RU2773528C2 - Reaction processing device - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: reaction device 100 includes: reaction vessel 10 including channel 12, first inlet air communication hole 24 and second inlet air communication hole 26; temperature control system 102 that provides low temperature area 38 and high temperature area 36 in channel 12; and liquid supply system 120 that moves sample 50 inside channel 12. Liquid supply system 120 includes: pump 121 having outlet 121a; first air duct 129 that connects outlet 121a and first inlet air communication hole 24; second air duct 130 that connects outlet 121a and second inlet air communication hole 26; first three-way valve 123 capable of switching between a state, in which first inlet air communication hole 24 communicates with outlet 121a, and a state, in which first inlet air communication hole 24 is open to atmospheric pressure; second three-way valve 124 capable of switching between a state, in which second inlet air communication hole 26 communicates with outlet 121a, and a state, in which second inlet air communication hole 26 is open to atmospheric pressure; as well as central processing unit (CPU) 105, which controls these components.

EFFECT: obtaining a reaction processing device.

8 cl, 1 tbl, 1 ex, 14 dwg

Description

The field of technology to which the invention relates

The present invention relates to reaction processing devices used for polymerase chain reactions (PCR, PCR).

State of the art

Genetic testing is widely used for research in various fields of medicine, identification of agricultural products and pathogenic microorganisms, food safety assessment, and even for research on pathogenic viruses and various infectious diseases. In order to detect with high sensitivity a small amount of DNA, methods are known for analyzing a result obtained by amplifying a portion of DNA. First of all, the method that PCR uses is an exceptional technology in which a certain portion of a very small amount of DNA collected from an organism or a similar source is selectively amplified.

In PCR, a given thermal cycle is applied to a sample in which a biological sample containing DNA and a PCR reagent consisting of primers, enzymes, and the like are mixed in such a way as to cause denaturation, annealing, and elongation reactions to be repeated, resulting in a certain part of the DNA is selectively amplified.

It is common practice to perform PCR by placing a predetermined amount of the target sample into a PCR tube or reaction vessel such as a microplate (microwell) in which a plurality of holes are formed. However, in recent years, PCR has been practiced using a reaction vessel (also referred to as a "chip") provided with a microchannel which is formed on a substrate (for example, Patent Document 1).

[Patent Document 1] Japanese Patent Application Publication No. 2009-232700

Disclosure of invention

[Problem solved by the invention]

When performing PCR using a reaction vessel provided with a channel, a thermal cycle is applied to a sample by setting temperature regions such as a high temperature region and a low temperature region in the channel, and moving the sample by reciprocating within the channel.

As a method of reciprocating movement of the sample inside the channel of the reaction vessel, a method using two liquid supply mechanisms can be considered. Liquid supply mechanisms are, for example, pumps or the like. A pump is connected to each end of the channel and the sample is moved by adjusting the pressure within the channel.

However, there may be individual differences in the characteristics of these pumps. In the method using two or more pumps, there is a problem that it is difficult to accurately control the pressure inside the duct when there are individual differences in the characteristics of these pumps.

Against this background, it is an object of the present invention to provide a reaction device that can easily control the movement of a sample within the channel of a reaction vessel.

Remedy for the problem

A reaction apparatus according to one embodiment of the present invention includes: a reaction vessel including a passage in which a sample travels, and a pair consisting of a first air communication inlet and a second air communication inlet, which are provided at the respective ends of the channel; a temperature control unit that provides a first temperature region maintained at a first temperature and a second temperature region maintained at a second temperature higher than the first temperature between the first air communication inlet and the second air communication inlet in the duct; and a fluid delivery system that moves and stops the sample in the channel. The fluid supply system includes: a pump capable of releasing air from an outlet; a first air passage that connects an outlet of the pump and a first inlet of an air communication port of the reaction vessel; a second air passage that connects the pump outlet and the second air communication inlet of the reaction vessel; the first switching valve, which is located in the first air passage and can switch between the state in which the first air communication hole is connected to the outlet, and the state in which the first air communication hole is open to atmospheric pressure; a second switching valve that is located in the second air passage and is capable of switching between a state in which the second air communication port communicates with the outlet port and a state in which the second air communication port is open to atmospheric pressure; and a control unit that controls the operation of the pump, the operation of the first switching valve, and the operation of the second switching valve.

The first temperature region may be located on the side of the first air communication inlet, and the second temperature region may be located on the side of the second air communication inlet. The control unit may be configured to: bleed air from the pump and switch the first switching valve so that it is in a state in which the first air communication inlet port communicates with the outlet, and the second switch valve switches so that it is in a state in which the second air inlet port is open to atmospheric pressure when the sample moves from the first temperature region to the second temperature region; and releasing air from the pump and switching the first switching valve to a state in which the first air communication inlet port is open to atmospheric pressure, and the second switching valve is switched so that it is in a state in which the second air communication inlet port communicates with the outlet opening when the sample moves from the second temperature region to the first temperature region.

The control unit may stop venting the pump when the sample stops inside the channel.

The pressure on the primary side and the pressure on the secondary side can become equal in the pump when the pump is stopped. When the sample stops inside the channel, the control unit may switch the first switching valve to a state in which the first air communication inlet is open to atmospheric pressure, and the second switching valve switches to a state in which the second air communication inlet is open to atmospheric pressure.

The first switching valve and the second switching valve may be three-way valves.

Another embodiment of the present invention also relates to a reaction device. This apparatus includes: a reaction vessel including a passage in which a sample is moved and a pair of a first air communication inlet and a second air communication inlet provided at respective ends of the channel; a temperature control unit that provides a first temperature region maintained at a first temperature and a second temperature region maintained at a second temperature higher than the first temperature between the first air communication inlet and the second air communication inlet in the duct; and a fluid delivery system that moves and stops the sample in the channel. The liquid supply system includes: a pressure chamber, the internal pressure of which is maintained at a level higher than atmospheric pressure in the environment of the reaction apparatus; a pump, which is located inside the pressure chamber and is able to release air from the outlet; a first air passage that connects an outlet of the pump and a first inlet of an air communication port of the reaction vessel; a second air passage that connects the pump outlet and the second air communication inlet of the reaction vessel; a first switching valve that is located in the first air passage and is capable of switching between a state in which the first air communication inlet is connected to the outlet and a state in which the first air communication inlet is opened to the inside of the plenum chamber; a second switching valve that is located in the second air passage and is capable of switching between a state in which the second air communication inlet is connected to the outlet and a state in which the second air communication inlet is opened to the inside of the plenum chamber; and a control unit that controls the operation of the pump, the operation of the first switching valve, and the operation of the second switching valve.

The first temperature region may be located on the side of the first air communication inlet, and the second temperature region may be located on the side of the second air communication inlet. The control unit may be configured to: bleed air from the pump and switch the first switching valve so that it is in a state in which the first inlet air communication hole communicates with the outlet, and the second switching valve should be in a state in which the second the inlet air communication opening opens into the interior of the plenum chamber when the sample moves from the first temperature region to the second temperature region; and also venting the pump and making the first changeover valve into a state in which the first air communication inlet is opened to the inside of the plenum chamber, and the second changeover valve is put into a state in which the second air communication inlet is in communication with the outlet when the sample moves from the second temperature region to the first temperature region.

The control unit can stop the release of air from the pump when the sample stops inside the channel.

The pressure on the primary side and the pressure on the secondary side can become equal in the pump when the pump is stopped. When the sample is stopped inside the channel, the control unit may switch the first switching valve to a state in which the first air communication inlet is open to the inside of the pressure chamber, and the second switching valve is in a state in which the second air communication inlet is open to the inside of the chamber. increased pressure.

The first switching valve and the second switching valve may be three-way valves.

Positive Effects

According to the present invention, a reaction apparatus can be provided that can easily control the movement of a sample within a channel of a reaction vessel.

Brief description of the drawings

Fig. 1A and 1B are block diagrams explaining the operation of a reaction vessel used in a reaction apparatus according to an embodiment of the present invention;

fig. 2 shows a cross-sectional view of the reaction vessel shown in FIG. 1A, which is cut along the line A-A;

fig. 3 shows a plan view of the support provided in the reaction vessel;

fig. 4 is a flowchart schematically showing a state in which a sample is introduced into a reaction vessel;

fig. 5 is a schematic block diagram showing the operation of a reaction apparatus according to an embodiment of the present invention;

fig. 6 shows a table for explaining the control method of the pump, the first three-way valve, and the second three-way valve in the reaction apparatus shown in FIG. 5;

fig. 7 shows a table for explaining another method for controlling the pump, the first three-way valve, and the second three-way valve in the reaction apparatus shown in FIG. 5;

fig. 8 shows a graph showing the result of PCR amplification by a reaction apparatus according to an embodiment of the present invention;

fig. 9 shows a flowchart explaining the operation of a reaction apparatus according to another embodiment of the present invention;

fig. 10 shows a block diagram explaining the operation of a reaction apparatus according to another embodiment of the present invention;

fig. 11 shows a table explaining another method for controlling the pump, the first three-way valve, and the second three-way valve in the reaction apparatus shown in FIG. ten;

fig. 12 shows a flowchart explaining the operation of a reaction apparatus according to another embodiment of the present invention;

fig. 13 shows a table explaining the control method of the pump, the first three-way valve, the second three-way valve, and the electromagnetic valve in the reaction apparatus shown in FIG. 12; and

fig. 14 shows a table explaining another method for controlling the pump, the first three-way valve, the second three-way valve, and the electromagnetic valve in the reaction apparatus shown in FIG. 12.

Method for carrying out the invention

Next, an explanation will be given regarding a reaction apparatus according to an embodiment of the present invention. The same or equivalent constituent elements, components, and processes illustrated in each drawing are identified by the same reference numerals, and duplicate explanations will be accordingly omitted. Furthermore, the embodiments do not limit the invention and are shown for illustrative purposes, and not all features and combinations thereof described in the embodiments are necessarily essential to the invention.

