liquid delivery mechanism (6) flows toward the first four-way valve (7) and the second four-way valve (8). On the side of the second four-way valve (8), the second two-way valve (10) is closed and the sample liquid (30) cannot escape beyond that. On the other hand, on the side of the first four-way valve (7), the end is open to the outside of the flow path via the second flow path (15), the first curved flow path (23), the intermediate flow path (25), and the second curved flow path (24) in the nucleic acid amplification chip (2), the third flow path (16), the second four-way valve (8), and the ninth flow path (22), so that the sample liquid (30) moves to the second heater (5) side due to the air pressure from the liquid delivery mechanism (6).

[0035] As shown in Fig. 5, when the movement of the sample liquid (30) is stopped in step 3, the first four-way valve (7) is controlled to communicate the first flow path (14) with the fourth flow path (17) and the second flow path (15) with the eighth flow path (21), and the second four-way valve (8) is controlled to communicate the first flow path (14) with the fifth flow path (18) and the third flow path (16) with the ninth flow path (22). In this case, at least one of the first two-way valve (9) and the second two-way valve (10) is opened. As a result, the residual pressure in the chip flow path in the nucleic acid amplification chip (2) and the second flow path (15) and the third flow path (16) in the nucleic acid amplification device (1) is released through the eighth flow path (21) and the ninth flow path (22), so that the undesired movement of the sample liquid (30) due to the residual pressure in the flow paths can be suppressed. Furthermore, since the air sent out from the liquid delivery mechanism (6) is discharged from the first flow path (14) to the outside of the flow path via the fourth flow path (17) and the sixth flow path (19), or also via the fifth flow path (18) and the seventh flow path (20), the pressure in the first flow path (14) does not become excessive, and damage and deterioration of the liquid delivery mechanism (6) can be avoided. Among the embodiments of the present invention, the embodiment using a four-way valve is preferable in that liquid delivery can be controlled only by controlling the valve and not by controlling the liquid delivery mechanism (6).

[0036] As shown in Fig. 6, in step 3, when the sample liquid (30) is moved from the second heater (5) side to the first heater (4) side, the second four-way valve (8) is controlled to communicate the first flow path (14) with the third flow path (16) and the fifth flow path (18) with the ninth flow path (22). In this case, the second two-way valve (10) may be opened or closed. On the other hand, the first four-way valve (7) is controlled to communicate the first flow path (14) with the fourth flow path (17) and the second flow path (15) with the eighth flow path (21), and the first two-way valve (9) is closed. Air sent from the liquid sending mechanism (6) flows toward the first four-way valve (7) and the second four-way valve (8). On the side of the first four-way valve (7), the first two-way valve (9) is closed and the sample liquid (30) cannot escape beyond that. On the other hand, on the side of the second four-way valve (8), the end is open to the outside of the flow path via the third flow path (16), the second curved flow path (24) in the nucleic acid amplification chip (2), the intermediate flow path (25) and the first curved flow path (23), the second flow path (15), the first four-way valve (7), and the eighth flow path (21), so that the sample liquid (30) moves to the first heater (4) side due to the air pressure from the liquid delivery mechanism (6).

[0037] Although typical embodiments of the present invention have been described above with reference to the drawings, the present invention is not limited to the above embodiments. For example, the chip flow channel of the nucleic acid amplification chip (2) may have filter regions (31, 32), a sample injection port (33), a reverse transcription reaction region, etc.

[0038] In the present invention, the nucleic acid amplifier (1) may further include a fluorescence detector (not shown) capable of measuring the fluorescence intensity of the sample liquid (30) passing through the intermediate flow path.

[0039] Furthermore, in a preferred embodiment of the present invention, in step 3, an electrical signal related to the movement of the sample liquid is sent from the fluorescence detector to the control device (11), and the control device detects the passage of the sample liquid (30) through the intermediate flow path (25) based on the electrical signal, and controls the switching of the first four-way valve (7), the second four-way valve (8), the first two-way valve (9), and the second two-way valve (10). Furthermore, the fluorescence detector for detecting the passage of the sample liquid (30) through the intermediate flow path (25) may be provided separately from the fluorescence detector for measuring the nucleic acid amplification of the sample liquid (30).

