Chip for nucleic acid amplification, nucleic acid amplification device, and nucleic acid amplification method

Abstract

Provided is a chip for nucleic acid amplification, which does not perform measurement of solution movement by fluorescence detection, can more easily achieve high-speed movement of a solution, and can suppress the generation of bubbles. The chip for nucleic acid amplification has a flow path that has, between a first connection tube having a first opening and a second connection tube having a second opening: a first convex tube that is in a convex shape in a first direction and communicates with the first connection tube; a second convex pipe which is in a convex shape in the first direction and communicates with the second connecting pipe; and a tubular bridge portion in fluid communication with the first convex tube and the second convex tube.

Description

Technical Field

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

[0002] The detection of nucleic acids has become central in various fields such as pharmaceutical research and development, forensic medicine, clinical examinations, and the identification of crop or pathogenic microorganism species. The ability to detect various diseases including cancer, microbial infections, gene markers based on molecular system analysis, etc. has become a common technique for disease and disease risk diagnosis, marker search, safety evaluation in food or the environment, criminal evidence, and many other techniques.

[0003] The PCR method is a powerful technique for selectively amplifying a specific region of DNA. When using PCR, for a target DNA sequence in template DNA, millions of replicated DNA fragments can be generated from a single template DNA. PCR repeats three-phase or two-phase temperature conditions called thermal cycling, thereby sequentially repeating reactions such as denaturation of DNA into single strands, annealing of the denatured DNA single strands with primers, and extension of the primers using a thermostable DNA polymerase.

[0004] Thus, although the PCR method is a powerful method for amplifying genes exponentially by thermal cycling, there are the following problems: The general thermal cycler device used in PCR has a large heat capacity in the aluminum block part as a heater, so the temperature control is slow, and a PCR operation of 30 to 40 cycles takes time. Therefore, high-speed PCR operation is desired, and various methods have been developed to achieve high speed.

[0005] For example, for performing a PCR operation on a chip having a microchannel, it is considered that the heat capacity becomes smaller by reducing the sample amount, and high-speed thermal cycling can be achieved, so various studies have been conducted. However, in the case of using a microchannel, if the desired amount of solution is not accurately transferred to a specified temperature region provided with a heater or the like, it causes insufficient heating or conversely overheating.

[0006] The present applicant has proposed a nucleic acid amplification apparatus that includes a liquid feeding mechanism using a micro blower in a chip having a microchannel and can perform real-time PCR while detecting the movement of the solution by fluorescence detection (Patent Document 1).

[0007] Prior Art Documents

[0008] Patent Documents

[0009] Patent Document 1: Japanese Patent No. 6226284 Summary of the Invention

[0010] Problems to be Solved by the Invention

[0011] However, according to methods such as the Sanger method and other PCR methods, only DNA amplification is required, and a fluorescent probe for quantification is not needed. Therefore, the movement of the solution cannot be detected by fluorescence detection. If the movement of the solution is to be detected by fluorescence detection, unnecessary fluorescent dyes need to be added, or another detection method for confirming the speed of the solution is required.

[0012] In addition, in a microchannel, when bubbles are generated due to the entrainment of air when the fluid flows into the microchannel or the temperature during heating, a stagnant portion is generated in the microchannel, which hinders the movement of the solution.

[0013] In view of the above circumstances, an object of the present invention is to provide a nucleic acid amplification chip that does not measure the movement of a solution by fluorescence detection, can more easily achieve high-speed movement of the solution, and suppresses the generation of bubbles.

[0014] Solutions for Solving the Problems

[0015] To solve the above problems, the nucleic acid amplification chip of the present invention has a flow path that includes: a first convex tube that is convex in a first direction and communicates with the first connection tube; a second convex tube that is convex in the first direction and communicates with the second connection tube; and a tubular bridging portion that fluidly connects the first convex tube and the second convex tube, between a first connection tube having a first opening and a second connection tube having a second opening.

