nucleic acid amplification method, nucleic acid amplification apparatus, and nucleic acid amplification chip

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

[0006] , , , ,

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

Background Art

[0002] The detection of nucleic acids is central in various fields such as the medical field (pharmaceutical research and development, clinical examinations, forensic medicine, etc.), the agricultural field (identification of crop and pathogenic microorganism species, etc.). The PCR method for selectively amplifying a specific region of DNA is a very useful technique for nucleic acid detection.

[0003] There has been a problem that the PCR reaction takes a long time due to the large heat capacity in general-purpose thermal cyclers for PCR and real-time PCR. To address this, a technique has been developed to speed up thermal cycling by repeatedly feeding a liquid onto multiple temperature zones using a flow channel chip (Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems 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 feeding control mechanism.

Means for Solving the Problems

[0006] The inventors of the present invention considered controlling the sample liquid feeding of the flow channel chip using a switching valve instead of a pump. However, when opening and closing the switching valve while driving the pump, there is no escape path for air in the flow channel directly connected to the pump, resulting in excessive internal pressure in the flow channel and causing pump damage and deterioration. Therefore, the inventors have found that in a nucleic acid amplification device having one liquid delivery mechanism, an increase in pressure within the flow path can be avoided by using a two-way valve in addition to a four-way valve.

[0007] Furthermore, based on these new findings, the inventors completed the present invention by appropriately modifying the arrangement of the flow path connecting the liquid delivery mechanism and the two types of valves.

[0008] Therefore, one aspect of the present invention provides the following: Item 1. A nucleic acid amplification method using a nucleic acid amplification apparatus (1) comprising a nucleic acid amplification chip (2) and a substrate (3) on which the nucleic acid amplification chip (2) can be mounted, comprising the following steps: Step 1: A step of placing a nucleic acid amplification chip (2) on the substrate (3) of the nucleic acid amplification device (1), The nucleic acid amplification device (1) A first heater (4) capable of forming a denaturation temperature range, A second heater (5) capable of forming an extension / annealing temperature range, A liquid transfer mechanism (6) that enables the movement of the sample liquid (30) between the denaturation temperature range and the extension / annealing temperature range, First four-way valve (7) and second four-way valve (8), First two-way valve (9) and second two-way valve (10), A control device (11) that 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. A first connection part (12) that can be connected to the nucleic acid amplification chip (2), A second connection part (13) that can be connected to the nucleic acid amplification chip (2), A first flow path (14) connecting the liquid delivery mechanism with the first four-way valve and the second four-way valve, A second flow path (15) is connected at one end to the first four-way valve (7) and at the other end to the first connection part (12), A third flow path (16) is connected at one end to the second four-way valve (8) and at the other end to the second connection part (13), A fourth flow path (17) is connected at one end to the first four-way valve (7) and at the other end to the first two-way valve (9), A fifth flow path (18) is connected at one end to the second four-way valve (8) and at the other end to the second two-way valve (10), A sixth flow path (19) has 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) has one end connected to the second two-way valve (10) and the other end open to the outside of the flow path. An eighth passage (21) whose one end is connected to the first four-way valve (7) and whose other end is open to the outside of the passage, A ninth flow path (22) has one end connected to the second four-way valve (8) and the other end open to the outside of the flow path. This reciprocal flow type nucleic acid amplification device (1) is characterized by performing real-time PCR by measuring the fluorescence intensity for each thermal cycle, and is equipped with the following: The nucleic acid amplification chip (2) comprises a first curved channel (23) and a second curved channel (24) corresponding to the denaturation temperature range and the extension / annealing temperature range, 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 part (26); and a second connection channel (29) connecting the second curved channel (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: A step of performing thermal cycling by causing the sample liquid to reciprocate through the first curved channel (23) and the second curved channel (24) via the intermediate channel (25) using the liquid delivery mechanism, and However, when moving the sample liquid (30) from the first heater (4) side to the second heater (5) side in step 3, the first four-way valve (7) is controlled to allow the sample liquid to pass through the first channel (14) and the second channel (15), and through the fourth channel (17) and the eighth channel (21), and the second four-way valve (8) is controlled to allow the sample liquid to pass through the first channel (14) and the fifth channel (18), and through the third channel (16) and the ninth channel (22), and the second two-way valve (10) is closed, and the first two-way valve (9) is either closed or open. In step 3, when moving the sample liquid (30) from the second heater (5) side to the first heater (4) side, the second four-way valve (8) is controlled to allow the sample liquid to pass through the first channel (14) and the third channel (16), and through the fifth channel (18) and the ninth channel (22), and the first four-way valve (7) is controlled to allow the sample liquid to pass through the first channel (14) and the fourth channel (17), and through the second channel (15) and the eighth channel (21), and the first two-way valve (9) is closed, and the second two-way valve (10) is closed or open. In step 3, when stopping the movement of the sample liquid (30), the first four-way valve (7) is controlled to allow the sample liquid to pass through the first channel (14) and the fourth channel (17) and through the second channel (15) and the eighth channel (21), the second four-way valve (8) is controlled to allow the sample liquid to pass through the first channel (14) and the fifth channel (18) and through the third channel (16) and the ninth channel (22), and the first two-way valve and / or the second two-way valve are opened.