[0040] [II] Nucleic acid amplification method using a three-way valve FIG. 8 shows an embodiment of a nucleic acid amplification device (1') and a nucleic acid amplification chip (2') used in the nucleic acid amplification of the present invention. As shown in FIG. 8, the nucleic acid amplification device (1') includes: A substrate (3') on which a nucleic acid amplification chip can be mounted; A first heater (4') capable of forming a denaturation temperature zone; A second heater (5') capable of forming an elongation / annealing temperature zone; a liquid transfer mechanism (6') that enables the transfer of a sample liquid (30') between the denaturation temperature zone and the extension / annealing temperature zone; A first three-way valve (7') and a second four-way valve (8'), a control device (11') that controls the operation of the first three-way valve (7'), the second three-way valve (8'), and the liquid delivery mechanism (6'); A first connection part (12') connectable to the nucleic acid amplification chip (2'); A second connection part (13') connectable to the nucleic acid amplification chip (2'); a first flow path (14') connecting the liquid delivery mechanism (6') to a first three-way valve (7') and a second three-way valve (8'); a second flow path (15') having one end connected to the first three-way valve (7') and the other end connected to the first connection part (12'); a third flow path (16') having one end connected to the second three-way valve (8') and the other end connected to the second connection part (13'); a fourth flow path (21"), one end of which is connected to the first three-way valve (7') and the other end of which is open to the outside of the flow path; and and a fifth flow path (22'') having one end connected to the second three-way valve (8') and the other end open to the outside of the flow path.

[0041] In the present invention, the first heater (4') is not particularly limited as long as it can form a denaturation temperature zone. The second heater (5') is not particularly limited as long as it can form an extension / annealing temperature zone. Specific examples of the first heater (4') and the second heater (5') include a cartridge heater, a film heater, a Peltier heater, etc. The denaturation temperature zone is maintained at a temperature required for the DNA denaturation reaction in PCR. The temperature in the extension / annealing temperature zone is preferably about 90 to 100°C, more preferably about 93 to 98°C. The extension / annealing temperature zone is maintained at a temperature required for the annealing reaction and extension reaction of DNA in PCR. The temperature in the extension / annealing temperature zone is preferably about 40 to 75°C, more preferably about 55 to 65°C.

[0042] The liquid delivery mechanism (6') is not particularly limited as long as it can move the sample liquid via the movement of air in the flow channel and the chip flow channel, and examples of the mechanism include a microblower, a fan, and a syringe pump.

[0043] A microblower (also called a piezoelectric microblower) is a known device that draws in and discharges air, and is characterized by not having a sealed structure (having no check valve). In a typical microblower, the diaphragm is bent and deformed by applying a voltage to a piezoelectric element, thereby achieving the drawing in and discharge of air. As a microblower, for example, those manufactured by Murata Manufacturing Co., Ltd. can be used (MZB1001T02, MZB3004T04, MZB3005T06, MZB4001T05).

[0044] A fan is a device that blows air by the rotational motion of an impeller. Due to the structural characteristics of the impeller, the flow path is not a closed system.

[0045] In the present invention, a three-way valve means, for example, as shown in Fig. 8, when a three-way valve connects flow paths A, B, and C, the three-way valve can be controlled to switch from a state in which flow paths A and B are connected to a state in which flow paths A and C are connected. In addition, for example, a valve that connects one flow path so that opening the other flow path closes the other flow path, as shown in Fig. 9, is also included in the "three-way valve".

[0046] A three-way solenoid valve is preferable, and an example of the valve is LVM095R (manufactured by SMC Corporation). The control device (11') may include a CPU for controlling the operation of the first three-way valve (7'), the second three-way valve (8'), and the liquid delivery mechanism (6'), and the like.