[0016] In addition, in other embodiments of the present invention, there is provided a nucleic acid amplification apparatus that includes the nucleic acid amplification chip according to an embodiment of the present invention, a first heater, a second heater, and a gas transfer device.

[0017] Furthermore, in other embodiments of the present invention, there is provided a nucleic acid amplification method that uses the nucleic acid amplification apparatus according to an embodiment of the present invention.

[0018] Advantages of the Invention

[0019] The present invention can provide a nucleic acid amplification chip that does not measure the movement of a solution by fluorescence detection, can more easily achieve high-speed movement of the solution, and suppresses the generation of bubbles. Brief Description of the Drawings

[0020] Figure 1 It is a schematic diagram of a nucleic acid amplification apparatus according to an embodiment of the present invention.

[0021] Figure 2This is a modified example of the nucleic acid amplification chip of the present invention.

[0022] Figure 3 This is a modified example of the nucleic acid amplification chip of the present invention.

[0023] Figure 4 This is a modified example of the nucleic acid amplification chip of the present invention.

[0024] Figure 5 This is a modified example of the nucleic acid amplification chip of the present invention.

[0025] Figure 6 This is a diagram showing nucleic acid amplification performed using the nucleic acid amplification device according to an embodiment of the present invention. Detailed Embodiment

[0026] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited thereto.

[0027] (Nucleic Acid Amplification Device)

[0028] Figure 1 This is a schematic diagram of a nucleic acid amplification device 1000 according to an embodiment of the present invention. As Figure 1 shown, the nucleic acid amplification device 1000 includes a nucleic acid amplification chip 100, a first heater 14, a second heater 24, and a gas transfer device (not shown). The nucleic acid amplification chip 100 includes a flow path 200, and the flow path 200 includes a first connection pipe 12 having a first opening 11, a first convex pipe (also simply referred to as "first convex pipe") 13 convex in the first direction, a second connection pipe 22 having a second opening 21, a second convex pipe (also simply referred to as "second convex pipe") 23 convex in the first direction, and a bridging portion 30. In the nucleic acid amplification device 1000, the first direction of the nucleic acid amplification chip 100 coincides with the direction of gravity. Hereinafter, with reference to Figure 1 the nucleic acid amplification device 1000, each structure will be described in detail.

[0029] (Nucleic Acid Amplification Chip)

[0030] The nucleic acid amplification chip 100 is a chip that holds a PCR solution containing a gene to be amplified in the flow path 200 and amplifies the gene exponentially by repeating thermal cycles.

[0031] The nucleic acid amplification chip 100 includes a flow path 200, and the flow path 200 includes a first connection pipe 12 having a first opening 11, a first convex pipe 13 convex in the first direction, a second connection pipe 22 having a second opening 21, a second convex pipe 23 convex in the first direction, and a bridging portion 30.

[0032] The first connecting pipe 12 has a first opening 11, which communicates with one end of the first convex pipe 13.

[0033] The first opening 11 is provided on the first connecting pipe 12, opening the flow path 200 to the outside of the nucleic acid amplification chip 100. Liquids such as gases and PCR solutions can be injected into the flow path 200 from the first opening 11 using a gas transfer device, dispenser, etc. described later. Conversely, gases and liquids in the flow path 200 can be discharged from the first opening 11.

[0034] The connection angle R1 between the first connecting pipe 12 and the first convex pipe 13 is preferably 90° or more. In addition, the first connecting pipe 12 is preferably extended parallel along the first direction. With such a structure, when the nucleic acid amplification chip 100 is set in the nucleic acid amplification device 1000 with the first direction as the gravity direction, the liquid injected into the first connecting pipe 12 flows into the first convex pipe 13 along the gravity direction. In addition, when there is gas in the flow path 200, the bubbles reach the first opening 11 through the first connecting pipe 12 and are discharged.

[0035] The first convex pipe 13, which is convex in the first direction, is a part that holds the PCR solution, is heated to the first temperature range by the first heater 14 described later, and performs the first PCR reaction.