Fig. 1A and 1B are block diagrams explaining the operation of a reaction vessel 10 used in a reaction apparatus according to an embodiment of the present invention. In FIG. 1A is a plan view of the reaction vessel 10, and FIG. 1B shows a front view of a reaction vessel 10. In FIG. 2 is a sectional view of the reaction vessel 10 shown in FIG. 1A, section along line A-A. Fig. 3 is a plan view of the support 14 provided in the reaction vessel 10.

The reaction vessel 10 comprises a resin-containing substrate 14 having a groove-like channel 12 formed on its bottom surface 14a, a channel sealing film 16 which is attached to the bottom surface 14a of the channel sealing substrate 14, and two sealing films (a first sealing film). film 18 and the second sealing film 20) attached to the upper surface 14b of the substrate 14.

The substrate 14 is preferably made of a material that is stable to changes in temperature and resistant to the sample solution used. In addition, the substrate 14 is preferably made of a material that has good formability, good transparency and barrier properties, and low self-fluorescent properties. As such a material, an inorganic material such as glass, silicon (Si) or the like, a resin such as acrylic, polyester, silicone or the like, and especially a cycloolefin resin (CR) are preferable. An example of substrate 14 dimensions includes a long side of 76 mm, a short side of 26 mm, and a thickness of 4 mm.

A channel 12 in the form of a groove is formed on the bottom surface 14a of the substrate 14. In the reaction vessel 10, the majority of the channel 12 is formed in the form of a groove open on the bottom surface 14a of the substrate. 14. This makes it easy to perform injection molding using a metal mold or the like. In order to seal this groove for using the groove as a channel, a sealing film 16 for sealing the channel is attached to the bottom surface 14a of the substrate 14. An example of channel dimensions 12 includes a width of 0.7 mm and a depth of 0.7 mm.

The channel sealing film 16 may be adhesive on one of its main surfaces, or may have a functional layer that exhibits adhesiveness or adhesiveness when pressed, irradiated with ultraviolet rays or the like, when heated, etc., formed on one of main surfaces. Thus, the channel sealing film 16 has the function of easily merging with the bottom surface 14a of the substrate 14 while being in intimate contact with the bottom surface 14a. The channel sealing film 16 is desirably formed from a material including an adhesive that has poor self-fluorescent properties. In this respect, a transparent film made from a resin such as a cycloolefin polymer, polyester, polypropylene, polyethylene, or acrylic is suitable, but not limited thereto. In addition, the channel sealing film 16 may be formed from plate glass or resin. Since stiffness can be expected in this case, the channel sealing film 16 is useful in preventing warping and deformation of the reaction vessel 10 .

The first air communication inlet 24 is formed at the location of one end 12a of the channel 12 in the substrate 14. The second air communication inlet 26 is formed at the location of the other end 12b of the channel 12 in the substrate 14. A pair of holes, the first air communication hole 24 and the second air communication hole 26 , are formed to be exposed on the upper surface 14b of the substrate 14.

A first filter 28 is provided between the first air communication inlet 24 and one end 12a of the channel 12 in the substrate 14. A second filter 30 is provided between the second air communication inlet and the other end 12b of the channel 12 in the substrate 14. A pair of filters, the first filter 28 and the second filter The 30s provided at the respective ends of the channel 12 have good impurity removal characteristics and also allow only air to pass through to prevent contamination, so that target DNA amplification and target DNA detection are not interrupted. As a filter material, for example, a material obtained by treating polyethylene with a water-repellent impregnation can be used. Alternatively, a known material may be selected, provided that the material performs the above function. With regard to the dimensions of the first filter 28 and the second filter 30, the first filter 28 and the second filter 30 are formed to fit without any gaps in the filter installation space formed in the substrate 14, and may have a diameter of 4 mm, for example. and 2 mm thick.

As shown in FIG. 1A, between the pair of the first air communication inlet 24 and the second air communication inlet 26, the channel 12 includes a thermal cycle region 32 for applying a thermal cycle to a sample and a dispensing region 34 for performing so-called dispensing when a predetermined sample quantity. The thermal cycle area 32 is located on the side of the second air communication hole 26 in the channel 12. The metering area 34 is located on the side of the first air communication hole 24 in the channel 12. The thermal cycle area 32 and the metering area 34 communicate with each other. By moving the sample dispensed in the dispensing region 34 to the thermal cycle region 32, whereby the sample continuously reciprocates between the reaction regions maintained at a predetermined temperature, which are included in the thermal cycle region 32, the thermal cycle can be applied in regarding the sample.

When the reaction vessel 10 is installed on the reaction apparatus described hereinafter, the thermal cycle region 32 of the channel 12 includes a reaction region (hereinafter referred to as "low temperature region 38") maintained at a relatively low temperature (about 60°C) , a reaction region (hereinafter referred to as "high temperature region 36") maintained at a higher temperature (about 95°C), and a junction region 40 connecting the high temperature region 36 and the low temperature region 38. The low temperature region 38 is located on the side of the first air inlet communication hole 24 (in other words, on the side of the metering area 34), and the high temperature area 36 is located on the side of the second air inlet 26.

The high temperature region 36 and the low temperature region 38 include a serpentine-shaped channel in which rotation is continuously performed by combining curved portions and straight portions. In the case where the serpentine-shaped passage as described above is used, the effective area which is limited so that the area of the heater or the like constituting the temperature control means described below can be effectively used, and there are advantages in that the change in temperature in the reaction area is easily reduced, and that the actual size of the reaction vessel can be reduced to help reduce the size of the reaction apparatus. Connection area 40 may be a line channel.

The dosing area 34 of the channel 12 is located between the low temperature area 38 in the thermal cycle area 32 and the first filter 28. As described above, the dosing area 34 performs the function of dosing a predetermined amount of the sample to be subjected to PCR. The dosing area 34 includes a dosing channel 42 for determining a predetermined amount of sample, two branching channels (first branching channel 43 and a second branching channel 44) branching off from the dosing channel 42, a first sample inlet 45 located at the end of the first channel 43 ramifications and a second sample inlet 46 located at the end of the second ramification channel 44. The first inlet 45 for introducing the sample communicates with the dosing channel 42 through the first channel 43 branching. The second inlet 46 for introducing the sample communicates with the dosing channel 42 through the second channel 44 branching. The dosing channel 42 is a serpentine-shaped channel for dosing a predetermined amount of sample using a minimum area. The first sample inlet 45 and the second sample inlet 46 are formed to be open on the upper surface 14b of the substrate 14. The first sample inlet 45 is formed to have a relatively small diameter, and the second inlet The sample entry 46 is shaped to have a relatively large diameter. When the branching point at which the first branching passage 43 branches off from the dispensing passage 42 is determined as the first branching point 431, and the branching point at which the second branching passage 44 exits from the dispensing passage 42 is determined as the second branching point 441, the volume of the sample to be PCR is practically determined by the volume inside the dosing channel 42 between the first branching point 431 and the second branching point 441.

In the reaction vessel 10, a distribution region 34 is provided between the thermal cycle region 32 and the first filter 28. However, the location of the distribution region 34 is not limited to this, and the distribution region 34 may be provided between the thermal cycle region 32 and the second filter 30. As long as the dosing may be performed accurately using a pipette or similar device, the channels may be formed without providing the dispensing area 34 or formed in such a way that the sample can be introduced directly into the thermal cycle area 32 or the like.

The first air communication inlet 24, the second air communication inlet 26, the first filter 28, the second filter 30, the first sample inlet 45 and the second sample inlet 46 are open on the top surface 14b of the substrate 14. Therefore, to seal the first of the air communication inlet 24, the second air communication inlet 26, the first filter 28 and the second filter 30, the first sealing film 18 is attached to the upper surface 14b of the substrate 14. To seal the first sample inlet 45 and the second sample inlet 46, the second sealing film 20 is attached to the upper surface 14b of the substrate 14. In the state where the first sealing film 18 and the second sealing film 20 are attached to the surfaces, the entire channel forms a closed space.

The first sealing film 18 used is of a size that allows the first air communication inlet 24, the second air communication inlet 26, the first filter 28 and the second filter 30 to be sealed at the same time. The fluid supply system (described below) is connected to the first air communication inlet 24 and the second air communication inlet 26 by perforating the respective portions of the first sealing film 18, which correspond to the first air communication inlet 24 and the second air communication inlet 26, through a hollow needle. (with a needle of a syringe with a sharp tip) provided in the fluid supply system. Therefore, the first sealing film 18 is preferably a film made of a material that is easily perforated by a needle and/or has a thickness that is easily perforated by a needle. In the reaction vessel 10, a sealing film having a size capable of sealing the first air communication inlet 24, the second air communication inlet 26, the first filter 28 and the second filter 30 at the same time is described. However, air inlets and filters can be sealed separately. In addition, the film sealing the first air communication inlet 24 and the second air communication inlet 26 can be peeled off so as to be connected to the liquid supply system.

As the second sealing film 20, a sealing film having a size capable of sealing the first sample inlet 45 and the second sample inlet 46 is used. Introduction of the sample into the channel 12 through the first sample port 45 and the second sample port 46 is performed by removing the second sealing film 20 from the substrate 14 once, and after introducing a predetermined amount of sample, the second sealing film 20 is again applied and attached to the top surface 14b of the substrate. 14. Therefore, as the second sealing film 20, it is desirable to use a film that is tacky enough to withstand several cycles of attachment and peeling. Alternatively, a new film may be attached as the second sealing film 20 after the introduction of the sample. In this case, the importance of the property relating to repeated attachment and peeling can be reduced.