[0009] Item 2. The method described in Item 1, wherein the nucleic acid amplification device (1) is the intermediate channel (25) The device further includes a fluorescence detector capable of measuring the fluorescence intensity of the sample solution (30) passing through it. The method further comprises step 4: measuring the fluorescence intensity of the sample solution (30) for each thermal cycle using 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 from the fluorescence detector regarding the movement of the sample liquid (30) is sent to the control device, and based on this electrical signal, the control device senses the passage of the sample liquid (30) in 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.

[0011] Item 4. A substrate (3) on which a nucleic acid amplification chip (2) can be placed, A first heater (4) capable of forming a denaturation temperature zone, A second heater (5) capable of forming an extension / annealing temperature zone, A liquid delivery mechanism (6) that enables the movement of the 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 the 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) that can be connected to the nucleic acid amplification chip (2), A second connection part (13) that can be connected to the nucleic acid amplification chip (2), A first flow path (14) that connects the liquid delivery mechanism (6) to the first four-way valve and the second four-way valve, a second flow path (15) with 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) with one end connected to the second four-way valve (8) and the other end connected to the second connection part (13), A fourth flow path (17) with 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) with 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) with 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) with 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, A reciprocal flow type nucleic acid amplification device (1) characterized by performing real-time PCR by measuring the fluorescence intensity for each thermal cycle.

[0012] In addition, the present inventor has found that in a nucleic acid amplification device having one liquid feeding mechanism, by using a three-way valve and adjusting the sample speed by a pump, it is possible to avoid an increase in the internal pressure of the flow path.