[0036] The first convex pipe 13 is formed with a convex part (convex portion) along the first direction, and the PCR solution injected from the first connecting pipe 12 can be retained in the convex portion. The convex portion can be, for example, U-shaped or V-shaped.

[0037] Since the PCR solution is retained in the first convex pipe 13 and heated to react, the size of the first convex pipe 13 is preferably such that it can hold the solution required for the desired PCR reaction.

[0038] The second opening 21, the second connecting pipe 22, and the second convex pipe 23 are the same as the first opening 11, the first connecting pipe 12, and the first convex pipe 13 respectively, except that the temperature of the PCR reaction performed in the second convex pipe 23 is different from the first temperature range. The second opening 21, the second connecting pipe 22, and the second convex pipe 23 can be in a shape symmetrical to the first opening 11, the first connecting pipe 12, and the first convex pipe 13, or can be in different shapes respectively. As described later, since the PCR solution is transferred alternately between the first convex pipe 13 and the second convex pipe 23, when the first direction is the gravity direction, it is preferable that the heights of the first convex pipe 13 and the second convex pipe 23 in the gravity direction are the same.

[0039] The bridging portion 30 is tubular, enabling fluid communication between the first convex tube 13 and the second convex tube 23. The bridging portion 30 is configured to extend away from the first convex tube and the second convex tube in a direction opposite to the first direction. When the nucleic acid amplification chip 100 is arranged with the first direction as the gravitational direction, the bridging portion 30 sandwiched between the adjacent first convex tube 13 and second convex tube 23 is configured to have a height in the gravitational direction higher than that of these two convex tubes. Therefore, when the PCR solution is transferred from one convex tube and crosses the bridging portion 30, it can flow into the other convex tube along the gravity.

[0040] The bridging portion 30 may be in the shape of a bridge extending in the vertical direction along the first direction ( Figure 2 and Figure 3 ), and may also be in a convex shape in the direction opposite to the first direction ( Figure 1 and Figure 4 ). For example, it may be in the shape of a mountain convex in the direction opposite to the first direction ( Figure 1 ), and may also have a semi-circular shape convex in the direction opposite to the first direction ( Figure 4 ). When the bridging portion 30 is in a convex shape in the direction opposite to the first direction, if the solution is transferred to the middle point (the inflection point of the convex portion) of the bridging portion 30, it will automatically move to the other convex tube, so it is preferred.

[0041] In addition, as long as it has a shape with a bridging portion between adjacent convex tubes, more than three convex tubes may also be provided between the first connecting tube 12 and the second connecting tube 22. In Figure 5 , a first convex tube 13, a bridging portion 30, a second convex tube 23, a second bridging portion 40, and a third convex tube 63 are sequentially arranged between the first connecting tube 12 and the second connecting tube 22. Adjacent convex tubes are respectively heated to different temperature ranges and can respectively perform PCR reactions. Non-adjacent convex tubes, for example, the first convex tube 13 and the third convex tube 63, may be heated to different temperature ranges, or may also be heated to the same temperature range.

[0042] The connection angles between the bridging portion 30 and the first convex tube 13 and the second convex tube 23, and between the second bridging portion 40 and the second convex tube 23 and the third convex tube 63 are preferably 90° or more. By adopting such an angle, air bubbles will not remain in the connection part, and air bubbles can be removed from the solution.

[0043] The flow path 200 of the nucleic acid amplification chip 100 is preferably formed in a plane parallel to the first direction. Thus, when the first direction is the gravitational direction, the solution in the flow path 200 can move along the gravity.

[0044] The shape of the inner cavity cross-section perpendicular to the central axis of the flow path 200 of the nucleic acid amplification chip 100 (hereinafter, simply referred to as "cross-sectional shape") can also be a circular or elliptical cylindrical shape, and this cross-sectional shape can also be a polygonal tube. In addition, the cross-sectional shape of the flow path 200 can also be a shape combining a curved portion and a polygonal portion in this cross-sectional shape. The cross-sectional shape of the flow path 200 is preferably consistent within the flow path.