As with the channel sealing film 16, the first sealing film 18 and the second sealing film 20 may have an adhesive layer or functional layer exhibiting tackiness or pressure-adhesiveness that is formed on one of the main surfaces of the film. As an example, a transparent film made from a resin such as a cycloolefin polymer, polyester, polypropylene, polyethylene, or acrylic is suitable, but is not limited thereto. As described above, properties such as stickiness or the like are desirably not degraded to such an extent that the use of the film is affected even after being repeatedly attached and peeled off. However, in the case where a new film is attached after peeling and introducing a sample or the like, or after connecting to a pressure pump, the importance of this bonding and peeling property can be reduced.

Next, an explanation will be given regarding the method of using the reaction vessel 10 formed in the manner described above. First, a sample is prepared for amplification in a thermal cycle. The sample includes, for example, samples obtained by adding a thermostable enzyme and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) as PCR reagents. To this is added a primer and a fluorescent probe, which react specifically with the DNA subjected to the reaction process. Commercially available real-time PCR kits and the like can also be used for this purpose. In addition, a mixture containing one or two or more types of DNA to be subjected to a reaction process (eg, subjected to PCR amplification) is added.

Then, the second sealing film 20 is removed from the substrate 14, whereby the first sample inlet 45 and the second sample inlet 46 are opened.

The sample is then introduced into the sample entry port using a dropper, syringe, or similar device. In FIG. 4 schematically shows the state when the sample 50 is introduced into the reaction vessel 10. The sample 50 is introduced into the dosing channel 42 through one of the first sample inlet 45 and the second sample inlet 46. The administration method is not limited to this. Alternatively, for example, a suitable amount of sample 50 may be administered directly using a pipette or dropper. In the case where the sample is introduced using a pipette, the sample 50 is introduced through the first sample inlet 45, which has a relatively small diameter. In this case, the sample 50 is loaded into the metering channel 42 towards the second sample inlet 46. When the sample 50 is injected using a dropper, the sample 50 is injected through the second sample port 46, which has a relatively large diameter. In this case, the sample 50 is loaded into the metering channel 42 towards the first sample inlet 45. The excess portion of the sample injected through one of the sample inlets, which exceeds the volume of the branch channel, accumulates in the other one of the sample inlets. Therefore, in order to use the sample inlet portion as a reservoir, the sample inlet portion can be provided with a certain space. As will be described later, the sample 50 loaded into the metering channel 42 between the first junction 431 and the second junction 441 is subjected to PCR by pressurizing from the first air communication inlet 24 and the second air communication inlet 26. Thus, the metering region 34 the reaction vessel 10 has the function of dispensing a predetermined amount of sample.

Then, the second sealing film 20 is again attached to the substrate 14, whereby the first sample inlet 45 and the second sample inlet 46 are sealed. In place of the second sealing film 20 that has been removed, a new second sealing film 20 may be attached. This completes the introduction of the sample 50 into the reaction vessel 10.

The aforementioned dosing function in the reaction vessel should not prevent the introduction of the sample when accurately dosing the sample with only one pipette.

Fig. 5 is a schematically shown block diagram for explaining the operation of a reaction apparatus 100 according to an embodiment of the present invention.

The reaction apparatus 100 according to the present embodiment of the present invention includes a vessel setting unit (not shown) in which the reaction vessel 10 is mounted, a temperature control system 102, and a CPU 105. As shown in FIG. . 5, with respect to the reaction vessel 10 installed in the vessel mounting unit, the temperature control system 102 is configured to accurately maintain and control the temperature of the high temperature region 36 in the channel 12 of the reaction vessel 10 to be about 95°. C, and the temperature in the region of 38 low temperatures should be about 60°C.

The temperature control system 102 is designed to control the temperature of each temperature region of the thermal cycle region, and is specifically provided with a high temperature heater 104 for heating the high temperature region 36 of the channel 12, a low temperature heater 106 for heating the low temperature region 38 of the channel 12, a temperature sensor (not shown), such as a thermocouple or the like for measuring the actual temperature of each temperature region, a high temperature heater actuator 108 for controlling the temperature of the high temperature heater 104, and a low temperature heater actuator 110 for controlling the temperature of the low temperature heater 106. In addition, the reaction apparatus 100 may include includes a metering heater (not shown) for heating the metering area of the channel 12, and a metering heater drive (not shown). The actual temperature information sensed by the temperature sensor is sent to the CPU 105. Based on the actual temperature information in each temperature region, the CPU 105 controls each heater driver so that the temperature of each heater becomes a predetermined temperature. Each heater may be, for example, a resistance heating element, a Peltier element, or the like. The temperature control system 102 may be further equipped with other components to improve temperature controllability in each temperature region.

The reaction apparatus 100 according to an embodiment of the present invention is further provided with a fluorescent detector 140. As described above, a predetermined fluorescent probe is added to the sample 50. Since the intensity of the fluorescence signal emitted by sample 50 increases as DNA amplification continues, the value of the fluorescent signal intensity can be used as a guideline to decide whether the PCR reaction is proceeding or not.

The fluorescent detector 140 can use the FLE-510 optical fiber type fluorescent detector manufactured by Nippon Sheet Glass Co., Ltd., which is a very compact optical system that can quickly measure and detect fluorescence regardless of whether the place is illuminated. or dark place. This optical fiber type fluorescent detector allows the excitation/fluorescence wavelength characteristic to be adjusted so that the wavelength characteristic is suitable for the characteristic of the fluorescence emitted by the sample 50, and thus provides an optimal optical and detection system for a sample having different characteristics, which must be provided. In addition, the fiber type fluorescent detector is suitable for detecting fluorescence from a sample existing in a small or narrow area such as a channel due to the small diameter of the light beam brought by the fiber type fluorescent detector and also due to its excellent response speed.

The optical fiber type fluorescent detector 140 is provided with an optical head 142, a fluorescence detector driver 144, and an optical fiber 146 connecting the optical head 142 and the fluorescence detector driver 144. The fluorescence detector driver 144 includes a light source for excitation of light (LED, laser, or light source tuned to emit at other specific wavelengths), an optical fiber multiplexer/demultiplexer, and a photoelectric conversion device (PD, APD, or light detector such as a photomultiplier). ) (none of which is shown in the figure), and a similar device from which the drive is formed, or a similar device for controlling these components. The optical head 142 is formed from an optical system such as a lens and has the function of irradiating a sample with excitation light and collecting fluorescence emitted from the sample. The collected fluorescence is separated from the excitation radiation by an optical fiber multiplexer/demultiplexer inside the fluorescence detector driver 144 via the optical fiber 146 and converted into an electrical signal by a photoelectric conversion element.

In the reaction apparatus 100 according to an embodiment of the present invention, the optical head 142 is positioned such that fluorescence from the sample 50 in the channel connecting the high temperature region 36 and the low temperature region 38 can be detected. Since the reaction proceeds when the sample 50 is repeatedly reciprocated in the channel, thereby amplifying the predetermined DNA contained in the sample 50, by monitoring the change in the amount of detected fluorescence, the progress of DNA amplification in real time can be known. In addition, in the reaction apparatus 100 according to the present embodiment, the output value from the fluorescence detector 140 is used to control the movement of the sample 50. The fluorescence detector is not limited to an optical fiber type fluorescence detector, as long as the fluorescence detector performs a detection function. fluorescence in the sample.

The reaction apparatus 100 according to the present embodiment of the present invention is further provided with a fluid supply system 120 for moving and stopping the sample 50 within the channel 12 of the reaction vessel 10. The fluid supply system 120 includes a pump 121, a pump driver 122 for driving the pump 121, a first three-way valve 123, and a second three-way valve 124. The pump driver 122, the first three-way valve 123, and the second three-way valve 124 are controlled by a central processing unit (CPU) 105.

The pump 121 may discharge air from the outlet 121a. The pump 121 may be, for example, a blowdown micropump containing a diaphragm pump. As the pump 121, for example, a micro jet pump (Model MZB1001 T02) manufactured by Murata Manufacturing Co., Ltd. can be used. or similar equipment. Although this blowdown micropump can increase the secondary side pressure to be higher than the primary side during operation, the primary side pressure and the secondary side pressure become equal when the pump is stopped or when the pump is stopped. . The Central Processing Unit (CPU) 105 controls the air supply and pressurization from the pump 121 through the pump drive 122.

The outlet 121a of the pump 121 is connected to the first air communication inlet 24 of the reaction vessel 10 through the first air passage 129. The first three-way valve 123 is located in the middle of the first air passage 129. The outlet 121a of the pump 121 is connected to the second air communication inlet 26 of the vessel. 10 to carry out the reaction with the second air passage 130. The second three-way valve 124 is located in the middle of the second air passage 130.

Each of the first three-way valve 123 and the second three-way valve 124 is a three-way valve having an inlet A, an inlet B, and an inlet C, and can switch between the state in which the inlet A and the inlet C cooperate with each other (while inlet B and inlet C do not interact with each other) and the state in which inlet B and inlet C cooperate with each other (inlet A and inlet C do not interact with each other) under the control of the CPU CPU 105. As the first three-way valve 123 and the second three-way valve 124, for example, a 3-way solenoid valve (LVM095R-6A) manufactured by SMC Corporation or the like can be used.

The inlet A of the first three-way valve 123 is connected to the outlet 121a of the pump 121 via the first tube 125. The inlet C of the first three-way valve 123 is connected to the first air inlet 24 of the reaction vessel 10 through the second tube 126. The first tube 125 and the second tube 126 constitutes the first air passage 129. The packing material 134 or airtight seal is preferably located at the junction of one end of the second tube 126 and the first air inlet 24. The inlet B of the first three-way valve 123 is open to atmospheric pressure.

The first three-way valve 123, disposed as described herein, can switch between a state in which the first air communication inlet 24 of the reaction vessel 10 communicates with an outlet 121a of the pump 121 and a state in which the first air communication inlet 24 of the vessel 10 for the reaction is open to atmospheric pressure. When the first air communication inlet 24 of the reaction vessel 10 communicates with the outlet 121a of the pump 121, the first three-way valve 123 is controlled such that the inlet A and the inlet C communicate with each other. On the other hand, when the first air communication inlet 24 of the reaction vessel 10 is opened, the first three-way valve 123 is controlled such that the inlet B and the inlet C communicate with each other.