[0047] In this embodiment, the first flow path (14'), the second flow path (15'), the third flow path (16'), the fourth flow path (21"), and the fifth flow path (22") are tubular members that connect the various components in the nucleic acid amplifier (1'). The materials constituting these flow paths are not particularly limited, but examples thereof include silicon and various plastics. The cross-sectional shape of each of these flow paths is not particularly limited, and may be a semicircular shape, a circular shape, a rectangular shape, a wedge shape, a polygonal shape, or the like. When the cross section of each flow path is approximately circular, the diameter is not particularly limited, but may be designed to be, for example, in the range of 1 to 6 mm, and preferably 2 to 5 mm.

[0048] The nucleic acid amplification chip (2') includes a first curved channel (23') and a second curved channel (24') corresponding to the denaturation temperature zone and the extension / annealing temperature zone, respectively, an intermediate channel (25') connecting the first curved channel (23') and the second curved channel (24'), two connection parts (26', 27') connectable to the nucleic acid amplification device (1'), a first connection channel (28) connecting the first curved channel (23') and the connection part (26'), and a second connection channel (29') connecting the second curved channel (24') and the connection part (27'). In this specification, the first curved channel (23'), the second curved channel (24'), the intermediate channel (25'), the first connection channel (28'), and the second connection channel (29') in the nucleic acid amplification chip (2') are collectively referred to simply as chip channels.

[0049] The materials constituting these chip channels are not particularly limited, but may be, for example, various thermosetting or photosetting materials such as glass, quartz, silicon, and cycloolefin polymer (COP). Resins and the like can be mentioned. The shape of the cross section of these chip channels is not particularly limited, and can be semicircular, circular, rectangular, trapezoidal, wedge-shaped, polygonal, and the like. The cross section of the chip channel can be, for example, a rectangular or trapezoidal shape with a width of about 100 to 1000 μm and a depth of about 100 to 1000 μm. It is preferably a rectangular or trapezoidal shape with a width of about 100 to 1000 μm and a depth of about 100 to 1000 μm. The width and depth of the chip channel can be constant, or the width or depth can vary partially. Therefore, in this specification, unless otherwise specified, for each chip channel, the "width" refers to the average value of the width of the chip channel. Similarly, in this specification, unless otherwise specified, for each chip channel, the "depth" refers to the average value of the depth of the chip channel.

[0050] The length of each of the first curved channel (23') corresponding to the denaturation temperature range and the second curved channel (24') corresponding to the extension / annealing temperature range is preferably 20 mm or more.

[0051] Next, a step is performed to connect the connection parts (26', 27') in the nucleic acid amplification chip to the first connection part (12') and the second connection part (13') in the nucleic acid amplification device (1'). This step enables the air flow from the nucleic acid amplification device (1') to be sent to the nucleic acid amplification chip (2'). Note that the connection between the nucleic acid amplification chip (2') and the nucleic acid amplification device (1') may be performed simultaneously with the placement of the nucleic acid amplification chip (2') on the substrate (3') of the nucleic acid amplification device (1').

[0052] Then, a step of performing thermal cycling is performed by moving the sample liquid (30') back and forth between the first curved flow path (23') and the second curved flow path (24') by the liquid delivery mechanism (6'). As shown in FIG. 10, in step 3', when the sample liquid (30') is moved from the first heater (4') side to the second heater (5') side, the first three-way valve (7') is controlled so that the first flow path (14') and the second flow path (15') are connected and the fourth flow path (21") is not connected to the first flow path (14') and the second flow path (15'). On the other hand, the second three-way valve (8') is controlled so that the third flow path (16') and the fifth flow path (22") are connected and the first flow path (14') is not connected to the third flow path (16') and the fifth flow path (22"). Liquid Delivery Mechanism The air sent out from (6') flows toward the first three-way valve (7') and the second three-way valve (8'). The air cannot escape beyond the second three-way valve (8'). On the other hand, on the first three-way valve (7') side, the ends are open to the outside of the flow paths via the second flow path (15'), the first curved flow path (23'), the intermediate flow path (25') and the second curved flow path (24') in the nucleic acid amplification chip (2'), the third flow path (16'), the second three-way valve (8'), and the fifth flow path (22"), so that the sample liquid (30') moves toward the second heater (5') due to the pressure of the air from the liquid sending mechanism (6').