[0013] Furthermore, based on such new findings, the present inventors have completed one of the present inventions by setting the arrangement of the flow path connecting the liquid feeding mechanism and the valve, the sample speed by the pump, and the stop condition of the pump. Therefore, one of the present inventions provides the following items: Item 5. A nucleic acid amplification method using a nucleic acid amplification device (1') comprising a nucleic acid amplification chip (2') and a substrate (3') on which the nucleic acid amplification chip (2') can be placed, the method including the following steps: Step 1: A step of placing the nucleic acid amplification chip (2') on the substrate (3') of the nucleic acid amplification device (1'), where the nucleic acid amplification device (1') has 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 feeding mechanism (6') that enables the movement of the sample liquid (30') between the denaturation temperature zone and the elongation / annealing temperature zone, A first three-way valve (7') and a second three-way valve (8'), A control device (11') that controls the driving of the first three-way valve (7'), the second three-way valve (8'), and the liquid feeding mechanism (6'), A first connection part (12') that can be connected to the nucleic acid amplification chip (2'), A second connection part (13') that can be connected to the nucleic acid amplification chip (2'), A first flow path (14') connecting the liquid delivery mechanism to the first three-way valve (7') and the second three-way valve (8'), A second flow path (15') has 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') is connected at one end to the second three-way valve (8') and at the other end to the second connection part (13'), A fourth flow path (21") is connected at one end to the first three-way valve (7') and has the other end open to the outside of the flow path, and A fifth flow path (22") has one end connected to the second three-way valve (8') and the other end open to the outside of the flow path. This is a reciprocal flow type nucleic acid amplification device (1') characterized by performing real-time PCR by measuring the fluorescence intensity for each thermal cycle, The nucleic acid amplification chip (2') comprises a first curved channel (23') and a second curved channel (24') corresponding to the denaturation temperature range and the extension / annealing temperature range, 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 part; and a second connection channel (29') connecting the second curved channel (24') and the connection part. Step 2: A step of 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 (1'). Step 3: A step of performing thermal cycling by moving the sample liquid back and forth between the first curved channel (23') and the second curved channel (24') via the intermediate channel (25') using the liquid delivery mechanism, provided that in step 3 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 it passes through the first channel (14') and the second channel (15'), and the fourth channel (21") is not connected to the first channel (14') and the second channel (15'), the second three-way valve (8') is controlled so that it passes through the third channel (16) and the fifth channel (22"), and the first channel (14') is not connected to the third channel (16') and the fifth channel (22"), and the liquid delivery mechanism (6') is controlled so that the speed at which the sample liquid passes through the intermediate channel (25') is 100 mm / s or less. In step 3, when moving the sample liquid (30') from the second heater (5') side to the first heater (4') side, the second three-way valve (8') is controlled so that the first channel (14') and the third channel (16') are connected, and the fifth channel (22") is not connected to the first channel (14') and the third channel (16'). The first three-way valve (7') is controlled so that the second channel (15') and the fourth channel (21") are connected, and the first channel (14') is not connected to the second channel (15') and the fourth channel (21"). The liquid delivery mechanism (6') is controlled so that the speed at which the sample liquid (30') passes through the intermediate channel (25') is 100 mm / s or less. In step 3, when stopping the movement of the sample liquid (30'), the first three-way valve (7') is controlled so that the second channel (15') and the fourth channel (21") are connected, and the first channel (14') is not connected to the second channel (15') and the fourth channel (21"), and the second three-way valve (8') is controlled so that the third channel (16') and the fifth channel (22") are connected, and the first channel (14') is not connected to the third channel (16') and the fifth channel (22"), and the liquid delivery mechanism (6') is stopped within 2 seconds after controlling both the first three-way valve (7') and the second three-way valve (8') to the above state.

[0014] Item 6. The method described in Item 5, wherein the nucleic acid amplification device (1') is equipped with a fluorescence detector capable of measuring the fluorescence intensity of the sample solution (30') passing through the intermediate channel (25'), The method comprises the step of measuring the fluorescence intensity of the sample solution for each thermal cycle using 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 from the fluorescence detector regarding the movement of the sample liquid (30') is sent to the control device (11'), and based on the electrical signal, the control device senses the passage of the sample liquid (30') through the intermediate channel (25') and controls the driving 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 internal cross-section of the first channel (14') is larger than the internal cross-sections of the first curved channel (23'), the second curved channel (24'), and the intermediate channel (25'). [Brief explanation of the drawing]

[0017] [Figure 1] An embodiment of the nucleic acid amplification device (1) and nucleic acid amplification chip (2) according to the present invention is shown. [Figure 2] This section outlines the four-way valve. [Figure 3] An overview of a four-way valve in another embodiment is shown. [Figure 4] The general flow of the sample solution and air in step 3 is shown. [Figure 5] The general flow of the sample solution and air in step 3 is shown. [Figure 6] The general flow of the sample solution and air in step 3 is shown. [Figure 7] The diagram shows an overview of a two-way valve. (a) represents a valve with two ports on different sides, and (b) represents a valve with two ports on the same side. [Figure 8] An embodiment of the nucleic acid amplification device (1') and nucleic acid amplification chip (2') according to the present invention is shown. [Figure 9] The following shows an overview of a three-way valve. (a) represents a valve with two ports on one side and one port on the opposite side, and (b) represents a valve with three ports on the same side. [Figure 10] The general flow of the sample solution and air in step 3' is shown. [Figure 11] The general flow of the sample solution and air in step 3' is shown. [Figure 12] The general flow of the sample solution and air in step 3' is shown. [Figure 13] An embodiment of a flow channel chip is shown. [Modes for carrying out the invention]