The inlet A of the second three-way valve 124 is connected to the outlet 121a of the pump 121 by a third tube 127. The inlet C of the second three-way valve 124 is connected to the second air inlet 26 of the reaction vessel 10 by a fourth tube 128. The third tube 127 and the fourth tube 128 defines the second air passage 130. The packaging material 136 or airtight seal is preferably located at the junction of one end of the fourth tube 128 and the second air inlet 26. The inlet B of the second three-way valve 124 is open to atmospheric pressure.

The second three-way valve 124, positioned as described herein, can switch between a state in which the second air communication inlet 26 of the reaction vessel 10 communicates with the outlet 121a of the pump 121 and a state in which the second air communication inlet 26 of the vessel for carrying out the reaction 10 is open to atmospheric pressure. When the second air communication inlet 26 of the reaction vessel 10 communicates with the outlet 121a of the pump 121, the second three-way valve 124 is controlled such that the inlet A and the inlet C communicate with each other. On the other hand, when the second air communication inlet 26 of the reaction vessel 10 is open to atmospheric pressure, the second three-way valve 124 is controlled such that the inlet B and the inlet C communicate with each other.

In the reaction apparatus 100 according to the present embodiment of the present invention, by controlling the operation of the pump driver 122, the operation of the first three-way valve 123, and the operation of the second three-way valve 124, the sample 50 is reciprocated within the channel so that the sample can repeatedly be exposed to each temperature region of the channel 12 of the reaction vessel 10, and as a result, a thermal cycle can be applied to the sample 50. More specifically, the target DNA in the sample 50 is selectively amplified by repeatedly applying a denaturation step in the high temperature region 36 and an annealing and extension step in areas 38 of low temperature. In other words, the high temperature region 36 may be considered the denaturation temperature region, and the low temperature region 38 may be considered the annealing and elongation temperature region. The residence time in each temperature region can be appropriately set by changing the time during which the sample 50 stops at a predetermined position in each temperature region.

In the reaction apparatus 100 according to the present embodiment, since only one pump is used in the liquid supply system 120, unlike the case where two pumps are used, there is no need to take into account individual differences in pump characteristics when controlling the operation of the pump. Since the operation of a three-way valve is based on whether a connection is established between inlet A and inlet C, or a connection between inlet B and inlet C, there is no difference in individual differences as long as a three-way valve with sufficiently small pressure losses is selected. Therefore, sample movement control is easier in the reaction apparatus 100 according to the present embodiment, compared with the case where two pumps are used. In addition, since three-way valves are generally less expensive than pumps, the cost of the reaction device 100 can be reduced.

In FIG. 6 is a table explaining the control method of the pump 121, the first three-way valve 123, and the second three-way valve 124 in the reaction apparatus 100 shown in FIG. 5. The control method explained with FIG. 6 uses as the pump 121 a pump that allows the pressure on the primary side and the pressure on the secondary side to be equal to each other when stopped.

In FIG. 6 and the following figures, the operating state of the pump 121 (i.e., the air-bleeding state) is indicated as "ON", and the non-working state of the pump 121 (that is, the air-bleeding state is stopped) is indicated as "OFF". ). With regard to the first three-way valve 123 and the second three-way valve 124, the state in which the inlet A and the inlet C are in communication with each other is referred to as "A-C", and the state in which the inlet B and the inlet C are in communication with each other denoted "B-C".

Step 1 shows the control status of the pump 121, the first three-way valve 123 and the second three-way valve 124 before moving the sample 50 from the low temperature region 38 to the high temperature region 36. In step 1, the pump 121 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 123 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 124 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM).

Step 2 shows the control status of the pump 121, the first three-way valve 123 and the second three-way valve 124 when the sample 50 is moved from the low temperature region 38 to the high temperature region 36. In step 2, the pump 121 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 123 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 124 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM). Thus, the first air communication inlet 24 of the reaction vessel 10 communicates with the outlet 121a of the pump 121, and the second air communication inlet 26 of the reaction vessel 10 is opened to atmospheric pressure. Thus, the first air inlet port 24 is under positive pressure due to the venting of air from the pump 121, while the sample 50 moves from the low temperature region 38 to the high temperature region 36.

Step 3 shows the control state of the pump 121, the first three-way valve 123, and the second three-way valve 124 when the sample 50 reaches the high temperature region 36. In step 3, the pump 121 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 123 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 124 is controlled such that the inlet B and the inlet C communicate with each other (B -C) Thus, both the first air communication inlet 24 and the second air communication inlet 26 are opened to atmospheric pressure, causing the sample 50 to stop in the high temperature region 36 .

Step 4 shows the control state of the pump 121, the first three-way valve 123, and the second three-way valve 124 when the sample 50 is in standby mode in the high temperature region 36. ). In addition, the first three-way valve 123 and the second three-way valve 124 are controlled to be either in a state where the inlet A is associated with the inlet C (A-C) or a state where the inlet B is associated with the inlet C ( B-C). Also at this time, since both the first air communication inlet 24 and the second air communication inlet 26 are open to atmospheric pressure, the sample 50 remains stopped in the high temperature region 36.

Step 5 shows the control status of the pump 121, the first three-way valve 123 and the second three-way valve 124 before moving the sample 50 from the high temperature region 36 to the low temperature region 38. In step 5, the pump 121 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 123 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 124 is controlled such that the inlet A and the inlet C communicate with each other (A-C ).

Step 6 shows the control status of the pump 121, the first three-way valve 123 and the second three-way valve 124 when the sample 50 is moved from the high temperature area 36 to the low temperature area 38. In step 6, the pump 121 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 123 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 124 is controlled such that the inlet A and the inlet C communicate with each other (A-C ). Thus, the first air communication inlet 24 of the reaction vessel 10 is opened to atmospheric pressure, and the second air communication inlet 26 of the reaction vessel 10 communicates with the outlet 121a of the pump 121. Thus, the second air communication inlet 26 is under positive pressure due to the release of air from the pump 121, and the sample 50 moves from the region 36 of high temperature to the region 38 of low temperature.

Step 7 shows the control status of the pump 121, the first three-way valve 123 and the second three-way valve 124 when the sample 50 reaches the low temperature region 38. In step 7, the pump 121 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 123 is controlled such that port B and port C communicate with each other (B-C), and the second three-way valve 124 is controlled such that inlet A and inlet C communicate with each other (A-C). Thus, both the first air communication inlet 24 and the second air communication inlet 26 are open to atmospheric pressure, causing the sample 50 to stop in the low temperature region 38.

Step 8 shows the control state of the pump 121, the first three-way valve 123 and the second three-way valve 124 when the sample 50 is in standby mode in the low temperature region 38. In step 8, the pump 121 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 123 and the second three-way valve 124 are controlled to be either in a state where the inlet A is associated with the inlet C (A-C) or a state where the inlet B is associated with the inlet C ( B-C). Also at this time, since both the first air communication inlet 24 and the second air communication inlet 26 are open to atmospheric pressure, the sample 50 remains stopped in the low temperature region 38.

By repeating steps 1-8 above, a thermal cycle can be applied to the sample 50 by continuously reciprocating the sample 50 between the low temperature region 38 and the high temperature region 36.

Fig. 7 shows a table for explaining another method for controlling the pump 121, the first three-way valve 123, and the second three-way valve 124 in the reaction apparatus shown in FIG. 5. Explained in FIG. 7, the control method as the pump 121 uses a pump that allows the pressure on the primary side and the pressure on the secondary side to be equal to each other when the pump is stopped.

Step 1 shows the control state of the pump 121, the first three-way valve 123, and the second three-way valve 124 before moving the sample 50 from the low temperature region 38 to the high temperature region 36. In step 1, the pump 121 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 123 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 124 is controlled such that the inlet B and the inlet C communicate with each other (B-C ).

Step 2 shows the control status of the pump 121, the first three-way valve 123 and the second three-way valve 124 when the sample 50 is moved from the low temperature region 38 to the high temperature region 36. In step 2, the pump 121 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 123 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 124 is controlled such that the inlet B and the inlet C communicate with each other (B-C ). Thus, the first air communication inlet 24 of the reaction vessel 10 communicates with the outlet 121a of the pump 121, and the second air communication inlet 26 of the reaction vessel 10 is opened to atmospheric pressure. Thus, the first air inlet port 24 is under positive pressure due to the venting of the pump 121 and the sample 50 moves from the low temperature region 38 to the high temperature region 36.

Step 3 shows the control state of the pump 121, the first three-way valve 123, and the second three-way valve 124 when the sample 50 reaches the high temperature region 36. In step 3, the pump 121 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 123 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 124 is controlled such that the inlet B and the inlet C communicate with each other (B-C .) Thus, both the first air communication inlet 24 and the second air communication inlet 26 are opened to atmospheric pressure, and the sample 50 is thus stopped in the high temperature region 36 .

Step 4 shows the control state of the pump 121, the first three-way valve 123, and the second three-way valve 124 when the sample 50 is in standby mode in the high temperature region 36. ). In addition, the first three-way valve 123 and the second three-way valve 124 are controlled to be either in a state where the inlet A is associated with the inlet C (A-C) or a state where the inlet B is associated with the inlet C ( B-C). Also at this time, since both the first air communication inlet 24 and the second air communication inlet 26 are open to atmospheric pressure, the sample 50 remains stopped in the high temperature region 36.

Step 5 shows the control status of the pump 121, the first three-way valve 123 and the second three-way valve 124 before moving the sample 50 from the high temperature region 36 to the low temperature region 38. In step 5, the pump 121 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 123 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 124 is controlled such that the inlet B and the inlet C communicate with each other (B-C .)