[0045] The flow path 200 is preferably a micro flow path. The maximum value of the length connecting two points on the outer periphery of the cross-sectional shape of the flow path 200 is preferably 0.5 mm or more. For example, when the cross-sectional shape is circular, its diameter is preferably 0.5 mm or more. In addition, when the cross-sectional shape is elliptical, its major axis radius is preferably 0.25 mm or more. In the case where the cross-sectional shape is polygonal, the maximum value of the diagonal length is preferably 0.5 mm or more. By having such a size, the gravity effect is greater than the frictional force between the solution and the inner wall of the flow path, and the solution can move to the desired position according to gravity. The moving speed in the gravity direction within the flow path 200 is preferably 5 mm / s or more.

[0046] In addition, it is preferably a micro flow path size capable of maintaining the solution in a plunger shape within the flow path 200. The maximum value of the length connecting two points on the outer periphery of the cross-sectional shape of the flow path 200 is preferably 1 mm or less. For example, when the cross-sectional shape is circular, its diameter is preferably 1 mm or less. In addition, when the cross-sectional shape is elliptical, its major axis radius is preferably 0.5 mm or less. In the case where the cross-sectional shape is polygonal, the maximum value of the diagonal length is preferably 1 mm or less.

[0047] The material used for the nucleic acid amplification chip 100 is preferably a material with relatively high thermal conductivity, stable within the temperature range required for PCR, difficult to be eroded by electrolyte solutions and organic solvents, and does not adsorb nucleic acids and proteins. For example, as the material, glass, quartz, silicon, resin, and metal can be used. Resins such as polymethyl methacrylate (PMMA) and cycloolefin polymer (COP) are preferred.

[0048] The nucleic acid amplification chip 100 can be formed by injection molding or MEMS processing.

[0049] (Heater)

[0050] The heater heats the solution in the convex tube to a specific temperature range. Figure 1 The nucleic acid amplification device 1000 is equipped with a first heater 14 and a second heater 24.

[0051] The first heater 14 is a heater that heats the solution in the first convex tube 13 to the first temperature range. To heat the first convex tube 13, the first heater 14 is arranged to cover the back surface of the first convex tube 13 of the nucleic acid amplification chip 100.

[0052] The second heater 24 is a heater that heats the solution in the second convex tube 23 to a second temperature range different from the first temperature. To heat the second convex tube 23, the second heater 24 is arranged to cover the back surface of the second convex tube 23 of the nucleic acid amplification chip 100.

[0053] The first temperature and the second temperature are determined according to the thermal cycle (denaturation temperature range and extension / annealing temperature range) of the PCR reaction. In the case of having three or more heaters as in Figure 5 adjacent heaters are set to different temperature ranges. Non - adjacent heaters, for example, the first heater 14 and the third heater 64, can be heated to different temperature ranges, or can also be heated to the same temperature range.

[0054] (Gas transfer device)

[0055] The nucleic acid amplification device 1000 includes at least one gas transfer device. The gas transfer device is connected to at least one of the first opening 11 and the second opening 21, and injects gas into the flow path 200 or discharges gas from the flow path 200. By injecting or discharging gas by the gas transfer device, the PCR solution in the flow path 200 can be moved.

[0056] The gas transfer device can be any device that can inject or discharge a desired amount of gas into or from the flow path 200, but when at rest, the pressure in the flow path 200 is released. For example, a micro - dispenser or a micro - blower can be used as the gas transfer device. For example, as the micro - dispenser, the micro - dosing unit Type - 7615 manufactured by Burkert can be used, and as the micro - blower, the micro - blower MZB100T02 of Murata Manufacturing Co., Ltd. can be used.

[0057] The gas transfer device can be connected to either the first opening 11 or the second opening 21, or can also be connected to both. In the case of being connected to both openings, the gas transfer device operates alternately in cooperation with the liquid feeding direction. Even when connected to both openings, when the gas transfer device is at rest, the pressure in the flow path 200 is released.