[0018] The present invention will be described in detail below with reference to the 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 Figure 1 shows one embodiment of a nucleic acid amplification device (1) and a nucleic acid amplification chip (2) used in nucleic acid amplification according to the present invention. As shown in Figure 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 range, A second heater (5) capable of forming an extension / annealing temperature range, A liquid transfer mechanism (6) that enables the movement of the sample liquid (30) between the denaturation temperature range and the extension / annealing temperature range, First four-way valve (7) and second four-way valve (8), First two-way valve (9) and second two-way valve (10), A control device (11) that 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), A first connection part (12) that can be connected to the nucleic acid amplification chip, A second connection part (13) that can be connected to the nucleic acid amplification chip, A first flow path (14) connecting the liquid delivery mechanism with the first four-way valve and the second four-way valve, A second flow path (15) has one end connected to the first four-way valve and the other end connected to the first connection part. A third flow path (16) is connected at one end to the second four-way valve and at the other end to the second connection part. A fourth flow path (17) is connected at one end to the first four-way valve and at the other end to the first two-way valve. A fifth flow path (18) is connected at one end to the second four-way valve and at the other end to the second two-way valve, A sixth passage (19) with one end connected to the first two-way valve and the other end open to the outside of the passage, a seventh passage (20) with one end connected to the second two-way valve and the other end open to the outside of the passage, an eighth passage (21) with one end connected to the first four-way valve and the other end open to the outside of the passage, and The system includes a ninth flow path (22), one end of which is connected to the second four-way valve and the other end of which is open to the outside of the flow path.

[0020] In the present invention, the first heater (4) is not particularly limited as long as it can form a denaturation temperature range. The second heater (5) is not particularly limited as long as it can form an extension / annealing temperature range. Specific examples of the first heater (4) and the second heater (5) include cartridge heaters, film heaters, Peltier heaters, and the like. The denaturation temperature range is maintained at the temperature necessary for the DNA denaturation reaction in PCR. The temperature of the denaturation temperature range is preferably around 90 to 100°C, and more preferably around 93 to 98°C. The extension / annealing temperature range is maintained at the temperature necessary for the DNA annealing and extension reactions in PCR. The temperature of the extension / annealing temperature range is preferably around 40 to 75°C, and more preferably around 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 path and tip flow path, and examples include micro blowers, fans, syringe pumps, etc.

[0022] A microblower (also called a piezoelectric microblower) is a known device that sucks in and discharges air, characterized by not having a sealed structure (not having a check valve). In a typical microblower, air is sucked in and discharged by bending and deforming a diaphragm by applying voltage to a piezoelectric element. 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 uses the rotational motion of an impeller to blow air. Due to the structural characteristics of the impeller, the airflow path is not a closed system.

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

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

[0026] Furthermore, in this invention, a two-way valve refers to a valve that connects two flow paths and can be opened or closed by control. For example, a "two-way valve" like the one shown in Figure 7 can be cited.

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

[0028] The control device (11) may include a CPU or the like for controlling 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). The control device (11) may also further control the fluid delivery mechanism (6).

[0029] In this embodiment, the first channel (14), second channel (15), third channel (16), fourth channel (17), fifth channel (18), sixth channel (19), seventh channel (20), eighth channel (21), and ninth channel (22) are tubular members that connect the components of the nucleic acid amplification device (1). The material that constitutes these channels is not particularly limited, but examples include silicon and various plastics. The cross-sectional shape of each of these channels is not particularly limited and can be semicircular, circular, rectangular, trapezoidal, wedge-shaped, polygonal, etc. When the cross-section of each channel is approximately circular, the diameter is not particularly limited, but can be designed 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 range and the extension / annealing temperature range, 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 the chip channel.

[0031] The materials constituting these chip channels are not particularly limited, but examples include glass, quartz, silicon, and various thermosetting or photocurable resins such as cycloolefin polymers (COP). The cross-sectional shape of these chip channels is not particularly limited and can be semicircular, circular, rectangular, trapezoidal, wedge-shaped, polygonal, etc. 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. Preferably, it is a rectangular or trapezoidal shape with a width of about 100 to 1000 μm and a depth of about 100 to 1000 μm. Furthermore, the width and depth of the chip channel can be constant, or the width or depth can vary in part. Accordingly, unless otherwise specified in this specification, "width" for each chip channel refers to the average width of the chip channel. Similarly, unless otherwise specified in this specification, "depth" for each chip channel refers to the depth of the chip channel. This shows the average value.