Step 6 shows the control status of the pump 121, the first three-way valve 123 and the second three-way valve 124 when the sample 50 is moved from the high temperature area 36 to the low temperature area 38. In step 6, the pump 121 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 123 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 124 is controlled such that the inlet A and the inlet C communicate with each other (A-C ). Thus, the first air communication inlet 24 of the reaction vessel 10 is opened to atmospheric pressure, and the second air communication inlet 26 of the reaction vessel 10 communicates with the outlet 121a of the pump 121. Thus, the second air communication inlet 26 is under positive pressure due to the release of air from the pump 121, and the sample 50 moves from the region 36 of high temperature to the region 38 of low temperature.

Step 7 shows the control status of the pump 121, the first three-way valve 123 and the second three-way valve 124 when the sample 50 reaches the low temperature region 38. In step 7, the pump 121 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 123 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 124 is controlled such that the inlet B and the inlet C communicate with each other ( B-C.) Thus, both the first air communication inlet 24 and the second air communication hole 26 are open to atmospheric pressure, and the sample 50 thus stops in the low temperature region 38.

Step 8 shows the control state of the pump 121, the first three-way valve 123 and the second three-way valve 124 when the sample 50 is in standby mode in the low temperature region 38. In step 8, the pump 121 is controlled such that it is in an idle state (OFF). In addition, the first three-way valve 123 and the second three-way valve 124 are controlled to be either in a state where the inlet A is associated with the inlet C (A-C) or a state where the inlet B is associated with the inlet C ( B-C). Also at this time, since both the first air communication inlet 24 and the air communication inlet 26 are open to atmospheric pressure, the sample 50 remains stopped in the low temperature region 38.

By repeating steps 1-8 above, a thermal cycle can be applied to the sample 50 by continuously reciprocating the sample 50 between the low temperature region 38 and the high temperature region 36.

In order to confirm the positive effect of the reaction device 100 formed as described above, an experiment was conducted in which a specific bacterial strain was amplified by PCR using the reaction device 100 according to an embodiment of the present invention. In this case, the control method described above with reference to FIG. 6. In an attempt to detect Vero toxin VT1, PCR reagents were prepared using the “KAPA3G Plant” PCR kit, which is a PCR enzyme from NIPPON Genetics Co., Ltd., in the manner shown in the table below.

Table 1

Final concentration Notes enzyme 0.1 U/μL (unit/μl) PCR kit KAPA 3G Plant (NIPPON Genetics Co., Ltd.) Primer F 720 nM 5'-GGA TAA TTT GTT TGC AGT TGA TGT C-3' (manufactured by NIHON GENE RESEARCH LABORATORIES Inc.) Primer R 720 nM 5'-CAA ATC CTG TCA CAT ATA AAT TAT TTC GT-3' (manufactured by NIHON GENE RESEARCH LABORATORIES Inc.) probe 240 nM 5'-CCG TAG ATT ATT AAA CCG CCC TTC CTC TGG A-3' FAM is used for fluorescent dye and dark type quencher (manufactured by NIHON GENE RESEARCH LABORATORIES Inc.) Extra Mg 1.25mm MgCl ₂ solution added to KAPA 3G Plant Buffer
+
purified water According to the KAPA 3G Plant manual, the buffer and water are mixed in such a way that the concentration of the buffer supplied with the kit is reduced to 1/2 of the total reagent.

On the other hand, a matrix of 20,000 copies/μl was prepared and subjected to dilutions to prepare aqueous solutions having matrix concentrations of 20,000, 1000, 100, and 10 copies/μl.

In addition, 1 µl of an aqueous matrix solution of each concentration was added to 19 µl of the KAPA 3G Plant solution prepared above to obtain 20 µl of a positive reagent (positive control).

For the sample of the above concentration in the reaction apparatus 100, the temperature of the high temperature region 36 was set to 96°C, the temperature of the low temperature region 38 was set to 62°C, the standby time in the high temperature region 36 was 3.5 seconds, the standby time in the low temperature area 38 was 15 seconds, and then spent 50 thermal cycle (50 Ct) for PCR. The liquid supply time was about one second. The waiting time in the high temperature area 36 in the first cycle was 15 seconds.

Fig. 8 shows the result of PCR amplification by the reaction apparatus 100 according to the present embodiment of the present invention. In FIG. 8, the horizontal axis represents the cycle counter (Ct) and the vertical axis represents the fluorescence signal intensity (arbitrary unit). Using the reaction apparatus 100 described above, the intensity of the fluorescence signal detected by the fluorescent detector 140 was measured relative to the number of cycles. As the study drug intensified in the sample, the intensity of the fluorescence signal increased.

Here, PCR was performed on samples containing study preparations having initial concentrations of 20,000 copies/μl, 1000 copies/μl, 100 copies/μl, and 10 copies μl, respectively. As shown in FIG. 8, the fluorescence signal intensity of the 20,000 copies/µl sample increases dramatically after about 30 cycles. In addition, the fluorescence signal intensity of the 1000 copies/µl sample increases dramatically after about 35 cycles. In addition, the fluorescence signal intensity of the 100 copies/μl sample increases dramatically after about 38 cycles. In addition, the fluorescence signal intensity of the 10 copies/µl sample increases dramatically after about 41 cycles. Such a sharp increase in the intensity of the fluorescence signal indicates amplification of the sample in the sample, and it can be found that a good PCR can be performed using the reaction apparatus 100 according to the present embodiment.

Fig. 9 shows a flowchart for explaining the operation of a reaction apparatus 200 according to another embodiment of the present invention. During the PCR process, the high temperature region is usually set at about 95°C. Because the sample is an aqueous solution, the boiling point decreases at higher altitudes. For example, the pressure is about 897hPa and the boiling point is 96.6°C according to the calculation at the place where the altitude is 1000m, if the pressure is 845hPa, then the boiling point is 95°C at the place where the altitude is 1500 m, if the pressure is 797 hPa, then the boiling point is 93.4°C at the place where the altitude is 2000 m. In such places, it is sometimes difficult to perform PCR because the sample boils and evaporates easily, and/ or foams in the high temperature region, or the sample evaporates significantly. The reaction apparatus 200 according to the present embodiment can perform PCR even in such a place having low atmospheric pressure while preventing the sample from boiling and bubbling.

In the reaction apparatus 200, the liquid supply system 220 is configured differently from the liquid supply system 120 of the reaction apparatus 100 shown in FIG. 5. As well as the fluid supply system 120, the fluid supply system 220 includes a pump 221, a pump driver 222 for driving the pump 221, a first three-way valve 223, and a second three-way valve 224. A pump driver 222, a first three-way valve 223, and the second three-way valve 224 is controlled by the central processing unit (CPU) 105.

The fluid supply system 220 according to an embodiment of the present invention further includes a plenum chamber 238, a plenum pump 244, and a plenum pump actuator 245 for controlling the plenum pump 244. In the present embodiment, the fluid pump 221 is located inside the plenum 238.

The pressure chamber 238 forms a space containing a certain volume. The plenum pump 244 is connected to the plenum 238. The plenum pump actuator 245 controls the plenum pump 244 so that the space inside the plenum chamber 238 has a predetermined pressure, in accordance with an instruction from the central processing unit (CPU) 105. A roller pump can be used as the plenum pump 244 * (Model: RSP08D-02RW) manufactured by OKEN SEIKO co., Ltd., or a similar pump, and a rubber ball, syringe, or similar pressurizer can be used as a simple means.

In the present embodiment, the pressure within the plenum chamber 238 is maintained at a value greater than the ambient pressure of the reaction device 200 (eg, 1.3 atm) during the reaction process. The ambient atmospheric pressure of the reaction apparatus 200 means the pressure (or atmospheric pressure) at the location where the reaction apparatus 200 is installed, the location where PCR is performed by the reaction apparatus 200, or when the reaction apparatus 200 is installed at the location , which is separated from the environment, i.e. in a separate place. The pressure inside the pressure chamber 238 should be such that significant vaporization of the sample and the formation of air bubbles or similar phenomena that affect the reaction process involving PCR can be prevented, even when the sample has been repeatedly exposed to high temperature (about 95°C). The higher the pressure inside the plenum chamber 238, the more the effect of sample evaporation and the like can be suppressed. However, on the other hand, the fluid supply system 220 becomes more complex or larger, including handling. Thus, a person skilled in the art can comprehensively evaluate the application, purpose, cost, impact, etc. processor to design the whole system.

In the plenum 238, a pressure release valve 248 is provided to atmospheric pressure. The atmospheric pressure relief valve 248 is adjusted such that the pressure of the liquid supply system 220 and the pressure of the reaction vessel 10 in the conduit 12 become equal to atmospheric pressure during the installation or removal of the reaction vessel 10. Thus, rapid movement and injection of the sample 50 can be prevented. In addition, during the reaction process, the pump 244 of the plenum chamber is stopped and the pressure relief valve 248 remains open, resulting in a reaction apparatus substantially equivalent to device 100 for carrying out the reaction.

Additionally, a pressure sensor (not shown) may be provided in the plenum 238 to continuously monitor the pressure inside the plenum. By sending the actual pressure value detected by the pressure sensor to the central processing unit (CPU) 105, the pressure inside the pressure chamber 238 can be appropriately controlled.

Further, in the present embodiment, a pump of the type in which the pressure on the primary side and the pressure on the secondary side become equal at the time of stopping can be used as the pump 221.

The outlet 221a of the pump 221 is connected to the first air communication inlet 24 of the vessel 10 to carry out the reaction through the first air passage 229. The first three-way valve 223 is located in the middle of the first air passage 229. The outlet 221a of the pump 121 is connected to the second air communication inlet 26 of the vessel 10 to carry out the reaction with the second air passage 230. The second three-way valve 224 is located in the middle of the second air passage 230.