[0053] As shown in FIG. 11, when the movement of the sample liquid (30') is stopped in step 3', the first three-way valve (7') is controlled so that the second flow path (15') and the fourth flow path (21") are connected and the first flow path (14') is not connected to the second flow path (15') and the fourth flow path (21"'), and the second three-way valve (8') is controlled so that the third flow path (16') and the fifth flow path (22") are connected and the first flow path (14') is not connected to the third flow path (16') and the fifth flow path (22"). As a result, the residual pressure in the chip flow paths in the nucleic acid amplification chip (2') and the second flow path (15') and the third flow path (16') in the nucleic acid amplification device (1') is released through the fourth flow path (21") and the fifth flow path (22"). This makes it possible to suppress undesired movement of the sample liquid (30') due to the residual pressure in the flow paths. Furthermore, by limiting the speed of the air sent from the liquid sending mechanism (6') in the intermediate flow path to 100 mm / s or less (preferably 30 to 70 mm / s) and controlling the first three-way valve (7') and the second three-way valve (8') to stop within 2 seconds after being controlled to the above-mentioned state, the pressure in the first flow path (14') does not become excessive, and damage and deterioration of the liquid sending mechanism (6') can be avoided. This is preferably within 1 second, and more preferably within 0.5 seconds, after the first three-way valve (7') and the second three-way valve (8') are controlled. The embodiment of the present invention that uses a three-way valve rather than a four-way valve consumes less electricity and is also preferred in terms of miniaturization of the device.

[0054] As shown in FIG. 12, in step 3', when the sample liquid (30') is moved from the second heater (5') side to the first heater (4') side, the second three-way valve (8') is controlled to connect the first flow path (14') and the third flow path (16') and to close the fifth flow path (22") to the first flow path (14') and the third flow path (16'). On the other hand, the first three-way valve (7') is controlled to connect the second flow path (15') and the fourth flow path (21") and to close the first flow path (14') to the second flow path (15') and the fourth flow path (21"). The air sent out from the liquid delivery mechanism (6') flows toward the first three-way valve (7') and the second three-way valve (8'). On the side of the first three-way valve (7'), the air cannot escape beyond that. On the other hand, on the side of the second three-way valve (8'), the end is open to the outside of the flow path via the third flow path (16'), the second curved flow path (24') in the nucleic acid amplification chip (2'), the intermediate flow path (25') and the first curved flow path (23'), the second flow path (15'), the first three-way valve (7), and the fourth flow path (21"), so that the sample liquid (30') moves to the first heater (4') side due to the air pressure from the liquid delivery mechanism (6').

[0055] In the present invention, the nucleic acid amplifier (1') may further include a fluorescence detector (not shown) capable of measuring the fluorescence intensity of the sample liquid (30') passing through the intermediate flow path.

[0056] In a preferred embodiment of the present invention, in step 3', an electrical signal related to the movement of the sample liquid is sent from the fluorescence detector to the control device (11'), and the control device detects the passage of the sample liquid (30') through the intermediate flow path (25') based on the electrical signal, and controls the operation of the first three-way valve (7'), the second three-way valve (8') and the liquid delivery mechanism (6'). Also, the fluorescence detector for detecting the passage of the sample liquid (30') through the intermediate flow path (25') may be provided separately from the fluorescence detector for measuring the nucleic acid amplification of the sample liquid (30'). EXAMPLES