[0032] The lengths 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 are preferably 20 mm or more.

[0033] Next, a step is performed to connect the connection parts (26, 27) on the nucleic acid amplification chip to the first connection part (12) and the second connection part (13) on the nucleic acid amplification device (1). This step allows airflow 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, the liquid delivery mechanism (6) is used to move the sample liquid (30) back and forth between the first curved channel (23) and the second curved channel (24) to perform thermal cycling. As shown in Figure 4, in step 3, when moving the sample liquid (30) from the first heater (4) side to the second heater (5) side, the first four-way valve (7) is controlled to allow the liquid to pass through the first channel (14) and the second channel (15) and through the fourth channel (17) and the eighth channel (21). In this case, the first two-way valve (9) may be open or closed. On the other hand, the second four-way valve (8) is controlled to allow the liquid to pass through the first channel (14) and the fifth channel (18) and through the third channel (16) and the ninth channel (22), and the second two-way valve (10) is closed. The air delivered from the liquid delivery mechanism (6) is directed towards the first four-way valve (7) and the second four-way valve (8). On the second four-way valve (8) side, the sample liquid (30) cannot pass through because the second two-way valve (10) is closed. On the other hand, on the first four-way valve (7) side, 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), the second curved flow path (24), the third flow path (16), the second four-way valve (8), and the ninth flow path (22) in the nucleic acid amplification chip (2), so 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 Figure 5, when stopping the movement of the sample liquid (30) in step 3, the first four-way valve (7) is controlled to allow the sample liquid (30) to pass through the first channel (14) and the fourth channel (17) to the second channel (15) and the eighth channel (21), and the second four-way valve (8) is controlled to allow the sample liquid (30) to pass through the first channel (14) and the fifth channel (18) to the third channel (16) and the ninth channel (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 channel in the nucleic acid amplification chip (2) and in the second channel (15) and the third channel (16) in the nucleic acid amplification device (1) is released through the eighth channel (21) and the ninth channel (22), thereby suppressing the unwanted movement of the sample liquid (30) due to residual pressure in the channels. Furthermore, since the air sent from the liquid delivery mechanism (6) is released 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 to the outside of the flow path via the fifth flow path (18) and the seventh flow path (20), the pressure inside the first flow path (14) does not become excessive, and damage and deterioration of the liquid delivery mechanism (6) can be avoided. The embodiment of the present invention that uses a four-way valve is preferable in that liquid delivery can be controlled solely by controlling the valve and not by controlling the liquid delivery mechanism (6).

[0036] As shown in Figure 6, in step 3, when moving the sample liquid (30) from the second heater (5) side to the first heater (4) side, the second four-way valve (8) is controlled to allow the liquid to pass through the first channel (14) and the third channel (16), and through the fifth channel (18) and the ninth channel (22). In this case, the second two-way valve (10) may be open or closed. On the other hand, the first four-way valve (7) is controlled to allow the liquid to pass through the first channel (14) and the fourth channel (17), and through the second channel (15) and the eighth channel (21), and the first two-way valve (9) is closed. The air sent from the liquid delivery mechanism (6) is directed towards 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, so the sample liquid cannot pass through to the other side. 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), 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) in the nucleic acid amplification chip (2), so 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, filter regions (31, 32), a sample inlet (33), a reverse transcription reaction region, etc., may be present in the chip channel of the nucleic acid amplification chip (2).

[0038] Furthermore, in the present invention, the nucleic acid amplification device (1) may further include a fluorescence detector (not shown) capable of measuring the fluorescence intensity of the sample solution (30) passing through the intermediate channel.

[0039] Furthermore, in a preferred embodiment of the present invention, in step 3, an electrical signal from the fluorescence detector regarding the movement of the sample solution is sent to the control device (11), and based on this electrical signal, the control device senses the passage of the sample solution (30) through the intermediate channel (25) 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). Alternatively, a fluorescence detector for sensing the passage of the sample solution (30) through the intermediate channel (25) may be installed separately from the fluorescence detector for measuring the nucleic acid amplification of the sample solution (30).