The inlet A of the first three-way valve 223 is connected to the outlet 221a of the pump 221 via the first tube 225. The inlet C of the first three-way valve 223 is connected to the first air inlet 24 of the reaction vessel 10 through the second tube 226. The first tube 225 and the second tube 226 form a first air passage 229. Port B of the first three-way valve 223 communicates with the interior of the plenum chamber 238 via a fifth tube 231.

The first three-way valve 223, positioned as previously described, can be switched between a state in which the first air communication inlet 24 of the reaction vessel 10 communicates with an outlet 221a of the pump 221 and a state in which the first air communication inlet 24 of the vessel 10 to carry out the reaction is open to the interior of the pressure chamber 238. When the first air communication inlet 24 of the reaction vessel 10 communicates with the outlet 221a of the pump 221, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other. On the other hand, when the first air communication inlet 24 of the reaction vessel 10 opens into the inside of the pressure chamber 238, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other.

The inlet A of the second three-way valve 224 is connected to the outlet 221a of the pump 221 by a third tube 227. The inlet C of the second three-way valve 224 is connected to the second air inlet 26 of the reaction vessel 10 through the fourth tube 228. The third tube 227 and the fourth the tube 228 form the second air passage 230. The inlet B of the second three-way valve 224 communicates with the interior of the plenum 238 via the sixth tube 232.

The second three-way valve 224, positioned as previously described, can switch between a state in which the second air communication inlet 26 of the reaction vessel 10 communicates with the outlet 221a of the pump 221, and a state in which the second air communication inlet 26 of the vessel 10 to carry out the reaction is open to the interior of the pressure chamber 238. When the second air communication inlet 26 of the reaction vessel 10 communicates with the outlet 221a of the pump 221, the second three-way valve 224 is controlled such that the inlet A and the inlet C communicate with each other. On the other hand, when the second air communication inlet 26 of the reaction vessel 10 opens into the interior of the pressure chamber 238, the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other.

In the reaction apparatus 200 according to an embodiment of the present invention, by controlling the operation of the pump driver 222, the operation of the first three-way valve 223, and the operation of the second three-way valve 224, the sample 50 is reciprocated within the channel so that the sample can be repeatedly exposed to each temperature region of the channel 12 of the reaction vessel 10, whereby a thermal cycle can be applied to the sample 50. In addition, the use of a single pump makes it easier to control the movement of the sample and can reduce the cost in the same way as in the above-described reaction device 100.

In addition, in the reaction apparatus 200 according to the present embodiment of the present invention, the pump 221 is located in the interior of the plenum 238 set to a pressure that is higher than the ambient pressure (for example, 1.3 atm), and the inlet B the first three-way valve 223 and the inlet B of the second three-way valve 224 are configured to open in the interior of the pressure chamber 238. Therefore, during the reaction process, the entire channel 12 is maintained at a pressure above ambient atmospheric pressure. For this reason, even under conditions of low atmospheric pressure such as high altitude, it is possible to prevent the boiling point of the sample 50 consisting mainly of an aqueous solution from lowering and the sample 50 from boiling and/or foaming, whereby a stable PCR reaction can be performed.

As a method for controlling the pump driver 222, the first three-way valve 223, and the second three-way valve 224 in the reaction apparatus 200, the method shown in FIG. 6. The control method will be described again with reference to FIG. 6. The pump 221 uses a pump that allows the pressure on the primary side and the pressure on the secondary side to become equal when the pump stops. In the reaction device 200, the pressure relief valve 248 is closed, the plenum pump 244 is operating, the pressure in the interior of the plenum chamber 238 is increasing, inside each tube and channel 12 is also increasing, all this happens in advance. The first three-way valve 223 and the second three-way valve 224 at this time are controlled such that they are either in the state that the inlet A is associated with the inlet C (A-C) or the state that the inlet B is associated with the inlet C ( B-C).

Step 1 shows the control status of the pump 221, the first three-way valve 223 and the second three-way valve 224 before moving the sample 50 from the low temperature area 38 to the high temperature area 36. In step 1, the pump 221 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM).

Step 2 shows the control status of the pump 221, the first three-way valve 223, and the second three-way valve 224 while moving the sample 50 from the low temperature region 38 to the high temperature region 36. In step 2, the pump 221 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM). Thus, the first air communication inlet 24 of the reaction vessel 10 communicates with the outlet 221a of the pump 221, and the second air communication inlet 26 of the reaction vessel 10 opens into the interior of the pressure chamber 238. In the present embodiment, since the pump 221 is located inside the plenum 238, when the air exits the pump 221, the pressure in the first air communication inlet 24 of the reaction vessel 10 becomes higher than in the second air communication inlet 26, and the sample 50 thus moves from the low temperature region 38 to the high temperature region 36 .

Step 3 shows the control state of the pump 221, the first three-way valve 223 and the second three-way valve 224 when the sample 50 reaches the high temperature region 36. In step 3, the pump 221 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM). Thus, both the first air communication inlet 24 and the second air communication inlet 26 open into the interior of the plenum chamber 238, resulting in the sample 50 stopping in the high temperature region 36 .

Step 4 shows the control state of the pump 221, the first three-way valve 223, and the second three-way valve 224 when the sample 50 is in standby mode in the high temperature area 36. In step 4, the pump 221 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 223 and the second three-way valve 224 are controlled to be either in a state where the inlet A is associated with the inlet C (A-C) or a state where the inlet B is associated with the inlet C ( B-C). Both the first air communication inlet 24 and the second air communication inlet 26 are open to the interior of the plenum chamber 238, and the sample 50 thus remains stopped in the high temperature region 36 also at this time.

Step 5 shows the control status of the pump 221, the first three-way valve 223 and the second three-way valve 224 before the sample 50 is moved from the high temperature region 36 to the low temperature region 38. In step 5, the pump 221 is controlled such that it is in an idle state (OFF) . In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet A and the inlet C communicate with each other (A-C ).

Step 6 shows the control status of the pump 221, the first three-way valve 223 and the second three-way valve 224 when the sample 50 is moved from the high temperature region 36 to the low temperature region 38. In step 6, the pump 221 is controlled so that it is in the operating state (ON) . In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet A and the inlet C communicate with each other (A-C ). Thus, the first air communication inlet 24 of the reaction vessel 10 opens into the interior of the plenum 238, and the second air communication inlet 26 of the reaction vessel 10 communicates with the outlet 221a of the pump 221. Therefore, when air exits the pump 221, the pressure in the second air communication inlet 26 of the reaction vessel 10 becomes higher than in the first air communication inlet 24, and as a result, the sample 50 moves from the high temperature region 36 to the low temperature region 38.

Step 7 shows the control status of the pump 221, the first three-way valve 223, and the second three-way valve 224 when the sample 50 reaches the low temperature region 38. In step 7, the pump 221 is controlled such that it is in an idle state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet A and the inlet C communicate with each other (A-C ). Thus, both the first air communication inlet 24 and the second air communication inlet 26 open into the interior of the pressure chamber 238, and the sample 50 thus stops in the low temperature region 38.

Step 8 shows the control state of the pump 221, the first three-way valve 223, and the second three-way valve 224 when the sample 50 is idle in the low temperature area 38. In step 8, the pump 221 is controlled such that it is in an idle state (OFF). In addition, the first three-way valve 223 and the second three-way valve 224 are controlled to be either in a state where the inlet A is associated with the inlet C (A-C) or a state where the inlet B is associated with the inlet C ( B-C). Both the first air communication inlet 24 and the second air communication inlet 26 are open to the interior of the pressure chamber 238, and the sample 50 thus remains stopped in the low temperature region 38 also at this time.

By repeating steps 1-8 above, a thermal cycle can be applied to the sample 50 by continuously reciprocating the sample 50 between the low temperature region 38 and the high temperature region 36.

The method shown in FIG. 7. The control method will be described again with reference to FIG. 7. This control method uses a pump as the pump 221, which allows the pressure on the primary side and the pressure on the secondary side to be equal to each other when the pump stops. In the same way, in the reaction apparatus 200, the pressure relief valve 248 is closed, the plenum pump 244 is operating, the pressure in the interior of the plenum chamber 238 is increased, and the inside of each tube and passage 12 is also increased, and all this performed in advance.

Step 1 shows the control state of the pump 221, the first three-way valve 223, and the second three-way valve 224 before moving the sample 50 from the low temperature region 38 to the high temperature region 36. In step 1, the pump 221 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM).

Step 2 shows the control status of the pump 221, the first three-way valve 223 and the second three-way valve 224 when the sample 50 is moved from the low temperature area 38 to the high temperature area 36. In step 2, the pump 221 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM). Thus, the first air communication inlet 24 of the reaction vessel 10 communicates with the outlet 221a of the pump 221, and the second air communication inlet 26 of the reaction vessel 10 opens into the interior of the pressure chamber 238. In the present embodiment, since the pump 221 is located inside the plenum 238, when the air exits the pump 221, the pressure in the first air communication inlet 24 of the reaction vessel 10 becomes higher than in the second air communication inlet 26, and the sample 50 thus moves from the low temperature region 38 to the high temperature region 36 .

Step 3 shows the control state of the pump 221, the first three-way valve 223 and the second three-way valve 224 when the sample 50 reaches the high temperature region 36. In step 3, the pump 221 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM). Thus, both the first air communication inlet 24 and the second air communication inlet 26 open into the interior of the plenum chamber 238, causing the sample 50 to stop in the high temperature region 36 .

Step 4 shows the control state of the pump 221, the first three-way valve 223, and the second three-way valve 224 when the sample 50 is in standby mode in the high temperature area 36. In step 4, the pump 221 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 223 and the second three-way valve 224 are controlled to be either in a state where the inlet A is in communication with the inlet C (A-C) or in a state where the inlet B is in communication with the inlet C ( B-C). Both the first air communication inlet 24 and the second air communication inlet 26 are open to the interior of the plenum chamber 238, and the sample 50 thus remains stopped in the high temperature region 36 also at this time.