[0057] Real-time PCR is performed using a nucleic acid amplification device (1) and a nucleic acid amplification chip (2) shown in Figure 1. The measurement conditions are as follows: Shape of each channel of the nucleic acid amplification device (1): Circular Diameter of each channel of the nucleic acid amplifier (1): 3 mm Shape of the chip channel of the nucleic acid amplification chip (2): Rectangular Width of the channel of the nucleic acid amplification chip (2): 0.5 mm Depth of the channel of the nucleic acid amplification chip (2): 0.5 mm Sample solution: 15μL Thermal cycle conditions for real-time PCR: Heat at 98°C for 30 seconds, followed by 45 cycles of 98°C for 2 seconds and 58°C for 4 seconds. By carrying out real-time PCR under the above conditions, it is possible to suppress the influence of residual pressure when the movement of the sample is stopped, and also to suppress damage to the pump due to excessive pressure in the first flow path (14). EXAMPLES

[0058] Real-time PCR is performed using a nucleic acid amplification device (1') and a nucleic acid amplification chip (2') whose outline is shown in Figure 8. The measurement conditions are as follows: Shape of each channel of the nucleic acid amplification device (1): Circular Diameter of each channel of the nucleic acid amplifier (1): 3 mm Shape of the chip channel of the nucleic acid amplification chip (2): Rectangular Width of the channel of the nucleic acid amplification chip (2): 0.5 mm Depth of the channel of the nucleic acid amplification chip (2): 0.5 mm Sample solution: 15μL The passing speed of the sample liquid through the intermediate flow path is set to 60 mm / s. In order to stop the sample liquid in the flow path, the pump is stopped within 0.4 seconds after the first and second three-way solenoid valves are controlled. Thermal cycle conditions for real-time PCR: Heat at 98°C for 30 seconds, followed by 45 cycles of 98°C for 2 seconds and 58°C for 4 seconds. By carrying out real-time PCR under the above conditions, it is possible to suppress the influence of residual pressure when the movement of the sample is stopped, and also to suppress damage to the pump due to excessive pressure in the first channel (14'). [Explanation of symbols]

[0059] REFERENCE SIGNS LIST 1 Nucleic acid amplification device, 2 Nucleic acid amplification chip, 3 Substrate, 4 First heater, 5 Second heater, 6 Liquid delivery mechanism, 7 First four-way valve, 8 Second four-way valve, 9 First two-way valve, 10 Second two-way valve, 11 Control device, 12 First connection part, 13 Second connection part, 14 First flow path, 15 Second flow path, 16 Third flow path, 17 4th flow path, 18···5th flow path, 19···6th flow path, 20···7th flow path, 21···8th flow path, 22···9th flow path, 23···1st curved flow path, 24···2nd curved flow path, 25···intermediate flow path, 26, 27···connection part, 28···1st connecting flow path, 29···2nd connecting flow path, 30···sample solution, 31···1st filter, 32···2nd filter, 33···sample injection port 1'···Nucleic acid amplification device, 2'···Nucleic acid amplification chip, 3'···Substrate, 4'···First heater, 5'···Second heater, 6'···Liquid delivery mechanism, 7'···First three-way valve, 8'···Second three-way valve, 11'···Control device, 12'···First connection part, 13'···Second connection part, 14'···First flow path, 15'···Second flow path, 16'···Third flow path, 21”···Fourth flow path, 22”···Fifth flow path, 23'···First curved flow path, 24'···Second curved flow path, 25'···Intermediate flow path, 26', 27'···Connection part, 28'···First connection flow path, 29'···Second connection flow path, 30'···Sample liquid