[0040] [II] Nucleic acid amplification method using a three-way valve Figure 8 shows one embodiment of the nucleic acid amplification device (1') and nucleic acid amplification chip (2') used in nucleic acid amplification according to the present invention. As shown in Figure 8, 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 range, A second heater (5') capable of forming an extension / annealing temperature zone, A liquid transfer mechanism (6') that enables the movement of the sample liquid (30') between the aforementioned denaturation temperature range and the aforementioned extension / annealing temperature range, First three-way valve (7') and second four-way valve (8'), A control device (11') that controls the drive of the first three-way valve (7'), the second three-way valve (8'), and the liquid delivery mechanism (6'), A first connection part (12') that can be connected to the nucleic acid amplification chip (2'), A second connection part (13') that can be connected to the nucleic acid amplification chip (2'), A first flow path (14') connecting the liquid delivery mechanism (6') to the first three-way valve (7') and the second three-way valve (8'), A second flow path (15') has 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') is connected at one end to the second three-way valve (8') and at the other end to the second connection part (13'), A fourth flow path (21") is connected at one end to the first three-way valve (7') and has the other end open to the outside of the flow path, and The system includes 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.

[0041] In the present invention, the first heater (4') is not particularly limited as long as it can form a denaturation temperature range. The second heater (5') is not particularly limited as long as it can form an extension / annealing temperature range. Specific examples of the first heater (4') and the second heater (5') include cartridge heaters, film heaters, Peltier heaters, etc. The denaturation temperature range is maintained at the temperature necessary for the DNA denaturation reaction in PCR. The temperature in the operating temperature range is preferably around 90-100°C, and more preferably around 93-98°C. The extension and annealing temperature range is maintained at the temperature necessary for the DNA annealing and extension reactions in PCR. The temperature in the extension and annealing temperature range is preferably around 40-75°C, and more preferably around 55-65°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 path and tip flow path, and examples include micro blowers, fans, syringe pumps, etc.

[0043] A microblower (also called a piezoelectric microblower) is a known device that sucks in and discharges air, characterized by not having a sealed structure (not having a check valve). In a typical microblower, air is sucked in and discharged by bending and deforming a diaphragm by applying voltage to a piezoelectric element. 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 uses the rotational motion of an impeller to blow air. Due to the structural characteristics of the impeller, the airflow path is not a closed system.

[0045] In the present invention, a three-way valve, for example as shown in Figure 8, connects flow paths A, B, and C, and by controlling the three-way valve, it is possible to switch from a state where flow paths A and B are connected to a state where flow paths A and C are connected. Furthermore, a valve that connects flow paths in such a way that opening one flow path closes the other, as shown in Figure 9, is also included in the definition of a "three-way valve".

[0046] Preferably, it is a three-way solenoid valve, for example, the 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 fluid delivery mechanism (6').

[0047] In this embodiment, the first channel (14'), second channel (15'), third channel (16'), fourth channel (21"), and fifth channel (22") are tubular members that connect the components of the nucleic acid amplification device (1'). The material constituting these channels is not particularly limited, but examples include silicon and various plastics. The cross-sectional shape of each of these channels is not particularly limited and can be semicircular, circular, rectangular, wedge-shaped, polygonal, etc. When the cross-section of each channel is approximately circular, the diameter is not particularly limited, but can be designed in the range of 1 to 6 mm, preferably 2 to 5 mm.

[0048] The nucleic acid amplification chip (2') comprises a first curved channel (23') and a second curved channel (24') corresponding to the denaturation temperature range and the extension / annealing temperature range, 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 the chip channel.

[0049] The materials that make up these chip channels are not particularly limited, but include various thermosetting or photocurable materials such as glass, quartz, silicon, and cycloolefin polymers (COP). Examples include resins. The cross-sectional shape of these chip channels is not particularly limited and can be semicircular, circular, rectangular, trapezoidal, wedge-shaped, polygonal, etc. The cross-section of the chip channel can also 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, it 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 can be constant, or the width or depth can vary in part. Therefore, unless otherwise specified in this specification, "width" for each chip channel refers to the average value of the width of the chip channel. Similarly, unless otherwise specified in this specification, "depth" for each chip channel refers to the average value of the depth of the chip channel.