Step 5 shows the control status of the pump 221, the first three-way valve 223, and the second three-way valve 224 before the sample 50 is moved from the high temperature region 36 to the low temperature region 38. In step 5, the pump 221 is controlled such that it is in the operating state (ON) . In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM).

Step 6 shows the control status of the pump 221, the first three-way valve 223 and the second three-way valve 224 when the sample 50 is moved from the high temperature region 36 to the low temperature region 38. In step 6, the pump 221 is controlled so that it is in the operating state (ON) . In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet A and the inlet C communicate with each other (A-C ). Thus, the first air communication inlet 24 of the reaction vessel 10 opens into the interior of the plenum 238, and the second air communication inlet 26 of the reaction vessel 10 communicates with the outlet 221a of the pump 221. Therefore, when air exits the pump 221, the pressure in the second air communication inlet 26 of the reaction vessel 10 becomes higher than in the first air communication inlet 24, and thus the sample 50 moves from the high temperature region 36 to the low temperature region 38.

Step 7 shows the control status of the pump 221, the first three-way valve 223, and the second three-way valve 224 when the sample 50 reaches the low temperature region 38. In step 7, the pump 221 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM). Thus, both the first air communication inlet 24 and the second air communication inlet 26 open into the interior of the pressure chamber 238, and the sample 50 thus stops in the low temperature region 38.

Step 8 shows the control status of the pump 221, the first three-way valve 223 and the second three-way valve 224 when the sample 50 is in standby mode in the low temperature region 38. In step 8, the pump 221 is controlled such that it is in an idle state (OFF). In addition, the first three-way valve 223 and the second three-way valve 224 are controlled to be either in a state where the inlet A is in communication with the inlet C (A-C) or in a state where the inlet B is in communication with the inlet C ( B-C). Both the first air communication inlet 24 and the second air communication inlet 26 are open to the interior of the pressure chamber 238, and the sample 50 thus remains stopped in the low temperature region 38 also at this time.

By repeating steps 1-8 above, a thermal cycle can be applied to the sample 50 by continuously reciprocating the sample 50 between the low temperature region 38 and the high temperature region 36.

In FIG. 10 is a block diagram illustrating the operation of a reaction apparatus 300 according to yet another embodiment of the present invention. This reaction apparatus 300 is different from the reaction apparatus 100 shown in FIG. 5 in that a pump 321 is used in which the primary side pressure and the secondary side pressure do not become equal to each other when the pump in the fluid supply system 320 is stopped. As such a pump 321, for example, a piezo micropump (Model SDMP302 (306)) manufactured by Takasago Electric, Inc. can be used.

In FIG. 11 is a table explaining the control method of the pump 321, the first three-way valve 123, and the second three-way valve 124 in the reaction apparatus 300 shown in FIG. ten.

Step 1 shows the control status of the pump 321, the first three-way valve 123 and the second three-way valve 124 before moving the sample 50 from the low temperature region 38 to the high temperature region 36. In step 1, the pump 321 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 123 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 124 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM).

Step 2 shows the control status of the pump 321, the first three-way valve 123 and the second three-way valve 124 when the sample 50 is moved from the low temperature region 38 to the high temperature region 36. In step 2, the pump 321 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 123 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 124 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM).

Step 3 shows the control status of the pump 321, the first three-way valve 123, and the second three-way valve 124 when the sample 50 reaches the high temperature region 36. In step 3, the pump 321 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 123 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 124 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM).

Step 4 shows the control status of the pump 321, the first three-way valve 123, and the second three-way valve 124 when the sample 50 is in standby mode in the high temperature region 36. ). In addition, the first three-way valve 123 and the second three-way valve 124 are controlled so that they are both in a state in which the inlet A is in communication with the inlet C (A-C), or in a state in which the inlet B is in communication with the inlet C (B-C).

Step 5 shows the control status of the pump 321, the first three-way valve 123, and the second three-way valve 124 before the sample 50 is moved from the high temperature region 36 to the low temperature region 38. In step 5, the pump 321 is controlled such that it is in an idle state (OFF) . In addition, the first three-way valve 123 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 124 is controlled such that the inlet A and the inlet C communicate with each other (A-C ).

Step 6 shows the control status of the pump 321, the first three-way valve 123 and the second three-way valve 124 when the sample 50 is moved from the high temperature region 36 to the low temperature region 38. In step 6, the pump 321 is controlled such that it is in the operating state (ON) . In addition, the first three-way valve 123 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 124 is controlled such that the inlet A and the inlet C communicate with each other (A-C ).

Step 7 shows the control status of the pump 321, the first three-way valve 123, and the second three-way valve 124 when the sample 50 reaches the low temperature region 38. In step 7, the pump 321 is controlled such that it is in an idle state (OFF). In addition, the first three-way valve 123 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 124 is controlled such that the inlet A and the inlet C communicate with each other (A-C ).

Step 8 shows the control state of the pump 321, the first three-way valve 123, and the second three-way valve 124 when the sample 50 is idle in the low temperature region 38. In step 8, the pump 321 is controlled such that it is in an idle state (OFF). In addition, the first three-way valve 123 and the second three-way valve 124 are controlled so that both valves are in a state in which the inlet A is in communication with the inlet C (A-C), or in a state in which the inlet B is in communication with the inlet C (B-C).

By repeating steps 1-8 above, a thermal cycle can be applied to the sample 50 by continuously reciprocating the sample 50 between the low temperature region 38 and the high temperature region 36. Also, in the reaction apparatus 300 according to the present embodiment, since only one pump is used, sample movement control can be easily performed and the cost can be reduced.

Fig. 12 shows a block diagram explaining the operation of a reaction apparatus 400 according to another embodiment of the present invention. This reaction apparatus 400 is different from the reaction apparatus 200 shown in FIG. 9 in that a pump 421 is used in which the primary side pressure and the secondary side pressure do not become equal to each other when the pump in the fluid supply system 420 is stopped. As the pump 421, for example, a piezo micropump (Model SDMP302 (306)) manufactured by Takasago Electric, Inc. can be used.

The reaction apparatus 400 according to the present embodiment further includes a solenoid valve 401. This solenoid valve 401 is provided in a passage that connects the pressure chamber 238 to the first air passage 229 and the second air passage 230, and can be switched between an open state and a closed state according to an instruction from the central processing unit (CPU) 105. When the solenoid valve 401 is in the open state, the first air passage 229 and the second air passage 230 communicate with the interior of the plenum chamber 238. On the other hand, when the solenoid valve 401 is in the closed state, the first air passage 229 and the second air passage 230 do not communicate with the interior of the plenum 238. As the solenoid valve 401, for example, a solenoid valve (Model TDS-V05B) manufactured by TDS Co., Ltd. or the like can be used.

Fig. 13 shows a table explaining the control method of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 in the reaction apparatus 400 shown in FIG. 12.

Step 1 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 when the pressure in the interior of the plenum chamber 238 increases and the pressure in the inside of each tube and the inside of the channel 12 increases. In step 1, the pump 421 is controlled so that it is in a non-operating state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -C) In addition, the solenoid valve 401 is in the open state.

Step 2 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 before moving the sample 50 from the low temperature region 38 to the high temperature region 36. In step 2, the pump 421 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM). In addition, the solenoid valve 401 is in a closed state.

Step 3 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 while moving the sample 50 from the low temperature region 38 to the high temperature region 36. In step 3, the pump 421 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM). In addition, the solenoid valve 401 is in a closed state.

Step 4 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 when the sample 50 reaches the high temperature area 36. In step 4, the pump 421 is controlled such that it is in an idle state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -C) In addition, the solenoid valve 401 is in the closed state.

Step 5 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 when the sample 50 is idle in the high temperature region 36. In step 5, the pump 421 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 223 and the second three-way valve 224 are controlled to be in a state where the inlet A is in communication with the inlet C (A-C), or in a state where the inlet B is in communication with the inlet C (B). -FROM). In addition, the solenoid valve 401 is in the open state.

Step 6 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 before moving the sample 50 from the high temperature region 36 to the low temperature region 38. In step 6, the pump 421 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet A and the inlet C communicate with each other (A-C ). In addition, the solenoid valve 401 is in a closed state.

Step 7 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 while moving the sample 50 from the high temperature region 36 to the low temperature region 38. In step 7, the pump 421 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet A and the inlet C communicate with each other (A-C ). In addition, the solenoid valve 401 is in a closed state.

Step 8 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 when the sample 50 reaches the low temperature region 38. In step 8, the pump 421 is controlled such that it is in an idle state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet A and the inlet C communicate with each other (A-C ). In addition, the solenoid valve 401 is in a closed state.

Step 9 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 when the sample 50 is idle in the low temperature region 38. In step 9, the pump 421 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 223 and the second three-way valve 224 are controlled to be in a state where the inlet A is in communication with the inlet C (A-C), or in a state where the inlet B is in communication with the inlet C (B). -FROM). In addition, the solenoid valve 401 is in the open state.

By repeating steps 2-9 described above, a thermal cycle can be applied to the sample 50 by continuously reciprocating the sample 50 between the low temperature region 38 and the high temperature region 36.

As a method for controlling the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 in the reaction apparatus 400, the method shown in FIG. fourteen.

Step 1 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 when the pressure in the interior of the plenum chamber 238 increases and the pressure in the inside of each tube as well as the inside of the channel 12 increases. 1 pump 421 is controlled in such a way that it is in a non-working state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM). In addition, the solenoid valve 401 is in the open state.

Step 2 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 before moving the sample 50 from the low temperature region 38 to the high temperature region 36. In step 2, the pump 421 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM). In addition, the solenoid valve 401 is in a closed state.

Step 3 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 while moving the sample 50 from the low temperature region 38 to the high temperature region 36. In step 3, the pump 421 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -C) In addition, the solenoid valve 401 is in the closed state.