Claims

1. A method for amplifying nucleic acid using a nucleic acid amplifier (1) including a chip for nucleic acid amplification (2) and a substrate (3) on which the chip for nucleic acid amplification (2) can be mounted, the method comprising the steps of: Step 1: A step of placing a nucleic acid amplification chip (2) on a substrate (3) of a nucleic acid amplification device (1), The nucleic acid amplification device (1), A first heater (4) capable of forming a denaturing temperature zone; A second heater (5) capable of forming an elongation / annealing temperature zone; A liquid transfer mechanism (6) that enables the movement of a sample liquid (30) between the denaturation temperature zone and the extension / annealing temperature zone; A first four-way valve (7) and a second four-way valve (8); A first two-way valve (9) and a second two-way valve (10), a control device (11) for controlling switching of the first four-way valve, the second four-way valve, the first two-way valve, and the second two-way valve; A first connection part (12) connectable to the nucleic acid amplification chip (2); A second connection part (13) connectable to the nucleic acid amplification chip (2); a first flow path (14) connecting the liquid delivery mechanism to the first four-way valve and the second four-way valve; a second flow path (15) having one end connected to the first four-way valve (7) and the other end connected to the first connection portion (12); a third flow path (16) having one end connected to the second four-way valve (8) and the other end connected to the second connection portion (13); a fourth flow passage (17) having one end connected to the first four-way valve (7) and the other end connected to the first two-way valve (9); a fifth flow path (18) having one end connected to the second four-way valve (8) and the other end connected to the second two-way valve (10); a sixth flow passage (19) having one end connected to the first two-way valve (9) and the other end open to the outside of the flow passage; a seventh flow path (20) having one end connected to the second two-way valve (10) and the other end open to the outside of the flow path; an eighth flow path (21) having one end connected to the first four-way valve (7) and the other end open to the outside of the flow path; and a ninth flow passage (22) having one end connected to the second four-way valve (8) and the other end open to the outside of the flow passage; The reciprocal flow type nucleic acid amplifier (1) is characterized in that it performs real-time PCR by measuring the fluorescence intensity for each thermal cycle, a step in which the nucleic acid amplification chip (2) comprises a first curved flow path (23) and a second curved flow path (24) corresponding to the denaturation temperature zone and the extension / annealing temperature zone, respectively, an intermediate flow path (25) connecting the first curved flow path (23) and the second curved flow path (24), two connection parts (26, 27) connectable to the nucleic acid amplification device, a first connection flow path (28) connecting the first curved flow path (23) and the connection part (26), and a second connection flow path (29) connecting the second curved flow path (24) and the connection part (27); Step 2: a step of connecting the connection parts (26, 27) in the nucleic acid amplification chip (2) to the first connection part (12) and the second connection part (13) in the nucleic acid amplification device; Step 3: performing thermal cycling by causing the sample liquid to travel back and forth between the first curved flow path (23) and the second curved flow path (24) via the intermediate flow path (25) using the liquid delivery mechanism; and However, when the sample liquid (30) is moved from the first heater (4) side to the second heater (5) side in step 3, the first four-way valve (7) is controlled to communicate the first flow path (14) with the second flow path (15) and the fourth flow path (17) with the eighth flow path (21), and the first flow path (14) with the fifth flow path (18) and the third flow path (16) with the ninth flow path (22). the second four-way valve (8) is controlled so as to open the second two-way valve (10) and close the first two-way valve (9); In step 3, when the sample liquid (30) is moved from the second heater (5) side to the first heater (4) side, the second four-way valve (8) is controlled to communicate the first flow path (14) with the third flow path (16) and the fifth flow path (18) with the ninth flow path (22), the first four-way valve (7) is controlled to communicate the first flow path (14) with the fourth flow path (17) and the second flow path (15) with the eighth flow path (21), the first two-way valve (9) is kept in a closed state, and the second two-way valve (10) is kept in a closed or open state, When the movement of the sample liquid (30) is stopped in step 3, the first four-way valve (7) is controlled to communicate the first flow path (14) with the fourth flow path (17) and the second flow path (15) with the eighth flow path (21), the second four-way valve (8) is controlled to communicate the first flow path (14) with the fifth flow path (18) and the third flow path (16) with the ninth flow path (22), and the first two-way valve and / or the second two-way valve are opened.