[0050] The lengths 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 are preferably 20 mm or more.

[0051] Next, a step is performed to connect the connection parts (26', 27') on the nucleic acid amplification chip to the first connection part (12') and the second connection part (13') on the nucleic acid amplification device (1'). This step allows airflow 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, the liquid delivery mechanism (6') is used to move the sample liquid (30') back and forth between the first curved channel (23') and the second curved channel (24') to perform thermal cycling. As shown in Figure 10, in step 3', when moving the sample liquid (30') from the first heater (4') side to the second heater (5') side, the first three-way valve (7') is controlled so that the fourth channel (21") is not connected to the first channel (14') and the second channel (15') through the first channel (14') and the second channel (15'). On the other hand, the second three-way valve (8') is controlled so that the first channel (14') is not connected to the third channel (16') and the fifth channel (22") through the third channel (16') and the fifth channel (22"). Liquid delivery mechanism The air sent from (6') is directed towards the first three-way valve (7') and the second three-way valve (8'). On the second three-way valve (8') side, the air cannot escape beyond that point. On the other hand, the end of the first three-way valve (7') side 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 three-way valve (8'), and the fifth flow path (22"), so the sample liquid (30') moves to the second heater (5') side due to the pressure of the air from the liquid delivery mechanism (6').

[0053] As shown in Figure 11, when stopping the movement of the sample solution (30') in step 3', the first three-way valve (7') is controlled so that the first channel (14') is not connected to the second channel (15') and the fourth channel (21'') through the second channel (15') and the fourth channel (21''), and the second three-way valve (8') is controlled so that the first channel (14') is not connected to the third channel (16') and the fifth channel (22'') through the third channel (16') and the fifth channel (22''). As a result, the residual pressure in the chip channel in the nucleic acid amplification chip (2') and in the second channel (15') and the third channel (16') in the nucleic acid amplification device (1') is released through the fourth channel (21'') and the fifth channel (22''), thereby suppressing the unwanted movement of the sample solution (30') due to residual pressure in the channels. Furthermore, by limiting the air supplied from the liquid supply mechanism (6') to a velocity of 100 mm / s or less (preferably 30-70 mm / s) in the intermediate flow path, and by 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 state, the pressure in the first flow path (14') will not become excessive, and damage and deterioration of the liquid supply mechanism (6') can be avoided. Preferably, the The first three-way valve (7') and the second three-way valve (8') are controlled within 1 second, more preferably within 0.5 seconds. Embodiments of the present invention that use three-way valves instead of four-way valves are preferable in terms of lower electricity consumption and miniaturization of the device.

[0054] As shown in Figure 12, in step 3', when moving the sample liquid (30') from the second heater (5') side to the first heater (4') side, the second three-way valve (8') is controlled so that the fifth channel (22") does not communicate with the first channel (14') and the third channel (16') while the sample liquid (30') is moved through the first channel (14') and the third channel (16'). On the other hand, the first three-way valve (7') is controlled so that the first channel (14') does not communicate with the second channel (15') and the fourth channel (21") while the sample liquid (30') is moved through the second channel (15') and the fourth channel (21"). The air sent from the liquid delivery mechanism (6') is directed towards the first three-way valve (7') and the second three-way valve (8'). On the side of the first three-way valve (7'), air cannot escape beyond that point. 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'), 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") in the nucleic acid amplification chip (2'). As a result, the sample liquid (30') moves to the first heater (4') side due to the air pressure from the liquid delivery mechanism (6').

[0055] Furthermore, in the present invention, the nucleic acid amplification device (1') may further include a fluorescence detector (not shown) capable of measuring the fluorescence intensity of the sample solution (30') passing through the intermediate channel.