Step 4 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 when the sample 50 reaches the high temperature area 36. In step 4, the pump 421 is controlled such that it is in an idle state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet A and the inlet C communicate with each other (A-C), and the second three-way valve 224 is controlled such that the inlet B and the inlet C communicate with each other (B -FROM). In addition, the solenoid valve 401 is in a closed state.

Step 5 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 when the sample 50 is idle in the high temperature region 36. In step 5, the pump 221 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 223 and the second three-way valve 224 are controlled to be in a state where the inlet A is in communication with the inlet C (A-C), or in a state where the inlet B is in communication with the inlet C (B). -FROM). In addition, the solenoid valve 401 is in a closed state.

Step 6 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 before moving the sample 50 from the high temperature region 36 to the low temperature region 38. In step 6, the pump 421 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet A and the inlet C communicate with each other (A-C ). In addition, the solenoid valve 401 is in a closed state.

Step 7 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 while moving the sample 50 from the high temperature region 36 to the low temperature region 38. In step 7, the pump 421 is controlled so that it is in an operating state (ON). In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet A and the inlet C communicate with each other (A-C ). In addition, the solenoid valve 401 is in a closed state.

Step 8 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 when the sample 50 reaches the low temperature region 38. In step 8, the pump 421 is controlled such that it is in an idle state (OFF). In addition, the first three-way valve 223 is controlled such that the inlet B and the inlet C communicate with each other (B-C), and the second three-way valve 224 is controlled such that the inlet A and the inlet C communicate with each other (A-C ). In addition, the solenoid valve 401 is in a closed state.

Step 9 shows the control status of the pump 421, the first three-way valve 223, the second three-way valve 224, and the solenoid valve 401 when the sample 50 is idle in the low temperature region 38. In step 9, the pump 421 is controlled so that it is in an idle state (OFF). In addition, the first three-way valve 223 and the second three-way valve 224 are controlled to be in a state where the inlet A is in communication with the inlet C (A-C), or in a state where the inlet B is in communication with the inlet C (B). -FROM). In addition, the solenoid valve 401 is in a closed state.

By repeating steps 2-9 described above, a thermal cycle can be applied to the sample 50 by continuously reciprocating the sample 50 between the low temperature region 38 and the high temperature region 36.

In addition, in the reaction apparatus 400 according to the present embodiment, since only one pump is used, sample movement control can be easily performed and the cost can be reduced.

The above is an explanation based on embodiments of the present invention. These embodiments are for illustrative purposes only, and it will be apparent to those skilled in the art that various modifications to the constituent elements and processes may be devised, and that such modifications are also within the scope of the present invention.

Description of reference numerals

10 - a vessel for carrying out the reaction,

12 - channel,

14 - substrate,

16 - film for sealing channels,

18 - the first sealing film,

20 - second sealing film,

24 - the first air inlet communication hole,

26 - second inlet air communication hole,

28 - the first filter,

30 - second filter,

32 - thermal cycle area,

34 - dosing area,

36 - high temperature area,

38 - low temperature region,

40 - connection area (channel),

42 - dosing channel,

43 - the first branching channel,

44 - second branching channel,

45 - the first inlet for entering the sample,

46 - the second inlet for entering the sample,

50 - sample,

100, 200, 300, 400 - reaction devices,

102 - temperature control system,

104 - high temperature heater,

105 - central processing unit (CPU),

106 - low temperature heater,

108 - high temperature heater,

110 - low-temperature heater drive,

120, 220, 320, 420 - fluid supply system,

121, 221, 321, 421 - pump,

122, 222 - pump drive,

123, 223 - the first three-way valve,

124, 224 - the second three-way valve,

125, 225 - the first tube,

126, 226 - the second tube,

127, 227 - the third tube,

128, 228 - the fourth tube,

129, 229 - the first air channel,

130, 230 - second air channel,

134, 136 - packaging material,

140 - fluorescent detector,

142 - optical head,

144 - fluorescent detector drive,

146 - optical fiber,

231 - fifth tube,

232 - sixth tube,

238 - pressure chamber,

244 - pump for pressure chamber,

245 - pressure chamber pump drive,

248 - pressure relief valve to atmospheric level,

401 - solenoid valve,

431 - first branch point,

441 is the second branch point.

Industrial Applicability

The present invention is applicable to the polymerase chain reaction (PCR).

Sequence #1: PCR Reaction Forward Primer

Sequence #2: PCR Reaction Reverse Primer

Sequence #3: Probe

[Sequence Listing] NSG-70057WO

Sequence Listing.txt.

--->

SEQUENCE LIST

<110> NIPPON SHEET GLASS COMPANY, LIMITED

<120> REACTION TREATMENT DEVICE

<130> P0013519WO01

<150> JP 2018-004295

<151> 2018-01-15

<160> 3

<170>PatentIn version 3.5

<210> 1

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Forward PCR Primer

<400> 1

ggataatttg tttgcagttg atgtc 25

<210> 2

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Reverse PCR Primer

<400> 2

caaatcctgt cacatataaa ttatttcgt 29

<210> 3

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 3

ccgtagatta ttaaaccgcc cttcctctgg a 31

<---

Claims (41)

1. Device for carrying out the reaction, containing: a reaction vessel including a passage in which the sample moves and a pair of a first air communication inlet and a second air communication inlet provided at respective ends of the channel; a temperature control unit that provides a first temperature region located on the side of the first air communication inlet and maintained at a first temperature, and a second temperature region located on the side of the second air communication inlet and maintained at a second temperature exceeding the first temperature, between the first air a communication hole and a second air communication hole in the channel; and fluid supply system that moves and stops the sample in the channel, where the sample is thermally cycled by repeatedly reciprocating the sample between the first temperature region and the second temperature region, wherein the fluid supply system includes: one pump capable of releasing air from the outlet; a first air passage that connects an outlet of the pump and a first inlet of an air communication port of the reaction vessel; a second air passage that connects the pump outlet and the second air communication inlet of the reaction vessel; a first switching valve that is located in the first air passage and is capable of switching between a state in which the first air communication inlet is in communication with the outlet and a state in which the first air communication inlet is open to atmospheric pressure; a second switching valve that is located in the second air passage and is capable of switching between a state in which the second air communication inlet is in communication with the outlet and a state in which the second air communication inlet is open to atmospheric pressure; and a control unit that controls the operation of the pump, the operation of the first switching valve and the operation of the second switching valve, and wherein the control unit is configured to: releasing air from the pump and changing the state of the first switching valve to be in a state in which the first air communication inlet port communicates with the outlet port, and the second switching valve is in a state in which the second air communication inlet port is open to atmospheric air pressure, when the sample moves from the first temperature region to the second temperature region; and releasing air from the pump and changing the state of the first switching valve to be in a state in which the first air communication inlet port is open to atmospheric pressure, and the second switching valve in a state in which the second air communication inlet port communicates with the outlet port, when the sample moves from the second temperature region to the first temperature region. 2. The reaction apparatus of claim. 1, wherein the control unit stops the release of air from the pump when the sample stops inside the channel. 3. The device for carrying out the reaction according to claim 1, where the pump is a blowdown micropump, when the sample stops inside the channel, the control unit switches the first switching valve to the state in which the first air communication inlet is open to atmospheric pressure, and the second switching valve is in the state in which the second air communication inlet is open to atmospheric pressure. 4. A device for carrying out the reaction according to any one of paragraphs. 1-3, in which the first switching valve and the second switching valve are three-way valves. 5. Device for carrying out the reaction, containing: a reaction vessel including a passage in which the sample moves and a pair of a first air communication inlet and a second air communication inlet provided at respective ends of the channel; a temperature control unit that provides a first temperature region located on the side of the first air communication inlet and maintained at a first temperature, and a second temperature region located on the side of the second air communication inlet and maintained at a second temperature exceeding the first temperature, between the first inlet an air communication hole and a second inlet air communication hole in the channel; and fluid supply system that moves and stops the sample in the channel, where the sample is thermally cycled by repeatedly reciprocating the sample between the first temperature region and the second temperature region, wherein the fluid supply system includes: one pressure chamber in which the internal pressure is maintained above atmospheric pressure in the environment of the reaction device; one pump, which is located inside the pressure chamber and is able to release air from the outlet; a first air passage that connects an outlet of the pump and a first inlet of an air communication port of the reaction vessel; a second air passage that connects the pump outlet and the second air communication inlet of the reaction vessel; a first switching valve that is located in the first air passage and is capable of switching between a state in which the first air communication inlet is in communication with the outlet and a state in which the first air communication inlet is opened to the inside of the plenum chamber; a second switching valve which is located in the second air passage and is capable of switching between a state in which the second air communication inlet is in communication with the outlet and a state in which the second air communication inlet is opened to the interior of the plenum chamber; and a control unit that controls the operation of the pump, the operation of the first switching valve and the operation of the second switching valve, and wherein the control unit is configured to: releasing air from the pump and changing the state of the first switching valve to be in a state in which the first air communication inlet port communicates with the outlet port, and the second switching valve is in a state in which the second air communication inlet port is open to the interior of the boost chamber. pressure when the sample moves from the first temperature region to the second temperature region; and releasing air from the pump and changing the state of the first switching valve to be in a state in which the first air communication inlet port is open to the inside of the plenum chamber, and the second switching valve is in a state in which the second air communication inlet port communicates with the outlet opening when the sample moves from the second temperature region to the first temperature region. 6. The reaction device according to claim 5, wherein the control unit stops air from the pump when the sample stops inside the channel. 7. The device for carrying out the reaction according to claim 5, and where the pump is a blowdown micropump, at the same time, when the sample stops inside the channel, the control unit switches the first switching valve to a state in which the first air communication inlet is open to the interior of the pressure chamber, and the second switching valve is in a state in which the second air communication inlet is open to the interior pressure chambers. 8. A device for carrying out the reaction according to any one of paragraphs. 5-7, in which the first switching valve and the second switching valve are three-way valves.

Images