2. 2. The method according to claim 1, wherein the nucleic acid amplification device (1) further comprises a fluorescence detector capable of measuring a fluorescence intensity of the sample liquid (30) passing through the intermediate flow path (25), The method further comprises step 4: measuring the fluorescence intensity of the sample liquid (30) for each thermal cycle by the fluorescence detector at a predetermined position in the intermediate flow path (25).

3. 3. The method according to claim 2, wherein in step 3, an electrical signal from the fluorescence detector regarding the movement of the sample liquid (30) is sent to the control device, and based on the electrical signal, the control device detects the passage of the sample liquid (30) through the intermediate flow path and controls the switching of the first four-way valve, the second four-way valve, the first two-way valve and the second two-way valve.

4. A substrate (3) on which a nucleic acid amplification chip (2) can be mounted; A first heater (4) capable of forming a denaturing temperature zone; A second heater (5) capable of forming an elongation / annealing temperature zone; A liquid transfer mechanism (6) that enables the movement of a sample liquid (30) between the denaturation temperature zone and the extension / annealing temperature zone; A first four-way valve (7) and a second four-way valve (8); A first two-way valve (9) and a second two-way valve (10), a control device (11) for controlling switching of the first four-way valve, the second four-way valve, the first two-way valve, and the second two-way valve; A first connection part (12) connectable to the nucleic acid amplification chip (2); A second connection part (13) connectable to the nucleic acid amplification chip (2); a first flow path (14) connecting the liquid delivery mechanism (6) to the first four-way valve and the second four-way valve; a second flow path (15) having one end connected to the first four-way valve (7) and the other end connected to the first connection portion (12); a third flow path (16) having one end connected to the second four-way valve (8) and the other end connected to the second connection portion (13); a fourth flow path (17) having one end connected to the first four-way valve (7) and the other end connected to the first two-way valve (9); a fifth flow path (18) having one end connected to the second four-way valve (8) and the other end connected to the second two-way valve (10); a sixth flow passage (19) having one end connected to the first two-way valve (9) and the other end open to the outside of the flow passage; a seventh flow path (20) having one end connected to the second two-way valve (10) and the other end open to the outside of the flow path; an eighth flow path (21) having one end connected to the first four-way valve (7) and the other end open to the outside of the flow path; and a ninth flow passage (22) having one end connected to the second four-way valve (8) and the other end open to the outside of the flow passage; The reciprocal flow type nucleic acid amplifier (1) is characterized in that it performs real-time PCR by measuring the fluorescence intensity for each thermal cycle.