[0056] Furthermore, in a preferred embodiment of the present invention, in step 3', an electrical signal from the fluorescence detector regarding the movement of the sample solution is sent to the control device (11'), and based on this electrical signal, the control device senses the passage of the sample solution (30') through the intermediate channel (25') and controls the driving of the first three-way valve (7'), the second three-way valve (8'), and the liquid delivery mechanism (6'). Alternatively, a fluorescence detector for sensing the passage of the sample solution (30') through the intermediate channel (25') may be installed separately from the fluorescence detector for measuring the nucleic acid amplification of the sample solution (30'). [Examples]

[0057] Real-time PCR is performed using the nucleic acid amplification device (1) and nucleic acid amplification chip (2) outlined in Figure 1. The measurement conditions are as follows: Shape of each channel in the nucleic acid amplification device (1): circular Diameter of each channel in the nucleic acid amplification device (1): 3 mm Shape of the chip channel of the nucleic acid amplification chip (2): Rectangular prism Width of the chip channel of the nucleic acid amplification chip (2): 0.5 mm Depth of the chip channel of nucleic acid amplification chip (2): 0.5 mm Sample solution: 15 μL Thermal cycling conditions for real-time PCR: Heat at 98°C for 30 seconds, then repeat 45 cycles of heating at 98°C for 2 seconds and 58°C for 4 seconds. By performing real-time PCR under the above conditions, it is possible to suppress the effect of residual pressure when stopping sample movement while also preventing damage to the pump due to excessive pressure in the first channel (14). [Examples]

[0058] Real-time PCR is performed using the nucleic acid amplification device (1') and nucleic acid amplification chip (2') outlined in Figure 8. The measurement conditions are as follows: Shape of each channel in the nucleic acid amplification device (1): circular Diameter of each channel in the nucleic acid amplification device (1): 3 mm Shape of the chip channel of the nucleic acid amplification chip (2): Rectangular prism Width of the chip channel of the nucleic acid amplification chip (2): 0.5 mm Depth of the chip channel of nucleic acid amplification chip (2): 0.5 mm Sample solution: 15 μL Set the flow velocity of the sample solution through the intermediate channel to 60 mm / s. To stop the sample liquid in the flow path, the pump is stopped within 0.4 seconds after controlling the first and second three-way solenoid valves. Thermal cycling conditions for real-time PCR: Heat at 98°C for 30 seconds, then repeat 45 cycles of heating at 98°C for 2 seconds and 58°C for 4 seconds. By performing real-time PCR under the above conditions, it is possible to suppress the effect of residual pressure when stopping sample movement while also preventing damage to the pump due to excessive pressure in the first channel (14'). [Explanation of Symbols]

[0059] 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, 13. Second connection, 14. First channel, 15. Second channel, 16. Third channel, 17. ...Fourth channel, 18...Fifth channel, 19...Sixth channel, 20...Seventh channel, 21...Eighth channel, 22...Ninth channel, 23...First curved channel, 24...Second curved channel, 25...Intermediate channel, 26, 27...Connection section, 28...First connecting channel, 29...Second connecting channel, 30...Sample liquid, 31...First filter, 32...Second filter, 33...Sample inlet 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, 13'... second connection, 14'... first channel, 15'... second channel, 16'... third channel, 21"... fourth channel, 22"... fifth channel, 23'... first curved channel, 24'... second curved channel, 25'... intermediate channel, 26', 27'... connection, 28'... first connecting channel, 29'... second connecting channel, 30'... sample liquid

Claims

[Claim 1] A substrate (3') on which a nucleic acid amplification chip (2') can be mounted, A first heater (4') capable of forming a denaturation temperature range, A second heater (5') capable of forming an extension / annealing temperature range, A liquid transfer mechanism (6') that enables the movement of the sample liquid (30') between the denaturation temperature range and the extension / annealing temperature range, First three-way valve (7') and second three-way valve (8'), A control device (11') that controls the drive of the first three-way valve (7'), the second three-way valve (8'), and the fluid delivery mechanism (6'), A first connection part (12') that can be connected to the nucleic acid amplification chip (2'), A second connection part (13') that can be connected to the nucleic acid amplification chip (2'), A first flow path (14') connects the liquid delivery mechanism (6') to the first three-way valve (7') and the 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') is connected at one end to the second three-way valve (8') and at the other end 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") is provided, 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 amplification device (1') characterized by performing real-time PCR by measuring the fluorescence intensity for each thermal cycle.

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