[0058] In the case where the gas transfer device is connected to only one of the openings, the gas transfer device can cause the solution to move by flowing gas into the flow path 200, and can also cause the solution to move by sucking the gas in the flow path 200. In either case, the pressure is released when at rest, and the solution moves in the flow path 200 along gravity.

[0059] (Nucleic acid amplification method)

[0060] Next, a nucleic acid amplification method using the nucleic acid amplification device 1000 will be described. Hereinafter, an embodiment in which a gas transfer device is provided at the first opening 11 will be described, but the present invention is not limited thereto.

[0061] (1) The nucleic acid amplification device 1000 is set such that the first direction of the nucleic acid amplification chip 100 is the direction of gravity.

[0062] (2) Using a micropipette or the like, 5 to 25 μL of the PCR solution is measured and injected into the flow path 200 from the first opening 11. The PCR solution flows into the first convex tube 13 via the first connection tube 12 and is held ( Figure 6 in (a)). The PCR solution only needs to be able to amplify the gene sequence, and may not have a fluorescent dye. Alternatively, it may have a fluorescent dye. As an example of the PCR reagent, TaKaRa Bio Inc.'s SpeedSTAR (registered trademark) HS DNA polymerase with a final concentration of 0.025 U / μL can be used, and the attached FAST Buffer I and dNTP Mixture are mixed according to the concentrations specified in the manual, and further 0.5 μM of primers are mixed, and it can be used as a PCR premix. It should be noted that the composition of the PCR reagent is not limited thereto, as long as it is a reagent composition for PCR.

[0063] (3) The PCR solution in the first convex tube 13 is heated to the denaturation temperature (about 95 °C) by the first heater 14 to dissociate double-stranded DNA into single-stranded DNA.

[0064] (4) After the reaction is completed, gas is injected through the gas transfer device, and the solution is transferred so that the PCR solution crosses the bridging portion 30, and the gas transfer device is stopped. After the pressure in the flow path 200 of the solution that has crossed the bridging portion 30 is released, it moves into the second convex tube 23 under the action of gravity and is held ( Figure 6 in (b)).

[0065] (5) The PCR solution in the second convex tube 23 is heated to the extension / annealing temperature (50 to 70 °C) by the second heater 24 to bind the primers, and the region sandwiched by the two primer sequences is replicated from the single-stranded template DNA into double-stranded DNA.

[0066] (6) The gas in the flow path 200 is sucked through the gas transfer device, and the solution is transferred so that the PCR solution crosses the bridging portion 30 from the second convex tube 23, and the gas transfer device is stopped. Similar to the forward path, after the pressure in the flow path 200 of the solution that has crossed the bridging portion 30 is released, it moves into the first convex tube 13 under the action of gravity and is held.

[0067] Repeat operations (3) to (6) to change the temperature, whereby the DNA of the target sequence can be amplified in the form of an exponential function.

[0068] In the present embodiment, as described above, when the PCR solution crosses the bridging portion 30, the solution can flow into and be held in the convex tube at the transfer destination along the gravity, so that strict adjustment of solution delivery is not required. According to the present embodiment, by repeatedly injecting a gas in an amount that allows the solution to cross the bridging portion 30 into the flow path 200 or sucking a gas in an amount that allows the solution to cross the bridging portion 30, the solution can be transferred to a desired position and thermal cycling can be performed.

[0069] In addition, in the present embodiment, since the first direction of the nucleic acid amplification chip 100 is set as the gravity direction, the generated bubbles move in a direction opposite to the gravity direction. As a result, the bubbles can move toward the first opening 11, the second opening 21, or the bridging portion 30 and be removed from the flow path 200. By removing the bubbles, accidental errors in thermal cycling can be avoided.

[0070] In the above, various embodiments and modification examples have been described, but the present invention is not limited to these. Other solutions considered within the technical idea of the present invention are also included in the scope of the present invention.

[0071] In addition, one or more of the above-described embodiments and modification examples can be appropriately combined.