5. A method for amplifying nucleic acid using a nucleic acid amplifier (1') including a chip for nucleic acid amplification (2') and a substrate (3') on which the chip for nucleic acid amplification (2') can be mounted, the method comprising the steps of: Step 1: A step of placing a nucleic acid amplification chip (2') on a substrate (3') of a nucleic acid amplification device (1'), The nucleic acid amplification device (1') A first heater (4') capable of forming a denaturation temperature zone; A second heater (5') capable of forming an elongation / annealing temperature zone; a liquid transport mechanism (6') for enabling the movement of a sample liquid (30') between the denaturation temperature zone and the extension / annealing temperature zone; A first three-way valve (7') and a second three-way valve (8'); a control device (11') for controlling the operation of the first three-way valve (7'), the second three-way valve (8'), and the liquid delivery mechanism (6'); A first connection part (12') connectable to the nucleic acid amplification chip (2'); A second connection part (13') connectable to the nucleic acid amplification chip (2'); a first flow path (14') connecting the liquid delivery mechanism to a first three-way valve (7') and a second three-way valve (8'); a second flow path (15') having one end connected to the first three-way valve (7') and the other end connected to the first connection portion (12'); a third flow path (16') having one end connected to the second three-way valve (8') and the other end connected to the second connection portion (13'); a fourth flow path (21″) having one end connected to the first three-way valve (7′) and the other end open to the outside of the flow path; and A fifth flow path (22″) having one end connected to the second three-way valve (8′) and the other end open to the outside of the flow path. and a reciprocal flow type nucleic acid amplifier (1') characterized in that the real-time PCR is performed by measuring the fluorescence intensity for each thermal cycle. a step in which the nucleic acid amplification chip (2') is provided with a first curved flow path (23') and a second curved flow path (24') corresponding to the denaturation temperature zone and the extension / annealing temperature zone, respectively, an intermediate flow path (25') connecting the first curved flow path (23') and the second curved flow path (24'), two connection parts (26', 27') connectable to the nucleic acid amplification device, a first connection flow path (28') connecting the first curved flow path (23') and the connection part, and a second connection flow path (29') connecting the second curved flow path (24') and the connection part; Step 2: a step of connecting the connection parts (26', 27') in the nucleic acid amplification chip (2') to the first connection part (12') and the second connection part (13') in the nucleic acid amplification device (1'); step 3: performing thermal cycling by using the liquid delivery mechanism to move the sample liquid back and forth between the first curved flow path (23') and the second curved flow path (24') via the intermediate flow path (25'); and, in step 3, when the sample liquid (30') is moved from the first heater (4') side to the second heater (5') side, the first three-way valve (7') is controlled so that the first flow path (14') and the second flow path (15') are communicated and the fourth flow path (21") is not communicated with the first flow path (14') and the second flow path (15'), the second three-way valve (8') is controlled so that the third flow path (16) and the fifth flow path (22") are communicated and the first flow path (14') is not communicated with the third flow path (16') and the fifth flow path (22"), and the liquid delivery mechanism (6') is controlled so that the speed at which the sample liquid passes through the intermediate flow path (25') is 100 mm / s or less; In step 3, the sample liquid (30') is fed from the second heater (5') side to the first heater (4' ), the second three-way valve (8') is controlled so that the first flow path (14') and the third flow path (16') are connected and the fifth flow path (22") is not connected to the first flow path (14') and the third flow path (16'), the first three-way valve (7') is controlled so that the second flow path (15') and the fourth flow path (21") are connected and the first flow path (14') is not connected to the second flow path (15') and the fourth flow path (21"), and the liquid sending mechanism (6') is controlled so that the speed at which the sample liquid (30') passes through the intermediate flow path (25') is 100 mm / s or less, When the movement of the sample liquid (30') is stopped in step 3, the first three-way valve (7') is controlled so that the second flow path (15') and the fourth flow path (21") are communicated and the first flow path (14') is not communicated with the second flow path (15') and the fourth flow path (21"), the second three-way valve (8') is controlled so that the third flow path (16') and the fifth flow path (22") are communicated and the first flow path (14') is not communicated with the third flow path (16') and the fifth flow path (22"), and the liquid delivery mechanism (6') is stopped within 2 seconds after both the first three-way valve (7') and the second three-way valve (8') are controlled to the above states.

6. 6. The method according to claim 5, wherein the nucleic acid amplification device (1′) comprises a fluorescence detector capable of measuring a fluorescence intensity of the sample liquid (30′) passing through the intermediate flow path (25′), The method includes a step of measuring the fluorescence intensity of the sample liquid for each thermal cycle by the fluorescence detector at a predetermined position in the intermediate flow path (25').

7. 7. The method according to claim 6, wherein in step 3, an electrical signal relating to the movement of the sample liquid (30') is sent from the fluorescence detector to the control device (11'), and based on the electrical signal, the control device detects the passage of the sample liquid (30') through the intermediate flow path (25') and controls the operation of the first three-way valve (7'), the second three-way valve (8') and the liquid delivery mechanism (6').

8. 8. The method according to any one of claims 5 to 7, wherein the first flow path (14') has an internal cross section which is larger than the internal cross sections of the first curvilinear flow path (23'), the second curvilinear flow path (24') and the intermediate flow path (25').

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