[0072] Explanation of reference numerals

[0073] 11 First opening

[0074] 12 First connecting tube

[0075] 13 First convex tube

[0076] 14 First heater

[0077] 21 Second opening

[0078] 22 Second connecting tube

[0079] 23 Second convex tube

[0080] 24 Second heater

[0081] 30 Bridging portion

[0082] 100 Nucleic acid amplification chip

[0083] 200 Flow path

Claims

1. A chip for nucleic acid amplification, wherein, the chip for nucleic acid amplification has a flow path, the flow path is between a first connecting pipe having a first opening and a second connecting pipe having a second opening, and has: a first convex pipe that communicates with the first connecting pipe and is convex in a first direction; a second convex pipe that communicates with the second connecting pipe and is convex in the first direction; and a tubular bridging portion that fluidly connects the first convex pipe and the second convex pipe.

2. The chip for nucleic acid amplification according to claim 1, wherein, the bridging portion is arranged to be away from the first convex pipe and the second convex pipe in a direction opposite to the first direction.

3. The chip for nucleic acid amplification according to claim 1, wherein, the inner cavity cross-sections of the first connecting pipe, the first convex pipe, the second connecting pipe, the second convex pipe and the bridging portion perpendicular to the central axis of the flow path are of the same shape.

4. The chip for nucleic acid amplification according to claim 1, wherein, the connection angle of a group of the communicating pipes is 90° or more.

5. The chip for nucleic acid amplification according to claim 3, wherein, the maximum value of the length connecting two points on the outer periphery of the inner cavity cross-section of the flow path is 0.5 mm or more.

6. The chip for nucleic acid amplification according to claim 5, wherein, the maximum value of the flow path is 1 mm or less.

7. The chip for nucleic acid amplification according to claim 1, wherein, the bridging portion is convex in a direction opposite to the first direction.

8. The chip for nucleic acid amplification according to claim 1, which is provided in a nucleic acid amplification device, wherein, the first direction is the gravity direction, and the moving speed in the first direction in the flow path is 5 mm / s or more.

9. The chip for nucleic acid amplification according to claim 1, wherein, the chip for nucleic acid amplification further includes a third convex pipe and a second bridging portion.

10. A nucleic acid amplification device, wherein, the nucleic acid amplification device includes: the chip for nucleic acid amplification according to any one of claims 1 to 9; a first heater; a second heater; and a gas transfer device, the first heater heats the first convex pipe to a first temperature, the second heater heats the second convex pipe to a second temperature different from the first temperature, the gas transfer device transfers the gas in the flow path.

11. The nucleic acid amplification device according to claim 10, wherein, the gas transfer device is a micro dispenser or a micro blower.

12. The nucleic acid amplification device according to claim 10, wherein, in the chip for nucleic acid amplification, the first direction is the gravity direction.

13. A nucleic acid amplification device, wherein, the nucleic acid amplification device includes: the chip for nucleic acid amplification according to claim 9; a first heater; a second heater; a third heater; and a gas transfer device, the first heater heats the first convex pipe to a first temperature, the second heater heats the second convex pipe to a second temperature different from the first temperature, the third heater heats the third convex pipe to a third temperature, The gas transfer device transfers the gas in the flow path.

14. A nucleic acid amplification method, wherein, the nucleic acid amplification method includes the following steps: arranging the nucleic acid amplification chip in the nucleic acid amplification device according to claim 10 with the first direction as the gravity direction; injecting a nucleic acid amplification solution into the first convex tube from the first opening; heating the nucleic acid amplification solution in the first convex tube to a first temperature by the first heater; transferring the gas by the gas transfer device to transfer the nucleic acid amplification solution to a position where the nucleic acid amplification solution crosses the bridging portion, and causing the nucleic acid amplification solution to flow into the second convex tube; and heating the nucleic acid amplification solution in the second convex tube to a second temperature by the second heater.

15. The nucleic acid amplification method according to claim 14, wherein, the nucleic acid amplification method further includes the following steps: transferring the gas by the gas transfer device to transfer the nucleic acid amplification solution to a position where the nucleic acid amplification solution crosses the bridging portion, and causing the nucleic acid amplification solution to flow into the first convex tube.

Images